WO2016142694A1 - Desorption electrospray ionisation ("desi") imaging - Google Patents

Desorption electrospray ionisation ("desi") imaging Download PDF

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Publication number
WO2016142694A1
WO2016142694A1 PCT/GB2016/050624 GB2016050624W WO2016142694A1 WO 2016142694 A1 WO2016142694 A1 WO 2016142694A1 GB 2016050624 W GB2016050624 W GB 2016050624W WO 2016142694 A1 WO2016142694 A1 WO 2016142694A1
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WO
WIPO (PCT)
Prior art keywords
ion source
target
ionisation
ion
desorption
Prior art date
Application number
PCT/GB2016/050624
Other languages
French (fr)
Inventor
Steven Derek Pringle
Emrys Jones
Stephen O'brien
Zoltán TAKÁTS
Original Assignee
Micromass Uk Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB201503879A external-priority patent/GB201503879D0/en
Priority claimed from GB201503876A external-priority patent/GB201503876D0/en
Priority claimed from GBGB1503878.9A external-priority patent/GB201503878D0/en
Priority claimed from GB201503867A external-priority patent/GB201503867D0/en
Priority claimed from GB201503864A external-priority patent/GB201503864D0/en
Priority claimed from GB201503863A external-priority patent/GB201503863D0/en
Priority claimed from GB201503877A external-priority patent/GB201503877D0/en
Priority claimed from GBGB1516003.9A external-priority patent/GB201516003D0/en
Priority claimed from GBGB1518369.2A external-priority patent/GB201518369D0/en
Priority to GB1714122.7A priority Critical patent/GB2551926B/en
Priority to PCT/GB2016/050624 priority patent/WO2016142694A1/en
Application filed by Micromass Uk Limited filed Critical Micromass Uk Limited
Publication of WO2016142694A1 publication Critical patent/WO2016142694A1/en

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Definitions

  • the present invention relates generally to the analysis of a target (which may, for example, comprise ex vivo tissue) by ambient ionisation techniques such as desorption electrospray ionisation ("DESI"), methods of analysis and diagnosis and apparatus for analysing a target using an ambient ionisation ion source.
  • a target which may, for example, comprise ex vivo tissue
  • ambient ionisation techniques such as desorption electrospray ionisation ("DESI")
  • DESI desorption electrospray ionisation
  • analyte ions generated by an ambient ionisation ion source are then subjected either to: (i) mass analysis by a mass analyser such as a quadrupole mass analyser or a Time of Flight mass analyser; (ii) ion mobility analysis (IMS) and/or differential ion mobility analysis (DMA) and/or Field Asymmetric Ion Mobility Spectrometry (FAIMS) analysis; and/or (iii) a combination of firstly ion mobility analysis (IMS) and/or differential ion mobility analysis (DMA) and/or Field Asymmetric Ion Mobility Spectrometry (FAIMS) analysis followed by secondly mass analysis by a mass analyser such as a quadrupole mass analyser or a Time of Flight mass analyser (or vice versa).
  • Various embodiments also relate to an ion mobility spectrometer and/or mass analyser and a method of ion mobility spectrometry and/or method of mass analysis.
  • Rapid evaporative ionisation mass spectrometry (“REIMS”) is a relatively new technique that is useful for the analysis of many different samples including the
  • the known approach for analysing bacterial colonies by rapid evaporative ionisation mass spectrometry involves using bipolar electrosurgical forceps and an electrosurgical RF generator.
  • a bacterial colony is scraped from the surface of an agar layer using the bipolar electrosurgical forceps and a short burst of RF voltage from the electrosurgical RF generator is applied between the bipolar electrosurgical forceps.
  • the RF voltage which is applied to the electrosurgical forceps has the result of rapidly heating the particular portion of the bacterial colony which is being analysed due to its nonzero impedance.
  • the rapid heating of the microbial mass results in an aerosol being generated.
  • the aerosol is transferred directly into a mass spectrometer and/or ion mobility
  • the aerosol sample may then be analysed by the mass spectrometer and/or ion mobility spectrometer. It is known to utilise multivariate statistical analysis in order to help distinguish and identify different samples.
  • the resulting mass spectra are similar to normal electrospray mass spectra in that they show mainly singly or multiply charged molecular ions of the analytes.
  • the desorption electrospray ionisation phenomenon was observed both in the case of conductive and insulator surfaces and for compounds ranging from nonpolar small molecules such as lycopene, the alkaloid coniceine, and small drugs, through polar compounds such as peptides and proteins. Changes in the solution that is sprayed can be used to selectively desorb and ionize particular compounds, including those in biological matrices. In vivo analysis was also demonstrated.
  • FFPE formalin-fixed, paraffin-embedded
  • a tissue sample may by fixed for 12-24 hours in 10% neutral buffered formalin and then embedded in paraffin using standardised and automated procedures. Samples may be dehydrated in graded ethanol, cleared with xylene and then embedded in molten paraffin.
  • fresh frozen samples may be stored post-surgery in liquid nitrogen. Prior to analysis, fresh frozen samples may be dehydrated in a vacuum for 1 hour at room temperature. Whereas fresh frozen samples can only be preserved for a short period of time, formalin fixed paraffin embedded samples can be archived for many years allowing retrospective reassessment and analysis.
  • desorption electrospray ionisation ion imaging of a formalin fixed tissue sample is problematic due to the fact that the formalin results in a strong signal in the resulting mass spectrum and also because weaker analyte ion signals may be suppressed.
  • a reversal agent such as urea and optionally also subjecting the tissue sample to one or more cycles of heat treatment according to various embodiments enables the cross-linkages between proteins to be reversed and the formalin and any embedding material such as paraffin to be removed from the tissue sample.
  • ambient ionisation ion imaging may be performed on chemically modified targets (e.g., formalin fixed tissue samples) which have been exposed to a reversal agent (such as urea) with the result that significantly improved ion images can be obtained (compared to conventional ion images of formalin fixed tissue samples).
  • chemically modified targets e.g., formalin fixed tissue samples
  • a reversal agent such as urea
  • the first device may comprise or form part of an ambient ion or ionisation source or the first device may generate aerosol, smoke or vapour for subsequent ionisation by an ambient ion or ionisation source or other ionisation source.
  • the first device may comprise a desorption electrospray ionisation (“DESI”) ion source or a desorption electro-flow focusing (“DEFFI”) ion source.
  • DESI desorption electrospray ionisation
  • DEFFI desorption electro-flow focusing
  • the first device may comprise an ion source selected from the group consisting of: (i) a rapid evaporative ionisation mass spectrometry (“REIMS”) ion source; (ii) a laser desorption ionisation (“LDI”) ion source; (iii) a thermal desorption ion source; (iv) a laser diode thermal desorption (“LDTD”) ion source; (v) a dielectric barrier discharge (“DBD”) plasma ion source; (vi) an Atmospheric Solids Analysis Probe (“ASAP”) ion source; (vii) an ultrasonic assisted spray ionisation ion source; (viii) an easy ambient sonic-spray ionisation (“EASI”) ion source; (ix) a desorption atmospheric pressure photoionisation (“DAPPI”) ion source; (x) a paperspray (“PS”) ion source; (xi) a jet desorption ionisation (“JeDI”) ion source
  • the first target may comprise a chemically modified target.
  • the first target may comprise a fixed tissue sample.
  • the fixed tissue sample may be fixed with either: (i) formalin or a formaldehyde solution; or (ii) formalin or a formaldehyde solution with glycerol or another chemical agent.
  • the fixed tissue sample may comprise a formalin fixed, paraffin-embedded
  • FFPE tissue sample
  • the fixed tissue sample may be further embedded in an embedding medium selected from the group consisting of: (i) glycerol; (ii) optimum cutting temperature (“OCT") (RTM) medium; (iii) paraffin; (iv) one or more plastic polymers; (v) polyester wax; (vi) polyethylene glycol (“PEG”); (vii) polyfin (RTM); (viii) a tissue freezing medium (“TFM”); (ix) glycol methacrylate (“GMA”); (x) gelatin; and (xi) carboxy methyl cellulose (“SMC”).
  • an embedding medium selected from the group consisting of: (i) glycerol; (ii) optimum cutting temperature (“OCT") (RTM) medium; (iii) paraffin; (iv) one or more plastic polymers; (v) polyester wax; (vi) polyethylene glycol (“PEG”); (vii) polyfin (RTM); (viii) a tissue freezing medium (“TFM”); (ix) glycol
  • the first target may comprise a tissue sample wherein methylene bridge cross- linkages are formed between proteins.
  • the first target may comprise a tissue sample which has been dehydrated with an alcohol.
  • the first target may comprise a tissue sample which has been infiltrated with molten paraffin.
  • the first target and/or the modified target may be provided on a substrate.
  • the substrate may be selected from the group consisting of: (i) a microscope slide;
  • a transparent slide (ii) a transparent slide; (iii) a glass slide; (iv) a plastic slide; and (v) an indium-tin-oxide (ITO) coated glass or other slide.
  • ITO indium-tin-oxide
  • the step of applying the reversal agent to the first target may reverse cross-linked bonds of proteins present in the first target.
  • the reversal agent may be arranged to reverse cross-linked bonds of formalin fixed tissue proteins or formalin fixed paraffin embedded (“FFPE”) tissue proteins.
  • FFPE formalin fixed paraffin embedded
  • the reversal agent may comprise a formalin or formaldehyde scavenger.
  • the reversal agent may comprise water.
  • the reversal agent may comprise sodium metabisulfate or sodium metabisulfite (Na 2 S 2 0 5 ).
  • the reversal agent may comprise urea.
  • the reversal agent may be selected from the group consisting of: (i) at least one dithioxamide; (ii) at least one carbodiimide; (iii) at least one monomeric, oligomeric or polymeric carbodiimide; (iv) at least one aminoplastic, thermoplastic, binder, polymeric compound or copolymeric compound which are able to cleave or eliminate formalin or formaldehyde; (v) pyrogallol; (vi) 1 ,2 hexanediol; (vii) one or more polyhydric alcohols, such as diethylene glycol and sorbitol; (viii) a nitrogen containing compound such as urea, melamine, diazine, triazine and amine compounds; (ix) a nitrogenous compound such as urea, ethyleneurea or carbohydrazide; (x) Dimethyl-1 ,3-acetonedicarboxylate,
  • the reversal agent may comprise an organic solvent.
  • the reversal agent may comprise an organic compound having at least one active methylene hydrogen and a pKa (logarithmic acid dissociation constant) in the range 5-13.
  • the step of applying the reversal agent to the first target may comprise applying the reversal agent to the first target at a concentration of: (i) 0-5 % w/v; (ii) 5-10 % w/v; (iii) 10- 15 % w/v; (iv) 15-20% w/v; (v) 20-25% w/v; (vi) 25-30 % w/v; (vii) 30-35% w/v; (viii) 35-40 % w/v; (ix) 40-45 % w/v; (x) 45-50 % w/v; (xi) 50-55% % w/v; (xii) 55-60% w/v; (xiii) 60-65% w/v; (xiv) 65-70% w/v; (xv) 70-75%; and (xvi) > 75 % w/v.
  • the step of applying the reversal agent to the first target may comprise applying the reversal agent to the first target at a concentration of: (i) 0-1 M; (ii) 1-2 M; (iii) 2-3 M; (iv) 3-4 M; (v) 4-5 M; (vi) 5-6 M; (vii) 6-7 M; (viii) 7-8 M; (ix) 8-9 M; (x) 9-10 M; (xi) 10-11 M; (xii) 11- 12 M; (xiii) 12-13 M; (xiv) 13-14 M; (xv) 14-15 M; and (xvi) > 15 M.
  • the method may further comprise subjecting the first target or the modified target to one or more heat treatment cycles.
  • a or each heat treatment cycle may comprise heating the first target or the modified target at a temperature selected from the group consisting of: (i) 20-30 °C; (ii) 30-40 °C; (iii) 40-50 °C; (iv) 50-60 °C; (v) 60-70 °C; (vi) 70-80 °C; (vii) 80-90 °C; (viii) 90-100 °C; (ix) 100- 1 10 °C; (x) 110-120 °C; and (xi) > 120 °C.
  • a or each heat treatment cycle may comprise heating the first target or the modified target for a period of time selected from the group consisting of: (i) 0-1 minutes; (ii) 1-2 minutes; (iii) 2-3 minutes; (iv) 3-4 minutes; (v) 4-5 minutes; (vi) 5-6 minutes; (vii) 6-7 minutes; (viii) 7-8 minutes; (ix) 8-9 minutes; (x) 9-10 minutes; and (xi) > 10 minutes.
  • the reversal agent may be applied by the first device.
  • a method of ambient ionisation comprising a method as disclosed above.
  • DESI desorption electrospray ionisation
  • DEFFI desorption electroflow focusing ionisation
  • a method of ion imaging comprising a method as disclosed above.
  • a method of analysis comprising a method as disclosed above.
  • a non-surgical, non-therapeutic method of mass spectrometry and/or ion mobility spectrometry comprising a method as disclosed above.
  • a method of mass spectrometry and/or ion mobility spectrometry comprising a method as disclosed above.
  • analyte ions generated by the ambient ionisation ion source are then subjected either to: (i) mass analysis by a mass analyser or filter such as a quadrupole mass analyser or a Time of Flight mass analyser; (ii) ion mobility analysis (IMS) and/or differential ion mobility analysis (DMA) and/or Field Asymmetric Ion Mobility Spectrometry (FAIMS) analysis; and/or (iii) a combination of firstly ion mobility analysis (IMS) and/or differential ion mobility analysis (DMA) and/or Field Asymmetric Ion Mobility Spectrometry (FAIMS) analysis followed by secondly mass analysis by a mass analyser or filter such as a quadrupole mass analyser or a Time of Flight mass analyser (or vice versa).
  • Various embodiments also relate to an ion mobility spectrometer and/or mass analyser and a method of ion mobility spectrometry and/or method of mass
  • apparatus comprising:
  • a first device arranged and adapted to generate analyte or analyte ions from multiple regions of a modified target, wherein the modified target comprises a first target to which a reversal agent has been applied;
  • the first device may comprise or form part of an ambient ion or ionisation source or the first device may generate aerosol, smoke or vapour for subsequent ionisation by an ambient ion or ionisation source or other ionisation source.
  • the first device may comprise a desorption electrospray ionisation (“DESI”) ion source or a desorption electro-flow focusing (“DEFFI”) ion source.
  • DESI desorption electrospray ionisation
  • DEFFI desorption electro-flow focusing
  • the first device may comprise an ion source selected from the group consisting of: (i) a rapid evaporative ionisation mass spectrometry (“REIMS”) ion source; (ii) a laser desorption ionisation (“LDI”) ion source; (iii) a thermal desorption ion source; (iv) a laser diode thermal desorption (“LDTD”) ion source; (v) a dielectric barrier discharge (“DBD”) plasma ion source; (vi) an Atmospheric Solids Analysis Probe (“ASAP”) ion source; (vii) an ultrasonic assisted spray ionisation ion source; (viii) an easy ambient sonic-spray ionisation (“EASI”) ion source; (ix) a desorption atmospheric pressure photoionisation (“DAPPI”) ion source; (x) a paperspray (“PS”) ion source; (xi) a jet desorption ionisation (“JeDI”) ion source
  • the first target may comprise a chemically modified target.
  • the first target may comprise a fixed tissue sample.
  • the fixed tissue sample may be fixed with either: (i) formalin or a formaldehyde solution; or (ii) formalin or a formaldehyde solution with glycerol or another chemical agent.
  • the fixed tissue sample may comprise a formalin fixed, paraffin-embedded
  • FFPE tissue sample
  • the fixed tissue sample may be further embedded in an embedding medium selected from the group consisting of: (i) glycerol; (ii) optimum cutting temperature (“OCT") (RTM) medium; (iii) paraffin; (iv) one or more plastic polymers; (v) polyester wax; (vi) polyethylene glycol (“PEG”); (vii) polyfin (RTM); (viii) a tissue freezing medium (“TFM”); (ix) glycol methacrylate (“GMA”); (x) gelatin; and (xi) carboxy methyl cellulose (“SMC”).
  • an embedding medium selected from the group consisting of: (i) glycerol; (ii) optimum cutting temperature (“OCT") (RTM) medium; (iii) paraffin; (iv) one or more plastic polymers; (v) polyester wax; (vi) polyethylene glycol (“PEG”); (vii) polyfin (RTM); (viii) a tissue freezing medium (“TFM”); (ix) glycol
  • the first target may comprise a tissue sample wherein methylene bridge cross- linkages are formed between proteins.
  • the first target may comprise a tissue sample which has been dehydrated with an alcohol.
  • the first target may comprise a tissue sample which has been infiltrated with molten paraffin.
  • the first target and/or the modified target may be provided on a substrate.
  • the substrate may be selected from the group consisting of: (i) a microscope slide; (ii) a transparent slide; (iii) a glass slide; (iv) a plastic slide; and (v) an indium-tin-oxide (ITO) coated glass or other slide.
  • the reversal agent may be applied to the first target in order to reverse cross-linked bonds of proteins present in the first target.
  • the reversal agent may be arranged to reverse cross-linked bonds of formalin fixed tissue proteins or formalin fixed paraffin embedded (“FFPE”) tissue proteins.
  • FFPE formalin fixed paraffin embedded
  • the reversal agent may comprise a formalin or formaldehyde scavenger.
  • the reversal agent may comprise water.
  • the reversal agent may comprise sodium metabisulfate or sodium metabisulfite (Na 2 S 2 0 5 ).
  • the reversal agent may comprise urea.
  • the reversal agent may be selected from the group consisting of: (i) at least one dithioxamide; (ii) at least one carbodiimide; (iii) at least one monomeric, oligomeric or polymeric carbodiimide; (iv) at least one aminoplastic, thermoplastic, binder, polymeric compound or copolymeric compound which may be able to cleave or eliminate formalin or formaldehyde; (v) pyrogallol; (vi) 1 ,2 hexanediol; (vii) one or more polyhydric alcohol, such as diethylene glycol and sorbitol; (viii) a nitrogen containing compound such as urea, melamine, diazine, triazine and amine compounds; (ix) a nitrogenous compound such as urea, ethyleneurea or carbohydrazide; (x) Dimethyl-1 ,3-acetonedicarboxylate, diethyl malonate or ethylaceto
  • the reversal agent may comprise an organic solvent.
  • the reversal agent may comprise an organic compound having at least one active methylene hydrogen and a pKa (logarithmic acid dissociation constant) in the range 5-13.
  • the reversal agent may be applied to the first target at a concentration of: (i) 0-5 % w/v; (ii) 5-10 % w/v; (iii) 10-15 % w/v; (iv) 15-20% w/v; (v) 20-25% w/v; (vi) 25-30 % w/v; (vii) 30-35% w/v; (viii) 35-40 % w/v; (ix) 40-45 % w/v; (x) 45-50 % w/v; (xi) 50-55% % w/v; (xii) 55-60% w/v; (xiii) 60-65% w/v; (xiv) 65-70% w/v; (xv) 70-75%; and (xvi) > 75 % w/v.
  • the reversal agent may be applied to the first target at a concentration of: (i) 0-1 M;
  • the first target or the modified target may be subjected to one or more heat treatment cycles.
  • a or each heat treatment cycle may comprise heating the first target or the modified target at a temperature selected from the group consisting of: (i) 20-30 °C; (ii) 30-40 °C; (iii) 40-50 °C; (iv) 50-60 °C; (v) 60-70 °C; (vi) 70-80 °C; (vii) 80-90 °C; (viii) 90-100 °C; (ix) 100- 1 10 °C; (x) 110-120 °C; and (xi) > 120 °C.
  • a or each heat treatment cycle may comprise heating the first target or the modified target for a period of time selected from the group consisting of: (i) 0-1 minutes; (ii) 1-2 minutes; (iii) 2-3 minutes; (iv) 3-4 minutes; (v) 4-5 minutes; (vi) 5-6 minutes; (vii) 6-7 minutes; (viii) 7-8 minutes; (ix) 8-9 minutes; (x) 9-10 minutes; and (xi) > 10 minutes.
  • the reversal agent may be applied by the first device.
  • an ambient ionisation ion source comprising apparatus as disclosed above.
  • DESI desorption electrospray ionisation
  • DEFFI desorption electroflow focusing ionisation
  • an ion imager comprising apparatus as disclosed above.
  • analysis apparatus comprising apparatus as disclosed above.
  • a mass and/or ion mobility spectrometer comprising apparatus as disclosed above.
  • analyte ions generated by the ambient ionisation ion source are then subjected either to: (i) mass analysis by a mass analyser or filter such as a quadrupole mass analyser or a Time of Flight mass analyser; (ii) ion mobility analysis (IMS) and/or differential ion mobility analysis (DMA) and/or Field Asymmetric Ion Mobility Spectrometry (FAIMS) analysis; and/or (iii) a combination of firstly ion mobility analysis (IMS) and/or differential ion mobility analysis (DMA) and/or Field Asymmetric Ion Mobility Spectrometry (FAIMS) analysis followed by secondly mass analysis by a mass analyser or filter such as a quadrupole mass analyser or a Time of Flight mass analyser (or vice versa).
  • Various embodiments also relate to an ion mobility spectrometer and/or mass analyser and a method of ion mobility spectrometry and/or method of mass
  • apparatus comprising:
  • an ion source comprising a nozzle having an aperture
  • a centering device which is arranged and adapted to locate or position the solvent emitter substantially centrally within the aperture.
  • the ion source may comprise a Desorption Electrospray lonisation (“DESI”) ion source or a desorption electro-flow focusing (“DEFFI”) ion source.
  • DESI Desorption Electrospray lonisation
  • DEFFI desorption electro-flow focusing
  • the apparatus may further comprise a solvent emitter guide.
  • the solvent emitter may be located or positioned within the solvent emitter guide.
  • the centering device may be arranged and adapted to locate or position the solvent emitter guide substantially centrally within an internal volume of the nozzle.
  • the solvent emitter guide may be electrically conductive.
  • the solvent emitter guide may comprise metal.
  • the solvent emitter guide may comprise stainless steel.
  • the nozzle may be electrically conductive.
  • the nozzle may comprise metal.
  • the nozzle may comprise stainless steel.
  • the centering device may comprise a disc.
  • the centering device may comprise or may be coated with an insulator.
  • the centering device may comprise or may be coated with a ceramic.
  • the centering device may comprise or may be coated with a polymer.
  • the solvent emitter guide may be electrically insulating.
  • the solvent emitter guide may comprise a ceramic.
  • the solvent emitter guide may comprise a polymer.
  • the apparatus may further comprise a device for supplying a solvent to the solvent emitter.
  • the apparatus may further comprise a device for supplying a nebulising gas within the nozzle so that, in use, the nebulising gas exits the nozzle via the aperture.
  • an ambient ionisation ion source comprising apparatus as disclosed above.
  • DESI desorption electrospray ionisation
  • DEFFI desorption electroflow focusing ionisation
  • an ion imager comprising apparatus as disclosed above.
  • analysis apparatus comprising apparatus as disclosed above.
  • a mass and/or ion mobility spectrometer comprising apparatus as disclosed above.
  • an ion source comprising a nozzle having an aperture
  • a method of mass spectrometry and/or ion mobility spectrometry comprising a method as disclosed above.
  • apparatus comprising:
  • a first device arranged and adapted to emit a stream of electrically charged droplets towards a target in use
  • a transfer capillary or transfer device arranged and adapted to transfer ions generated from the target towards an ion analyser or mass and/or ion mobility
  • a heating device arranged and adapted to heat either: (i) a capillary of the first device; (ii) the stream of electrically charged droplets emitted from the first device; (iii) the target; or (iv) the transfer capillary or transfer device.
  • the first device may comprise a Desorption Electrospray lonisation (“DESI”) ion source or a desorption electroflow focusing ionisation (“DEFFI”) ion source.
  • DESI Desorption Electrospray lonisation
  • DEFFI desorption electroflow focusing ionisation
  • the heating device may comprise a heater.
  • the heater may comprise a wire heater.
  • the heater may comprise an inductive or eddy current heater.
  • the heating device may be arranged and adapted to heat the capillary of the first device, the stream of electrically charged droplets emitted from the first device, the target or the transfer capillary or transfer device to a temperature of at least 50 °C, 100 °C, 200 °C, 300 °C, 400 °C, 500 °C, 600 °C, 700 °C or 800 °C.
  • a mass and/or ion mobility spectrometer comprising apparatus as disclosed above.
  • the first device may comprise a Desorption Electrospray lonisation (“DESI”) ion source or a desorption electroflow focusing ionisation (“DEFFI”) ion source.
  • DESI Desorption Electrospray lonisation
  • DEFFI desorption electroflow focusing ionisation
  • the heating device may comprise a heater.
  • the heater may comprise a wire heater.
  • the heater may comprise an inductive or eddy current heater.
  • the method may comprise heating the capillary of the first device, the stream of electrically charged droplets emitted from the first device, the target or the transfer capillary or transfer device to a temperature of at least 50 °C, 100 °C, 200 °C, 300 °C, 400 °C, 500 °C, 600 °C, 700 °C or 800 °C.
  • a method of mass spectrometry and/or ion mobility spectrometry comprising a method as disclosed above.
  • apparatus comprising:
  • an ion source comprising a nozzle having an aperture
  • a fixing device which is arranged and adapted to fix the solvent emitter relative to the aperture.
  • the fixing device may comprise a centering device which is arranged and adapted to locate or position the solvent emitter substantially centrally within the aperture.
  • the fixing device comprises a device which is arranged and adapted to locate or position the solvent emitter substantially non-centrally within the aperture.
  • the fixing device may comprise a device which is arranged and adapted to locate or position the solvent emitter substantially non-centrally within the aperture, such that the solvent emitter is substantially closer to and/or oriented towards an inlet of a mass and/or ion mobility spectrometer.
  • the fixing device may be arranged and adapted such that gas may flow through the nozzle and/or aperture relatively unimpeded by the fixing device.
  • apparatus comprising:
  • an ion source comprising a nozzle having an aperture
  • a device which is arranged and adapted to monitor the solvent emitter position relative to the aperture.
  • the apparatus may comprise a device arranged and adapted to calibrate and/or correct the ion source and/or mass and/or ion mobility spectrometric data acquired using the ion source based on the monitoring.
  • a mass and/or ion mobility spectrometer comprising apparatus as described above.
  • an ion source comprising a nozzle having an aperture
  • the method may comprise using the fixing device to locate or position the solvent emitter substantially centrally within the aperture.
  • the method may comprise using the fixing device to locate or position the solvent emitter substantially non-centrally within the aperture.
  • the method may comprise using the fixing device to locate or position the solvent emitter substantially non-centrally within the aperture, such that the solvent emitter is substantially closer to and/or oriented towards an inlet of a mass and/or ion mobility spectrometer.
  • the method may comprise passing gas through the nozzle and/or aperture such that the gas flows relatively unimpeded by the fixing device.
  • an ion source comprising a nozzle having an aperture
  • the method may comprise calibrating and/or correct the ion source and/or mass and/or ion mobility spectrometric data acquired using the ion source based on the monitoring.
  • a method of mass spectrometry and/or ion mobility spectrometry comprising a method as described above.
  • the mass and/or ion mobility spectrometer may obtain data in negative ion mode only, positive ion mode only, or in both positive and negative ion modes.
  • Positive ion mode spectrometric data may be combined or concatenated with negative ion mode
  • Ion mobility spectrometric data may be obtained using different ion mobility drift gases and/or dopants. This data may then be combined or concatenated.
  • Various embodiments are contemplated which relate to generating smoke, aerosol or vapour from a target (details of which are provided elsewhere herein) using an ambient ionisation ion source.
  • the aerosol, smoke or vapour may then be mixed with a matrix and aspirated into a vacuum chamber of a mass spectrometer and/or ion mobility spectrometer.
  • the mixture may be caused to impact upon a collision surface causing the aerosol, smoke or vapour to be ionised by impact ionization which results in the generation of analyte ions.
  • the resulting analyte ions may then be mass analysed and/or ion mobility analysed and the resulting mass spectrometric data and/or ion mobility spectrometric data may be subjected to multivariate analysis or other mathematical treatment in order to determine one or more properties of the target in real time.
  • the first device for generating aerosol, smoke or vapour from the target may comprise a tool which utilises an RF voltage, such as a continuous RF waveform.
  • the first device for generating aerosol, smoke or vapour from the target may comprise an argon plasma coagulation ("APC") device.
  • An argon plasma coagulation device involves the use of a jet of ionised argon gas (plasma) that is directed through a probe. The probe may be passed through an endoscope.
  • Argon plasma coagulation is essentially a non-contact process as the probe is placed at some distance from the target. Argon gas is emitted from the probe and is then ionized by a high voltage discharge (e.g., 6 kV). High-frequency electric current is then conducted through the jet of gas, resulting in coagulation of the target on the other end of the jet.
  • the depth of coagulation is usually only a few millimetres.
  • the first device, surgical or electrosurgical tool, device or probe or other sampling device or probe disclosed in any of the aspects or embodiments herein may comprise a non-contact surgical device, such as one or more of a hydrosurgical device, a surgical water jet device, an argon plasma coagulation device, a hybrid argon plasma coagulation device, a water jet device and a laser device.
  • a non-contact surgical device such as one or more of a hydrosurgical device, a surgical water jet device, an argon plasma coagulation device, a hybrid argon plasma coagulation device, a water jet device and a laser device.
  • a non-contact surgical device may be defined as a surgical device arranged and adapted to dissect, fragment, liquefy, aspirate, fulgurate or otherwise disrupt biologic tissue without physically contacting the tissue. Examples include laser devices, hydrosurgical devices, argon plasma coagulation devices and hybrid argon plasma coagulation devices.
  • the procedure may be seen as relatively safe and can be used to treat delicate tissue having low intracellular bonds, such as skin or fat.
  • the mass spectrometer and/or ion mobility spectrometer may obtain data in negative ion mode only, positive ion mode only, or in both positive and negative ion modes.
  • Positive ion mode spectrometric data may be combined or concatenated with negative ion mode spectrometric data.
  • Negative ion mode can provide particularly useful spectra for classifying aerosol, smoke or vapour samples, such as aerosol, smoke or vapour samples from targets comprising lipids.
  • Ion mobility spectrometric data may be obtained using different ion mobility drift gases, or dopants may be added to the drift gas to induce a change in drift time of one or more species. This data may then be combined or concatenated.
  • the ambient ionisation ion source may comprise an ultrasonic ablation ion source or a hybrid electrosurgical -ultrasonic ablation source that generates a liquid sample which is then aspirated as an aerosol.
  • the ultrasonic ablation ion source may comprise a focused or unfocussed ultrasound.
  • the first device comprises or forms part of an ion source selected from the group consisting of: (i) a rapid evaporative ionisation mass spectrometry (“REIMS”) ion source; (ii) a desorption electrospray ionisation (“DESI”) ion source; (iii) a laser desorption ionisation (“LDI”) ion source; (iv) a thermal desorption ion source; (v) a laser diode thermal desorption (“LDTD”) ion source; (vi) a desorption electro-flow focusing (“DEFFI”) ion source; (vii) a dielectric barrier discharge (“DBD”) plasma ion source; (viii) an Atmospheric Solids Analysis Probe (“ASAP”) ion source; (ix) an ultrasonic assisted spray ionisation ion source; (x) an easy ambient sonic-spray ionisation (“EASI”) ion source; (xi) a desorption atmospheric pressure
  • Fig. 1A shows a desorption electrospray ionisation ion image for counts in the range 0-250 at m/z 726.5 obtained in negative ion mode from fresh pork liver embedded in a cryoprotective embedding medium OCT
  • Fig. 1 B shows a desorption electrospray ionisation image for counts in the range 0-12000 at m/z 766.5 obtained in negative ion mode from fresh pork liver embedded in a cryoprotective embedding medium OCT
  • Fig. 1 C shows a desorption electrospray ionisation image for counts in the range 0-1400 at m/z 740.5 obtained in negative ion mode from fresh pork liver embedded in a cryoprotective embedding medium OCT;
  • Fig. 2A shows a desorption electrospray ionisation mass spectrometry imaging (MSI) ion image obtained in negative ion mode of formalin fixed pork liver embedded in OCT and Fig. 2B shows a mass spectrum obtained in negative ion mode from formalin fixed pork liver embedded in OCT;
  • MSI mass spectrometry imaging
  • Fig. 3A shows a spectrum intensity in negative ion mode from frozen fresh pork liver at m/z 885.5 (characteristic of phosphatidylinositol (Pl(18:0/20:4)) and Fig. 3B shows a corresponding spectrum intensity from formalin fixed pork liver washed in LC-MS grade water at m/z 885.5 (characteristic of phosphatidylinositol (Pl(18:0/20:4));
  • Fig. 4A shows a desorption electrospray ionisation mass spectrum obtained in negative ion mode from fresh frozen pork liver and Fig. 4B shows a desorption electrospray ionisation mass spectrum from formalin fixed pork liver washed in LC-MS grade water showing a lower signal intensity;
  • Fig. 5A shows a spectrum intensity in negative ion mode from fresh frozen pork liver
  • Fig. 5B shows a spectrum intensity from formalin fixed pork liver
  • Fig. 5C shows a spectrum intensity from heat-treated formalin fixed pork liver
  • Fig. 6A shows a desorption electrospray ionisation mass spectrometry imaging (MSI) ion image obtained in negative ion mode from frozen fresh pork liver
  • Fig. 6B shows a desorption electrospray ionisation mass spectrometry imaging (MSI) ion image obtained from formalin fixed pork liver
  • Fig. 6C shows a desorption electrospray ionisation mass spectrometry imaging (MSI) ion image obtained from heat-treated formalin fixed pork liver;
  • Fig. 7 A shows spectra intensity obtained in negative ion mode from formalin fixed pork liver heat-treated with 15% (w/v) sodium metabisulfate and Fig. 7B shows a corresponding ion image for m/z 794.5;
  • Fig. 8A shows a microscope image from formalin fixed pork liver heat-treated with sodium metabisulfate prior to desorption electrospray ionisation mass spectrometry imaging (MSI) analysis and Fig. 8B shows a corresponding microscope image post- desorption electrospray ionisation mass spectrometry imaging (MSI) analysis;
  • Fig. 9A shows a spectral intensity obtained in negative ion mode of formalin fixed pork liver using sodium metabisulfate 15% (w/v) and one heat-treatment cycle as formalin scavengers
  • Fig. 9B shows a spectral intensity obtained in negative ion mode of formalin fixed pork liver using sodium metabisulfate 15% (w/v) and three heat-treatment cycles as formalin scavengers
  • Fig. 9C shows a spectral intensity obtained in negative ion mode of formalin fixed pork liver using sodium metabisulfate 40% (w/v) and one heat-treatment cycle as formalin scavengers;
  • Fig. 10A shows a desorption electrospray ionisation mass spectrometry imaging (MSI) ion image at m/z 794.53 from formalin fixed pork liver, heat-treated for 3 cycles with 15% (w/v) sodium metabisulfate after desorption electrospray ionisation mass spectrometry imaging (MSI) analysis in negative ion mode ion image and Fig. 10B shows a
  • Fig. 11 shows a comparison of spectra intensity obtained in negative ion mode from formalin fixed pork liver using either 1 M or 5M of urea and either 3 or 5 heat-treatment cycles as formalin scavengers;
  • Fig. 12A shows a microscope image of formalin fixed pork liver post-desorption electrospray ionisation mass spectrometry imaging (MSI) analysis after being subjected to 5M urea and 3 heat-treatment cycles using a SuperFrost slide
  • Fig. 12B shows a microscope image of formalin fixed pork liver post-desorption electrospray ionisation mass spectrometry imaging (MSI) analysis after being subjected to 5M urea and 3 heat-treatment cycles using a SuperFrostPlus slide
  • Fig. 12A shows a microscope image of formalin fixed pork liver post-desorption electrospray ionisation mass spectrometry imaging (MSI) analysis after being subjected to 5M urea and 3 heat-treatment cycles using a SuperFrostPlus slide
  • MSI formalin fixed pork liver post-desorption electrospray ionisation mass spectrometry imaging
  • FIG. 12C shows a microscope image of formalin fixed pork liver post-desorption electrospray ionisation mass spectrometry imaging (MSI) analysis after being subjected to 5M urea and 5 heat-treatment cycles on a Poly-Lysine slide;
  • MSI mass spectrometry imaging
  • Fig. 13A shows an ion image of formalin fixed pork liver heat-treated for 3 cycles with 5M urea after desorption electrospray ionisation mass spectrometry imaging (MSI) analysis in negative ion mode with ion image (m/z 794.53) using a SuperFrost slide and Fig. 13B shows an ion image (m/z 794.53) using a SuperFrostPlus slide;
  • MSI mass spectrometry imaging
  • Fig. 14 shows a desorption electrospray ionisation mass spectrometry imaging (MSI) image of a human lung biopsy after EVLP treatment wherein the image comprises a RGB image of PCs 1 , 2 and 5 for a human lung tissue sample (EXT8-T2-S1);
  • MSI mass spectrometry imaging
  • Fig. 15 shows a method of analysis that comprises building a classification model according to various embodiments
  • Fig. 16 shows a set of reference sample mass spectra obtained from two classes of known reference samples
  • Fig. 17 shows a multivariate space having three dimensions defined by intensity axes, wherein the multivariate space comprises plural reference points, each reference point corresponding to a set of three peak intensity values derived from a reference sample spectrum;
  • Fig. 18 shows a general relationship between cumulative variance and number of components of a PCA model
  • Fig. 19 shows a PCA space having two dimensions defined by principal component axes, wherein the PCA space comprises plural transformed reference points or scores, each transformed reference point corresponding to a reference point of Fig. 17;
  • Fig. 20 shows a PCA-LDA space having a single dimension or axis, wherein the
  • the LDA is performed based on the PCA space of Fig. 19, the PCA-LDA space comprising plural further transformed reference points or class scores, each further transformed reference point or class score corresponding to a transformed reference point or score of
  • Fig. 21 shows a method of analysis that comprises using a classification model according to various embodiments
  • Fig. 22 shows a sample spectrum obtained from an unknown sample
  • Fig. 23 shows the PCA-LDA space of Fig. 20, wherein the PCA-LDA space further comprises a PCA-LDA projected sample point derived from the peak intensity values of the sample spectrum of Fig. 22;
  • Fig. 24 shows a method of analysis that comprises building a classification library according to various embodiments
  • Fig. 25 shows a method of analysis that comprises using a classification library according to various embodiments
  • Fig. 26A shows RGB images of PCA components 1 , 2 and 3 in negative ion mode along with a respective histological image of a Grade II Invasive Ductal Carcinoma (IDC) and Fig. 26B shows RGB images of PCA components 1 , 2 and 3 in positive ion mode along with a respective histological image of a Grade II Invasive Ductal Carcinoma (IDC);
  • IDC Invasive Ductal Carcinoma
  • Fig. 27A shows PCA analysis of a Grade II Invasive Ductal Carcinoma in negative ion mode and Fig. 27B shows MMC analysis of a Grade II Invasive Ductal Carcinoma in negative ion mode;
  • Fig. 28A shows PCA analysis of a Grade II Invasive Ductal Carcinoma in positive ion mode and Fig. 28B shows MMC analysis of a Grade II Invasive Ductal Carcinoma in positive ion mode;
  • Fig. 29A shows leave one out cross validation of different tissue types in a Grade II Invasive Ductal Carcinoma in negative ion mode and Fig. 29B shows leave one out cross validation of different tissue types in a Grade II Invasive Ductal Carcinoma in positive ion mode; Fig. 30 shows histological images with annotated regions of interest by a histopathologist together with PCA analysis of these assigned regions and MMC
  • Fig. 31 A shows analysis of a combined dataset from multiple samples (negative ion mode) of PCA of identified regions
  • Fig. 31 B shows MMC supervised analysis
  • Fig. 31 C shows MMC analysis excluding the samples with outliers identified in Fig. 31 B
  • Fig. 31 D shows respective leave-one-region-per-patient-out cross validation
  • Fig. 32A shows PCA of identified regions
  • Fig. 32B shows MMC supervised analysis
  • Fig. 32C shows leave-one-region-per-patient-out cross validation for the positive ion mode data
  • Fig. 33A shows supervised MMC analysis of healthy ovary, borderline tumours and carcinomas and Fig. 33B shows corresponding leave one patient out cross validation for negative ion mode;
  • Fig. 34A shows supervised MMC analysis of healthy ovary and different epithelial carcinomas (endometrioid and serous) and Fig. 34B shows corresponding leave one patient out cross validation for negative ion mode;
  • Fig. 35A shows a sample with unknown histology used to predict different tissue types and Fig. 35B shows a cross validation of the prediction based on the histological annotation;
  • Fig. 36 shows a schematic of a desorption electroflow focusing ionisation ("DEFFI”) setup according to an embodiment
  • Fig. 37 shows a total ion count for desorption electroflow focusing ionisation ("DEFFI”) on a pork liver at different applied voltages in negative ion mode;
  • DEFFI desorption electroflow focusing ionisation
  • Fig. 38A shows a desorption electroflow focusing ionisation ("DEFFI”) mass spectrometric image of human breast cancer tissue and shows RGB images of the first three principal components from principal component analysis of all pixels using the full acquired m/z range from 150 to 1500
  • Fig. 38B shows base peak intensity (BPI) normalised example spectra for breast tissue for desorption electroflow focusing ionisation (“DEFFI”)
  • Fig. 38C shows desorption traces in line-scanning mode on rhodamine B-coated glass slides for desorption electroflow focusing ionisation (“DEFFI”)
  • FIG. 38D shows a desorption electrospray ionisation mass spectrometric image of human breast cancer tissue and shows RGB images of the first three principal components from principal component analysis of all pixels using the full acquired m/z range from 150 to 1500
  • Fig. 38E shows base peak intensity (BPI) normalised example spectra for breast tissue for desorption electrospray ionisation
  • Fig. 38F shows desorption traces in line-scanning mode on rhodamine B-coated glass slides for desorption electrospray ionisation;
  • Fig. 39A shows a RGB image for pork liver of the first three principal components of principal component analysis across the full m/z range for desorption electroflow focusing ionisation (“DEFFI”)
  • Fig. 39B shows a RGB image for pork liver of the first three principal components of principal component analysis across the full m/z range for desorption electrospray ionisation
  • Fig. 39C shows representative spectra from pork liver in negative ion mode for desorption electroflow focusing ionisation (“DEFFI”)
  • Fig. 39D shows representative spectra from pork liver in negative ion mode for desorption electrospray ionisation
  • Fig. 39E show desorption traces on rhodamine B for both methods for three different solvent flow rates;
  • Fig. 40 shows normalised base peak intensity for pork liver in negative ion mode for desorption electroflow focusing ionisation ("DEFFI”) and desorption electrospray ionisation at three different solvent flow rates;
  • DEFFI desorption electroflow focusing ionisation
  • Fig. 41 shows a diagram of a sprayer nozzle according to an embodiment wherein a stainless steel emitter guide and gas nozzle are electrically insulated from each other to avoid charging of the gas nozzle and wherein this is achieved by using a ceramic-coated centering disc;
  • Fig. 42A shows desorption electrospray ionisation-MS imaging of adjacent colorectal cancer sections at different spatial resolutions wherein RGB images for the first three components of principal component analysis of images were acquired at 100 ⁇
  • Fig. 42B shows RGB images for the first three components of principal component analysis of images which were acquired at 50 ⁇
  • Fig. 42C shows RGB images for the first three components of principal component analysis of images which were acquired at 20 ⁇ pixel size/ scan speed per second
  • Fig. 42D shows a trace on rhodamine B corresponding to the experimental conditions of Fig. 42A
  • Fig. 42E shows a trace on rhodamine B corresponding to the experimental conditions of Figs. 42B and 42C
  • Fig. 42F shows an optical image of section A after hematoxylin and eosin staining
  • Fig. 42G shows an example spectrum from section A;
  • Fig. 43 shows a Desorption Electrospray Ionisation ("DESI”) device according to another embodiment.
  • Fig. 44A shows a graph of intensity versus inlet capillary temperature for analysis of fatty acids using a Waters Synapt (RTM) mass spectrometer
  • Fig. 44B shows a graph of intensity versus inlet capillary temperature for analysis of fatty acids using a Waters Xevo (RTM) mass spectrometer
  • Fig. 44C shows a graph of intensity versus inlet capillary temperature for analysis of phospholipids using a Waters Synapt (RTM) mass
  • Fig. 44D shows a graph of intensity versus inlet capillary temperature for analysis of phospholipids using a Waters Xevo (RTM) mass spectrometer.
  • a target e.g., ex vivo tissue
  • an ambient ionisation ion source such as a desorption electrospray ionisation (“DESI”) ion source.
  • DESI desorption electrospray ionisation
  • a device is used to generate analyte ions from one or more regions of a target (e.g., ex vivo tissue).
  • the device may comprise an ambient ionisation ion source which is characterised by the ability to generate analyte aerosol, smoke or vapour from a native or unmodified target.
  • a target e.g., ex vivo tissue
  • the device may comprise an ambient ionisation ion source which is characterised by the ability to generate analyte aerosol, smoke or vapour from a native or unmodified target.
  • other types of ionisation ion sources such as Matrix Assisted Laser Desorption lonisation (“MALDI”) ion sources require a matrix or reagent to be added to the sample prior to ionisation.
  • MALDI Matrix Assisted Laser Desorption lonisation
  • ambient ionisation techniques are particularly advantageous since firstly they do not require the addition of a matrix or a reagent (and hence are suitable for the analysis of in vivo tissue) and since secondly they enable a rapid simple analysis of target material to be performed.
  • a number of different ambient ionisation techniques are known and are intended to fall within the scope of the present invention.
  • DAI Desorption Electrospray lonisation
  • DESI Desorption Electrospray lonisation
  • These ambient ionisation techniques differ in their precise ionisation method but they share the same general capability of generating gas-phase ions directly from native (i.e. untreated or unmodified) samples.
  • a particular advantage of the various ambient ionisation techniques which are intended to fall within the scope of the present invention is that the various ambient ionisation techniques do not require any prior sample preparation.
  • the various ambient ionisation techniques enable both in vivo tissue and ex vivo tissue samples to be analysed without necessitating the time and expense of adding a matrix or reagent to the tissue sample or other target material.
  • V-EASI Venturi easy ambient sonic-spray ionization AFAI Air flow-assisted ionization
  • the ambient ionisation ion source may comprise a rapid evaporative ionisation mass spectrometry ("REIMS”) ion source wherein a RF voltage is applied to one or more electrodes in order to generate an aerosol or plume of surgical smoke by Joule heating.
  • REIMS rapid evaporative ionisation mass spectrometry
  • the ambient ionisation ion source may comprise a laser ionisation ion source.
  • the laser ionisation ion source may comprise a mid-1 R laser ablation ion source.
  • the ambient ionisation ion source may comprise a laser ablation ion source having a wavelength close to 2.94 ⁇ on the basis of the high absorption coefficient of water at 2.94 ⁇ .
  • the laser ablation ion source may comprise a Er:YAG laser which emits radiation at 2.94 ⁇ .
  • a mid-infrared optical parametric oscillator may be used to produce a laser ablation ion source having a longer wavelength than 2.94 ⁇ .
  • OPO optical parametric oscillator
  • an Er:YAG pumped ZGP-OPO may be used to produce laser radiation having a wavelength of e.g. 6.1 ⁇ , 6.45 ⁇ or 6.73 ⁇ .
  • a Co:MgF2 laser may be used as a laser ablation ion source wherein the laser may be tuned from 1.75-2.5 ⁇ .
  • an optical parametric oscillator (“OPO") system pumped by a Nd:YAG laser may be used to produce a laser ablation ion source having a wavelength between 2.9-3.1 ⁇ .
  • a C0 2 laser having a wavelength of 10.6 ⁇ may be used to generate the aerosol, smoke or vapour.
  • the ambient ionisation ion source may comprise an ultrasonic ablation ion source which generates a liquid sample which is then aspirated as an aerosol.
  • the ultrasonic ablation ion source may comprise a focused or unfocussed source.
  • the first device may comprise a tool or probe which utilises a continuous RF waveform.
  • a tool or probe which utilises a continuous RF waveform.
  • radiofrequency tissue dissection tool or probe may be used which is arranged to supply pulsed plasma RF energy to the tool or probe.
  • the tool or probe may comprise, for example, a PlasmaBlade (RTM).
  • Pulsed plasma RF tools operate at lower temperatures than conventional electrosurgical tools (e.g. 40-170 °C c.f. 200-350 °C) thereby reducing thermal injury depth.
  • Pulsed waveforms and duty cycles may be used for both cut and coagulation modes of operation by inducing electrical plasma along the cutting edge(s) of a thin insulated electrode.
  • FFPE formalin-fixed, paraffin-embedded
  • a tissue sample may by fixed for 12-24 hours in 10% neutral buffered formalin and then embedded in paraffin using standardized and automated procedures. Samples may be dehydrated in graded ethanol, cleared with xylene and then embedded in molten paraffin.
  • fresh frozen samples may be stored post-surgery in liquid nitrogen. Prior to analysis, fresh frozen samples may be dehydrated in a vacuum for 1 hour at room temperature.
  • a method which involves applying a reversal agent to a chemically modified target (e.g., a FFPE tissue sample) in order to produce a modified target (e.g., a tissue sample which is no longer formalin fixed and/or embedded with paraffin).
  • a modified target e.g., a tissue sample which is no longer formalin fixed and/or embedded with paraffin.
  • the modified target e.g., unfixed tissue
  • an ambient ionisation ion source such as a Desorption Electrospray Ionisation (“DESI”) ion source.
  • DESI Desorption Electrospray Ionisation
  • Fresh pork liver was embedded in OCT, without any previous sample treatment, and was sectioned and imaged in negative ion mode.
  • Figs. 1A-C show a comparison of desorption electrospray ionisation mass spectrometry imaging (MSI) in negative ion mode for morphological structures for different signal intensities from fresh pork liver embedded in OCT.
  • MSI desorption electrospray ionisation mass spectrometry imaging
  • Fig. 1A shows a desorption electrospray ionisation image for counts in the range 0- 250 at m/z 726.5
  • Fig. 1 B shows a desorption electrospray ionisation image for counts in the range 0-12000 at m/z 766.5
  • Fig. 1 C shows a desorption electrospray ionisation image for counts in the range 0-1400 at m/z 740.5.
  • the fixed tissue sample were then embedded in 10% gelatin and in OCT, and were then sectioned onto glass slides.
  • Fig. 2A shows a desorption electrospray ionisation mass spectrometry imaging (MSI) in negative ion mode from formalin fixed pork liver embedded in OCT and Fig. 2B shows the signal intensity in negative ion mode from formalin fixed pork liver embedded in OCT.
  • MSI mass spectrometry imaging
  • Fresh frozen pork liver and formalin fixed pork liver were washed in LC-MS grade water.
  • the step of washing the tissue sample in water helped to remove any excess of formalin present in the tissue.
  • tissue sample were then analysed by desorption electrospray ionisation mass spectrometry imaging (MSI).
  • MSI desorption electrospray ionisation mass spectrometry imaging
  • Fig. 3A shows a comparison of spectra intensity in negative ion mode from frozen fresh pork liver and Fig. 3B shows from formalin fixed pork liver washed in LC-MS grade water.
  • Fig. 4A shows a comparison of desorption electrospray ionisation mass
  • spectrometry imaging in negative ion mode from frozen fresh pork liver and Fig. 4B shows from formalin fixed pork liver washed in LC-MS grade water.
  • Fig. 5A shows the spectrum intensity in negative ion mode of fresh frozen pork liver
  • Fig. 5B shows the spectrum intensity of formalin fixed pork liver
  • Fig. 5C shows the spectrum intensity of heat-treated formalin fixed pork liver.
  • Fig. 6A shows a comparison of desorption electrospray ionisation mass
  • spectrometry imaging in negative ion mode from frozen fresh pork liver
  • Fig. 6B shows a mass spectrum from formalin fixed pork liver
  • Fig. 6C shows a mass spectrum for heat-treated formalin fixed pork liver.
  • Fig. 7A shows spectra intensity in negative ion mode from formalin fixed pork liver heat-treated with 15% (w/v) sodium metabisulfate.
  • Fig. 7B shows a corresponding ion image for m/z 794.5. This phenomenon is surprising as before analyses, the large morphological features are visible to the naked eye. However, in the desorption electrospray ionisation-MS image, no morphological features are detected.
  • tissue sections were examined under the microscope. It was observed that prior to analysis morphological features could be detected in the tissue section, but after analysis these seem to have been destroyed.
  • Fig. 8A shows a microscope image of formalin fixed pork liver heat-treated with sodium metabisulfate prior to desorption electrospray ionisation mass spectrometry imaging (MSI) analysis and Fig. 8B shows a corresponding microscope image after desorption electrospray ionisation mass spectrometry imaging (MSI) analysis.
  • tissue not adhering as firmly to the glass slide as frozen tissue is that it may be due to the fact that formalin cross-linking of proteins was not reversed to a sufficient extent to promote enough ionic interactions between the proteins and the glass slide.
  • time for sample heat- treatment was increased (heat-treatment cycles), urea was used as a formalin scavenger and microscope adhesion slides were also tested.
  • Figs. 9A-C shows a comparison of spectral intensity in negative ion mode of formalin fixed pork liver using different sodium metabisulfate concentrations (15% and 40% (w/v)) and different heat-treatment cycles as formalin scavengers.
  • Fig. 9A shows the spectral intensity in negative ion mode of formalin fixed pork liver using 15% (w/v) sodium metabisulfate and one heat-treatment cycle
  • Fig. 9B shows the spectral intensity in negative ion mode of formalin fixed pork liver using 15% (w/v) sodium metabisulfate and three heat-treatment cycles
  • Fig. 9C shows the spectral intensity in negative ion mode of formalin fixed pork liver using 40% (w/v) sodium metabisulfate and one heat-treatment cycle.
  • Fig. 10A shows an ion image of formalin fixed pork liver, heat-treated for three cycles with 15% (w/v) sodium metabisulfate after desorption electrospray ionisation mass spectrometry imaging (MSI) analysis in negative ion mode with ion image m/z 794.53 being shown and Fig. 10B shows a microscope image of tissue after desorption electrospray ionisation mass spectrometry imaging (MSI) (square).
  • urea was tested as a formalin scavenger. Different concentrations were tested (1M, 5M and 10M). Different heat- treatment cycles and different microscope adhesion slides were also tested. It was observed that urea has greater effective formalin reversal properties than sodium metabisulfate as the fatty acid:phospholipid ratio was improved with all urea concentrations tested.
  • Fig. 11 shows a comparison of spectra intensity in negative ion mode of formalin fixed pork liver using different concentrations of urea and different numbers of heat- treatment cycles as formalin scavengers.
  • Fig. 12A shows a microscope image of formalin fixed pork liver post-desorption electrospray ionisation mass spectrometry imaging (MSI) analysis after being subjected to 5M urea and 3 heat-treatment cycles using a SuperFrost slide.
  • MSI mass spectrometry imaging
  • Fig. 12B shows a microscope image of formalin fixed pork liver post-desorption electrospray ionisation mass spectrometry imaging (MSI) analysis after being subjected to 5M urea and 3 heat-treatment cycles using a SuperFrostPlus slide.
  • MSI mass spectrometry imaging
  • Fig. 12C shows a microscope image of formalin fixed pork liver post-desorption electrospray ionisation mass spectrometry imaging (MSI) analysis after being subjected to 5M urea and 5 heat-treatment cycles using a Poly-Lysine slide.
  • MSI mass spectrometry imaging
  • Fig. 13A shows an ion image from formalin fixed pork liver heat-treated for 3 cycles with 5M Urea after desorption electrospray ionisation mass spectrometry imaging (MSI) analysis in negative ion mode with ion image m/z 794.53 using a SuperFrost slide and Fig. 13B shows an ion image m/z 794.53 using a SuperFrostPlus slide.
  • MSI mass spectrometry imaging
  • MSI Desorption electrospray ionisation mass spectrometry imaging
  • Fig. 14 shows a desorption electrospray ionisation mass spectrometry imaging (MSI) of human lung biopsy after EVLP treatment.
  • the RGB image is of PCs 1 , 2 and 5 for a human lung tissue sample (EXT8-T2-S1).
  • Formaldehyde scavengers Any formaldehyde scavenger known in the art may potentially be used in the reversal agent in accordance with various embodiments.
  • the action of the reversal agent may be increased by raising the temperature and/or lowering the pH.
  • Formaldehyde scavengers react with formaldehyde, thus reducing the
  • the formaldehyde scavenger may be selected from the group consisting of: (i) at least one dithiooxamide; (ii) at least one carbodiimide; (iii) at least one polyhydroxy compound; (iv) at least one nitrogen-containing compound such as an amide, amine or hydrazide compound; (v) at least one dicarbonyl compound; (vi) at least one water-soluble active methylene compound; (vii) at least one sulfur-containing compound and (viii) at least one metabisulfite salt.
  • Suitable dithiooxamide scavengers include those of formula:
  • R denotes a hydroxyalkyl group having from 1-4 carbon atoms and from 1-4 hydroxy groups, a heterocyclic group or a toluidinomethylene group.
  • Specific such compounds include N,N'-di-(2-hydroxyethyl) dithiooxamide, N,N'-di-(3-hydroxypropyl) dithiooxamide, ⁇ , ⁇ '-diglucityl dithiooxamide, ⁇ , ⁇ '-difurfuryl dithiooxamide, N,N'-di-(2- pyridylmethyl) dithiooxamide, N,N'-di-(3-pyridylmethyl) dithiooxamide, N,N'-di-(m- toluidinoethyl) dithiooxamide and N,N'-di-[2-(N"-ethyl-m-toluidino)-ethyl dithiooxamide.
  • Suitable metabisulfite salts include sodium metabisulfite (Na 2 S 2 0 5 ), potassium metabisulfite and ammonium metabisulfite.
  • Suitable carbodiimide scavengers include monomeric, oligomeric and/or polymeric carbodiimides.
  • R 2 represents aryl, aralkyl, R-NCO, R-NHCONHR 4 , R-NHCONR 4 R 5 R-HCOOR 6 , -NHCOS-R 4 , -COOR 4 , -OR 4 , -N(R 4 ) 2 , -SR 4 , -OH, -NH 2 , NHR4, or epoxy; and wherein R 3
  • R 4 and R 5 are the same or different and represent an alkyl, cycloalkyl or aralkyl radical, and R has one of the meanings of R 4 or represents a polyester or a polyamide radical, and n represents an integer from 1 to 5,000, such as from 2 to 500.
  • Suitable monomeric carbodiimide scavengers include dicyclohexylcarbodiimide, diisopropylcarbodiimide, dimethylcarbodiimide, diisobutylcarbodiimide, dioctylcarbodiimide, tert-butyl-isopropylcarbodiimide, 2,6-diisopropylphenylencarbodiimide,
  • diphenylcarbodiimide di-tert-butylcarbodiimide and di- ⁇ - naphthylcarbodiimide.
  • Suitable polymeric carbodiimide scavengers include
  • Suitable ⁇ -dicarbonyl scavengers include compounds of formula 1a and 1 b:
  • X ! and X 4 are C, N, S, or P;
  • X, X2, X3, X5 and X 6 are selected from the group consisting of H;
  • phenyl selected from phenyl, phenylene, naphthalene or other polyaromatic hydrocarbons, substituted by a polar group or 1-3 halogens.
  • X, X 3 and X 6 groups may be chemically linked to form cyclic or heterocyclic structures.
  • halogen denotes F, CI, Br or I.
  • polar group denotes O, OH, COOH, carbonyl, amide, amine, thiol, quaternary nitrogen, ethoxy or propoxy group, or combinations thereof.
  • ⁇ -dicarbonyl scavengers include
  • Nitrogen-containing formaldehyde scavengers include amides, amines and hydrazides. Hydrazides such as 2,4-dinitrophenzylhydrazine and adipic acid dihydrazide can react with formaldehyde to give hydrazones.
  • Suitable mono or di-amide scavengers include those of formula:
  • X 17 and X 18 are independently selected from the group consisting of H; - a Ci-22 straight chain, branched or cyclic hydrocarbon or aromatic moiety selected from phenyl, phenylene, naphthalene or other polyaromatic hydrocarbons, substituted by a polar group or 1-3 halogens;
  • Ci-22 hydrocarbon straight chain, branched or cyclic
  • aromatic moiety phenyl, phenylene, naphthalene or other polyaromatic hydrocarbon
  • Examples of mono- and di-amide scavengers are urea, ethylene urea, propylene urea, ⁇ -caprolactam, glycouril, hydantoin, 2- oxazolidinone, 2-pyrrolidinone, uracil, barbituric acid, thymine, uric acid, allantoin, polyamides, 4,5-dihydroxyethylene urea (DHEU), monomethylol-4-hydroxy-4-methoxy-5,5- dimethyl-propylurea, nylon 2-hydroxyethyl ethylene urea, 2-hydroxyethyl urea, L-citrulline, biotin, N-methyl urea, N-ethyl urea, N-butyl urea, N-phenyl urea, 4,5-dimethoxy ethylene urea, succinimide, 2-imidazolidinone and 1 ,3-dimethyl-2-imidazolidinone.
  • DHEU
  • Suitable amine scavengers include amines which form imines by reaction with formaldehyde as represented by the following formula:
  • X 27 -NH 2 wherein X 27 is selected from the group consisting of H;
  • amine scavengers include compounds of formula:
  • R A is a bond or is C(R 7 R 8 );
  • Ri is H, OH or Ci-Ce alkyl
  • R 2 is H, OH or Ci-Ce alkyl
  • R 3 , R 4 , R 5 , and R 6 are independently H, or C C 6 alkyl
  • R 7 and R 8 are independently H, OH or C C 6 alkyl
  • alkyl in R Rs is optionally independently substituted with OH, NR 9 R 19 , C C 6 alkyl, or phenyl, wherein R 9 and R 19 are independently H or C C 6 alkyl,
  • Suitable salts include hydrochloride, acetate, formate, oxalate, citrate, carbonate, sulfate, and phosphate.
  • Amine scavengers include 2-amino-1-butanol, 2- amino-2-ethyl-1 ,3-propanediol, 2-amino-2-methyl-1-propanol, 2-amino-1-methyl-1 ,3- propanediol, tris(hydroxymethyl)aminomethane, N-isopropylhydroxylamine, ethanolamine, diethanolamine, N-methylethanolamine, N-butylethanolamine, monoisopropanolamine, diisopropanolamine, mono-sec-butanolamine, di-sec-butanolamine, and salts thereof; polyvinyl amine), arginine, lysine, asparagine, proline, tryptophan; proteins such as casein, gelatin, collagen, whey protein, soy protein, and albumin; melamine, benzoguanamine, 4- aminobenzoic acid (PABA), 3-aminobenzoic acid, 2-aminobenzo
  • Another class of formaldehyde scavengers are polyhydroxy compounds such as those represented by the following structure: wherein X 33 and X 34 are independently selected from the group consisting of H;
  • polyvinylalcohol pyrogallol, 1 ,2-hexanediol, diethylene glycol, ascorbic acid, saccharides such as D-sorbitol, dextrose and sucrose, tannins/tannic acid, and polysaccharides such as starches, guar, xanthan, pectin, chemically-modified cellulose, chitosan, and mixtures thereof.
  • Sulfur containing compounds include 1 ,3,5- triazine-2,4,6-trithiol, glutathione and cysteine.
  • Water-soluble active methylene scavengers may include compounds of formula: wherein R and independently denote -CH 3 or H(0(CH 2 ) m )nO-;
  • n denotes 1-19;
  • n 1-3
  • Y denotes -CH 2 - or -CH 2 -C(0)-CH 2 -.
  • Active methylene scavengers may include bis(2-hydroxyethoxyethyl) malonate and (2-hydroxyethoxyethyl) acetoacetate, dimethyl-1 ,3-acetonedicarboxylate, diethyl malonate and ethylacetoacetate. Analysing sample spectra
  • SVM Support Vector Machines
  • KNN K-Nearest Neighbours
  • Quadratic Discriminant Analysis (QDA)
  • Combinations of the foregoing analysis approaches can also be used, such as PCA-LDA, PCA-MMC, PLS-LDA, etc.
  • Analysing the sample spectra can comprise unsupervised analysis for
  • Fig. 15 shows a method 1500 of building a classification model using multivariate analysis.
  • the method comprises a step 1502 of obtaining plural sets of intensity values for reference sample spectra.
  • the method then comprises a step 1504 of unsupervised principal component analysis (PCA) followed by a step 1506 of supervised linear discriminant analysis (LDA).
  • PCA-LDA unsupervised principal component analysis
  • LDA-MMC supervised linear discriminant analysis
  • the multivariate analysis such as this can provide a classification model that allows an aerosol, smoke or vapour sample to be classified using one or more sample spectra obtained from the aerosol, smoke or vapour sample.
  • the multivariate analysis will now be described in more detail with reference to a simple example.
  • Fig. 16 shows a set of reference sample spectra obtained from two classes of known reference samples.
  • the classes may be any one or more of the classes of target described herein. However, for simplicity, in this example the two classes will be referred as a left-hand class and a right-hand class.
  • Each of the reference sample spectra has been pre-processed in order to derive a set of three reference peak-intensity values for respective mass to charge ratios in that reference sample spectrum. Although only three reference peak-intensity values are shown, it will be appreciated that many more reference peak-intensity values (e.g., ⁇ 100 reference peak-intensity values) may be derived for a corresponding number of mass to charge ratios in each of the reference sample spectra. In other embodiments, the reference peak-intensity values may correspond to: masses; mass to charge ratios; ion mobilities (drift times); and/or operational parameters.
  • Fig. 17 shows a multivariate space having three dimensions defined by intensity axes.
  • Each of the dimensions or intensity axes corresponds to the peak-intensity at a particular mass to charge ratio.
  • the multivariate space comprises plural reference points, with each reference point corresponding to a reference sample spectrum, i.e., the peak-intensity values of each reference sample spectrum provide the co-ordinates for the reference points in the multivariate space.
  • the set of reference sample spectra may be represented by a reference matrix D having rows associated with respective reference sample spectra, columns associated with respective mass to charge ratios, and the elements of the matrix being the peak-intensity values for the respective mass to charge ratios of the respective reference sample spectra.
  • PCA may accordingly be carried out on the matrix D in order to calculate a PCA model that defines a PCA space having a reduced number of one or more dimensions defined by principal component axes.
  • the principal components may be selected to be those that comprise or "explain" the largest variance in the matrix D and that cumulatively explain a threshold amount of the variance in the matrix D.
  • Fig. 18 shows how the cumulative variance may increase as a function of the number n of principal components in the PCA model.
  • the threshold amount of the variance may be selected as desired.
  • the PCA model may be calculated from the matrix D using a non-linear iterative partial least squares (NIPALS) algorithm or singular value decomposition, the details of which are known to the skilled person and so will not be described herein in detail. Other methods of calculating the PCA model may be used.
  • NNIPALS non-linear iterative partial least squares
  • the resultant PCA model may be defined by a PCA scores matrix S and a PCA loadings matrix L.
  • the PCA may also produce an error matrix E, which contains the variance not explained by the PCA model.
  • the relationship between D, S, L and E may be:
  • Fig. 19 shows the resultant PCA space for the reference sample spectra of Figs. 16 and 17.
  • the PCA model has two principal components PC 0 and PCi and the PCA space therefore has two dimensions defined by two principal component axes.
  • a lesser or greater number of principal components may be included in the PCA model as desired. It is generally desired that the number of principal components is at least one less than the number of dimensions in the multivariate space.
  • the PCA space comprises plural transformed reference points or PCA scores, with each transformed reference point or PCA score corresponding to a reference sample spectrum of Fig. 16 and therefore to a reference point of Fig. 17.
  • the reduced dimensionality of the PCA space makes it easier to group the reference sample spectra into the two classes. Any outliers may also be identified and removed from the classification model at this stage.
  • supervised multivariate analysis such as multi-class LDA or maximum margin criteria (MMC) in the PCA space may then be performed so as to define classes and, optionally, further reduce the dimensionality.
  • MMC maximum margin criteria
  • multi-class LDA seeks to maximise the ratio of the variance between classes to the variance within classes (i.e., so as to give the largest possible distance between the most compact classes possible).
  • the details of LDA are known to the skilled person and so will not be described herein in detail.
  • the resultant PCA-LDA model may be defined by a transformation matrix U, which may be derived from the PCA scores matrix S and class assignments for each of the transformed spectra contained therein by solving a generalised eigenvalue problem.
  • Fig. 20 shows a PCA-LDA space having a single dimension or axis, wherein the LDA is performed in the PCA space of Fig. 19.
  • the LDA space comprises plural further transformed reference points or PCA-LDA scores, with each further transformed reference point corresponding to a transformed reference point or PCA score of Fig. 19.
  • each class in the PCA-LDA model may be defined by its transformed class average and covariance matrix or one or more hyperplanes (including points, lines, planes or higher order hyperplanes) or hypersurfaces or Voronoi cells in the PCA-LDA space.
  • the PCA loadings matrix L, the LDA matrix U and transformed class averages and covariance matrices or hyperplanes or hypersurfaces or Voronoi cells may be output to a database for later use in classifying an aerosol, smoke or vapour sample.
  • the transformed covariance matrix in the LDA space V' g for class g may be given by
  • V g are the class covariance matrices in the PCA space.
  • Fig. 21 shows a method 2100 of using a classification model.
  • the method comprises a step 2102 of obtaining a set of intensity values for a sample spectrum.
  • the method then comprises a step 2104 of projecting the set of intensity values for the sample spectrum into PCA-LDA model space.
  • Other classification model spaces may be used, such as PCA-MMC.
  • the sample spectrum is then classified at step 2106 based on the project position and the classification is then output in step 2108.
  • Fig. 22 shows a sample spectrum obtained from an unknown aerosol, smoke or vapour sample.
  • the sample spectrum has been pre-processed in order to derive a set of three sample peak-intensity values for respective mass to charge ratios.
  • sample peak-intensity values may correspond to: masses; mass to charge ratios; ion mobilities (drift times);
  • the sample spectrum may be represented by a sample vector d x , with the elements of the vector being the peak-intensity values for the respective mass to charge ratios.
  • Fig. 23 again shows the PCA-LDA space of Fig. 20.
  • the PCA-LDA space of Fig. 23 further comprises the projected sample point, corresponding to the transformed PCA-LDA vector z x , derived from the peak intensity values of the sample spectrum of Fig. 22.
  • the projected sample point is to one side of a hyperplane between the classes that relates to the right-hand class, and so the aerosol, smoke or vapour sample may be classified as belonging to the right-hand class.
  • the Mahalanobis distance from the class centres in the LDA space may be used, where the Mahalanobis distance of the point z x from the centre of class g may be given by the square root of:
  • each class may be treated as a multivariate Gaussian.
  • a probability of membership of the data vector to each class may be calculated.
  • Fig. 24 shows a method 2400 of building a classification library.
  • the method comprises a step 2402 of obtaining plural input reference sample spectra and a step 2404 of deriving metadata from the plural input reference sample spectra for each class of sample.
  • the method then comprises a step 2404 of storing the metadata for each class of sample as a separate library entry.
  • the classification library is then output, for example to electronic storage, in step 2406.
  • a classification library such as this allows an aerosol, smoke or vapour sample to be classified using one or more sample spectra obtained from the aerosol, smoke or vapour sample.
  • the library based analysis will now be described in more detail with reference to an example.
  • each entry in the classification library is created from plural pre- processed reference sample spectra that are representative of a class.
  • the reference sample spectra for a class are pre-processed according to the following procedure:
  • N chan is a selected value and
  • j denotes the nearest integer below x.
  • N chan is 2 12 or 4096.
  • a background subtraction process is performed.
  • a cubic spline with k knots is then constructed such that p% of the data between each pair of knots lies below the curve. This curve is then subtracted from the data.
  • k is 32.
  • p is 5.
  • a constant value corresponding to the q% quantile of the intensity subtracted data is then subtracted from each intensity. Positive and negative values are retained. In one example, q is 45.
  • the data are normalised to have mean y t .
  • y t 1.
  • An entry in the library then consists of metadata in the form of a median spectrum value ⁇ and a deviation value ⁇ ⁇ for each of the N chan points in the spectrum.
  • C is 3/2, which lies between Cauchy and Gaussian, so that the likelihood becomes: 3 1 1
  • the parameters are set to the median of the list of values in the i'th channel of the input reference sample spectra while the deviation D t is taken to be the interquartile range of these values divided by V2. This choice can ensure that the likelihood for the i'th channel has the same interquartile range as the input data, with the use of quantiles providing some protection against outlying data.
  • Fig. 25 shows a method 2500 of using a classification library.
  • the method comprises a step 2502 of obtaining a set of plural sample spectra.
  • the method then comprises a step 2504 of calculating a probability or classification score for the set of plural sample spectra for each class of sample using metadata for the class entry in the classification library.
  • the sample spectra are then classified at step 2506 and the classification is then output in step 2508.
  • an unknown sample spectrum y is the median spectrum of a set of plural sample spectra. Taking the median spectrum y can protect against outlying data on a channel by channel basis.
  • the likelihood L s for the input data given the library entry s is then given by: wherein and ⁇ ⁇ are, respectively, the library median values and deviation values for channel i.
  • the likelihoods L s may be calculated as log likelihoods for numerical safety.
  • the exponent (l/F) can soften the probabilities which may otherwise be too definitive.
  • F 100.
  • RMS classification scores R s may be calculated using the same median sample values and derivation values from the library:
  • the aerosol, smoke or vapour sample may then be classified as belonging to the class having the highest probability and/or highest RMS classification score. Prediction of tissue types in breast cancer biopsies
  • Desorption electrospray ionisation mass spectrometry imaging enables to visualise spatial distribution of lipid species across tissue sections allowing direct correlation with the histological features. Therefore, breast cancer tissues were analysed with desorption electrospray ionisation mass spectrometry imaging (MSI) to obtain lipidomic data. 45 samples were analysed in positive and negative ion mode.
  • Fig. 26A shows RGB images of PCA components 1 , 2 and 3 in negative ion mode and Fig. 26B shows in positive ion mode with the respective histological images of a Grade II Invasive Ductal Carcinoma (IDC).
  • IDC Invasive Ductal Carcinoma
  • An ovarian cancer dataset with different epithelial carcinomas (endometrioid, serous and clear cell carcinomas), borderline tumours, and healthy ovary and fallopian tube has been analysed.
  • a total of 109 samples were collected and acquired by desorption electrospray ionisation-MS in positive and negative ion mode.
  • Fig. 27A shows PCA analysis of Grade II IDC in negative ion mode.
  • Fig. 27B shows MMC analysis of Grade II IDC negative ion mode.
  • Fig. 28A shows PCA analysis of Grade II IDC in positive ion mode.
  • Fig. 28B shows MMC analysis of Grade II IDC positive ion mode.
  • Fig. 29A shows leave one out cross validation of different tissue types in a Grade II IDC in negative ion mode and Fig. 29B shows in positive ion mode.
  • the dataset was initially pre-processed and multivariate statistical analysis was performed on each individual sample's dataset in order to compile a database of histologically authentic lipidomic profiles.
  • the morphological regions of interest were assigned by a qualified histopathologist and automatically co-registered and aligned with the MSI dataset.
  • Fig. 30 shows histological images with the annotated regions of interest by histopathologist.
  • PCA principal component analysis
  • MMC maximum margin criteria
  • This profile database was also used to perform comparative analysis across multiple samples.
  • PCA was used to perform unsupervised tissue segmentation based on the lipidomic profiles, without taking into account histological assignment.
  • a supervised analysis was then performed and a respective leave-one-tissue-per-patient-out cross validation was calculated.
  • Fig. 31 A shows analysis of a combined dataset from multiple samples (negative ion mode using PCA of identified regions)
  • Fig. 31 B shows corresponding MMC supervised analysis
  • Fig. 31 C shows MMC analysis excluding the samples with outliers identified in Fig. 31 B
  • Fig. 31 D shows respective leave-one-region-per-patient-out cross validation.
  • PCA shows some separation between normal ovary, serous carcinoma, and serous carcinoma associated stroma.
  • the supervised MMC analysis shows good separation between all three tissue types with six outliers (circles in Fig. 31 B). All four misclassified normal samples were samples which were classified as normal ovary but were taken from an ovary with a tumour distant from the sampling area. This suggests that the biochemistry of this tissue is altered, even though this cannot be detected in a morphological
  • MMC analysis was repeated under exclusion of the outliers and leave-one-region- per-patient-out cross validation was performed, showing a complete separation of normal tissue and an overall accuracy of 85%.
  • Fig. 32A shows PCA of identified regions
  • Fig. 32B shows MMC supervised analysis
  • Fig. 32C shows leave-one-region-per-patient-out cross validation for the positive ion mode data.
  • the cross validation shows lower scores then in negative ion mode, the different tissue types being classified with an average cross validation accuracy of around 80%.
  • MSI mass spectrometry imaging
  • Fig. 33A shows supervised MMC analysis of healthy ovary, borderline tumours and carcinomas together with Fig. 33B showing leave one patient out cross validation.
  • Fig. 34A shows supervised MMC analysis of healthy ovary and different epithelial carcinomas (endometrioid and serous) and Fig. 34B leave one patient out cross validation.
  • Fig. 35A shows a sample with unknown histology used to predict the different tissue types. Cross validation of this prediction was based on the histological annotation.
  • the desorption electrospray ionisation data allowed an excellent prediction of the two tissue types present in the sample i.e. stroma and cancer.
  • a cross validation as shown in Fig. 35B was performed based on histological annotation performed after this analysis and a classification accuracy of almost 100% was achieved.
  • Desorption electroflow focussing ionisation is a recently developed ambient ionisation technique, in which an electroFlow Focusing (RTM) nebulizer is used to desorb ions from a sample surface. This nebulizer focusses the emitted electrospray through a small orifice in a grounded plate using a concentric gas flow. Unlike desorption
  • desorption electroflow focusing ionisation (DESI), which uses very high nebulising gas pressures (100 psi) and high electrospray voltages (4.5 to 5 kV), desorption electroflow focusing ionisation (“DEFFI”) has so far been operated at relatively low gas pressures (10 psi) and lower voltages (500 V), as higher voltages were reported to cause droplet discharge at the orifice and corona discharge.
  • DEFFI desorption electroflow focusing ionisation
  • Fig. 36 shows a schematic of Desorption Electroflow Focussing lonisation setup.
  • the desorption electroflow focusing ionisation (“DEFFI”) was initially tested on a mass spectrometer, with the solvent being supplied by the instrument's syringe pump. Initial operating conditions were 10 psi nitrogen gas pressure and 5 ⁇ /min solvent flow rate. This produced a spray with a relatively large diameter (approx. 1 mm) impact area on the sample surface, which was visualised by directing the spray at a glass slide coated with Rhodamine B.
  • Fig. 37 shows total ion count for desorption electroflow focusing ionisation
  • DEFFI desorption electroflow focusing ionisation
  • DEFFI desorption electrospray ionisation
  • Figs. 38A-F show a comparison of desorption electroflow focusing ionisation (“DEFFI”) and desorption electrospray ionisation for mass spectrometric imaging of human breast cancer tissue.
  • DEFFI desorption electroflow focusing ionisation
  • Figs. 38A and 38D show RGB images of the first three principal components from principal component analysis of all pixels using the full acquired m/z range from 150 to 1500.
  • Figs. 38B and Fig. 38E show base peak intensity (BPI) normalised example spectra for breast tissue for desorption electroflow focusing ionisation (“DEFFI”) and desorption electrospray ionisation respectively.
  • Figs. 38C and Fig. 38F show desorption traces in line-scanning mode on rhodamine B-coated glass slides for desorption electroflow focusing ionisation (“DEFFI”) and desorption electrospray ionisation, respectively.
  • BPI base peak intensity
  • DEFFI desorption electroflow focusing ionisation
  • Figs. 38C and Fig. 38F show desorption traces in line-scanning mode on rhodamine B-coated glass slides for desorption electroflow focusing ionisation (“DEFFI”) and desorption electrospray ionisation, respectively.
  • DEFFI desorption electroflow focusing ionisation
  • the solvent was delivered by a nanoflow pump, allowing for a more precise control of solvent flow rate, even under high backpressure conditions.
  • the solvent flow rate was gradually decreased from 4 to 1.5 ⁇ /min, while gas pressure was gradually increased from 10 to 30 psi, and the electrospray was found to be stable throughout.
  • the desorption trace on rhodamine was reduced to about 450 ⁇ in width.
  • Figs. 39A-E shows a comparison of desorption electroflow focusing ionisation ("DEFFI”) and desorption electrospray ionisation for mass spectrometric imaging of pork liver tissue.
  • DEFFI desorption electroflow focusing ionisation
  • Figs. 39A and 39B show RGB images for pork liver of the first three principal components of principal component analysis across the full m/z range for desorption electroflow focusing ionisation (“DEFFI”) and desorption electrospray ionisation, respectively.
  • DEFFI desorption electroflow focusing ionisation
  • Fig. 39C and 39D show representative spectra from pork liver in negative ion mode for desorption electroflow focusing ionisation (“DEFFI”) and desorption electrospray ionisation, respectively.
  • DEFFI desorption electroflow focusing ionisation
  • Fig. 39E show desorption traces on rhodamine B for both methods for 3 different solvent flow rates.
  • desorption electrospray ionisation and desorption electroflow focusing ionisation were compared for tissue imaging, in this case using pork liver sections and a pixel size of 100 ⁇ for both (Figs. 39A and 39B).
  • Overall intensity for desorption electroflow focusing ionisation (“DEFFI”) was slightly higher, but spectral composition was similar (Figs. 39C and 39D).
  • a comparison of desorption traces on rhodamine showed a similar desorption area of approximately 450 ⁇ in width, although the desorption electroflow focusing ionisation (“DEFFI”) trace still showed slightly diffuser edges.
  • both methods were tested on rhodamine using flow rates of 1 and 0.5 ⁇ /min.
  • Fig. 40 shows normalised base peak intensity for pork liver in negative ion mode for desorption electroflow focusing ionisation ("DEFFI”) and desorption electrospray ionisation at three different solvent flow rates.
  • DEFFI desorption electroflow focusing ionisation
  • desorption electroflow focusing ionisation (“DEFFI”) was shown to be highly suitable for imaging of biological tissues. Its performance in terms of spectral intensity was comparable to, or slightly better than, that of desorption electrospray ionisation. Its spatial resolution was slightly inferior to that of desorption electrospray ionisation. This may, however, be improved by altering the dimensions of the electroFlow Focusing (RTM) nebulizer.
  • RTM electroFlow Focusing
  • DEFFI desorption electroflow focusing ionisation
  • the sprayer In desorption electrospray ionisation, the sprayer is potentially one of the largest sources of reproducibility problems.
  • the solvent capillary In conventional desorption electrospray ionisation sprayers, the solvent capillary is not fixed relative to the gas capillary. This can result in reproducibility errors, e.g. due to the variable solvent capillary geometry.
  • an improved desorption electrospray ionisation geometry for imaging is provided.
  • a desorption electrospray ionisation sprayer wherein the solvent capillary is fixed relative to the gas capillary.
  • apparatus comprising an ion source comprising a nozzle having an aperture, a solvent emitter which extends through the aperture, and a fixing device which is arranged and adapted to fix the solvent emitter relative to the aperture.
  • the fixing device may comprise a centering device which is arranged and adapted to locate or position the solvent emitter substantially centrally within the aperture.
  • the fixing device may comprise a device which is arranged and adapted to locate or position the solvent emitter substantially non-centrally within the aperture.
  • the fixing device may comprise a device which is arranged and adapted to locate or position the solvent emitter substantially non-centrally within the aperture, such that the solvent emitter is substantially closer to and/or oriented towards an inlet, such as an inlet capillary, of a mass and/or ion mobility spectrometer, e.g. relative to the centre and/or central axis of the aperture.
  • the solvent capillary 302 and/or its outlet end 303 may be positioned centrally or non-centrally within sheath gas tube 312, and may be positioned such that the outlet end 303 is closer to the transfer capillary 330 relative to the centre of the tube outlet 316 and/or such that the solvent capillary 302 and/or its outlet end 303 is angled towards the transfer capillary 330 relative to the central axis of the tube 312.
  • the fixing device may be arranged and adapted such that gas may flow through the nozzle and/or aperture relatively unimpeded by the fixing device.
  • the fixing device may be arranged and adapted so as to avoid or reduce turbulence in gas flowing through the nozzle and/or aperture.
  • an ion source comprising a nozzle having an aperture, a solvent emitter which extends through the aperture, and a device which is arranged and adapted to monitor the solvent emitter position relative to the aperture.
  • the results of the monitoring may be used to calibrate and/or correct the ion source and/or mass and/or ion mobility spectrometric data acquired using the ion source.
  • any movements are corrected for, e.g. in post processing of the data and/or by adjusting one or more parameters of the ion source (such as gas flow rate, applied voltage, solvent flow rate, position, orientation, temperature, etc.), e.g. in real-time, e.g. using a feedback loop.
  • one or more parameters of the ion source such as gas flow rate, applied voltage, solvent flow rate, position, orientation, temperature, etc.
  • a sprayer in which the solvent capillary geometry was fixed by attaching it to the gas capillary, was tested to assess the impact of solvent capillary positioning on lipid signal.
  • a modified sprayer was produced, where cyanoacrylate glue was used to attach the inner solvent capillary to the outer gas capillary, in order ensure consistent alignment.
  • the sprayer with the fixed solvent capillary was mounted in four different orientations.
  • the orientations tested were: forward, directed towards the mass
  • a solvent capillary orientation straight towards the inlet of the mass and/or ion mobility spectrometer was found to be beneficial, while sideways rotations led to signal reductions of up to 50 % and orientation away from the mass and/or ion mobility spectrometer inlet led to a loss of signal.
  • Fixing the position of the solvent capillary within the gas capillary in accordance with various embodiments provides an improved, and reproducible sprayer geometry.
  • the obstructed gas flow due to the glue fixing was found to reduce the total ion intensity by around 50% to 65%.
  • the phenomenon was associated with the change in the gas flow pattern introduced by fixing the solvent capillary to the gas capillary.
  • the mounting point may block the gas flow, so that the electrospray is no longer surrounded by the nebulising gas, which can lead to a formation of larger, slower droplets and lower desolvation and desorption in that part of the primary electrospray. That is, fixing the solvent capillary directly to the gas capillary may introduce further turbulence in the proximity of the spray tip, which can contribute to poor reproducibility.
  • a more carefully engineered source is provided that does not disrupt the gas flow.
  • the solvent capillary is fixed without obstructing the nebulising gas flow.
  • the solvent capillary may be fixed centrally or with a forward orientation for a high and less variable lipid signal.
  • a desorption electrospray ionisation sprayer with a capillary fixing or centring device which allows fixing the solvent capillary geometry without obstructing the nebulising gas flow.
  • This provides improved absolute intensity repeatability and improvements in spectral pattern repeatability of up to an order of magnitude. For tissue imaging, spatial resolutions of 20 ⁇ are readily achieved.
  • a sprayer with a centred solvent capillary and relatively small inner diameter, and tapered solvent capillary is provided.
  • a desorption electrospray ionisation sprayer may be provided wherein the emitter guide and electrically conductive (e.g. stainless steel) gas nozzle are insulated from each other in order to avoid charging of the gas nozzle and consequently, loss of signal.
  • electrically conductive e.g. stainless steel
  • Fig. 41 shows a diagram of a sprayer nozzle according to an embodiment.
  • a stainless steel emitter guide and a gas nozzle are electrically insulated from each other to avoid charging of the gas nozzle. This may be achieved by using an insulating fixing or centering disc.
  • the fixing or centering disc may comprise or may be coated with a ceramic and/or a polymer.
  • the emitter guide may be made to be electrically insulating, e.g. the emitter guide may comprise a ceramic and/or a polymer. In this case, the fixing or centering disc need not be insulating.
  • This sprayer was tested for imaging using human colorectal cancer sections as shown in Figs. 42A-42G.
  • Fig. 42A shows desorption electrospray ionisation-MS imaging of adjacent colorectal cancer sections at different spatial resolutions wherein RGB images for the first three components of principal component analysis of images were acquired at 100 ⁇
  • Fig. 42B shows RGB images for the first three components of principal component analysis of images which were acquired at 50 ⁇
  • Fig. 42C shows RGB images for the first three components of principal component analysis of images which were acquired at 20 ⁇ pixel size/ scan speed per second
  • Fig. 42A was acquired using a solvent flow rate of 1.5 ⁇ /min and a gas pressure of 7 bars and corresponds with trace D on rhodamine B, wherein Figs.
  • Fig. 42B and 42C were acquired using a solvent flow rate of 0.5 ⁇ /min and a gas pressure of 6 bars and correspond with trace E and wherein Fig. 42F shows an optical image of section A after hematoxylin and eosin staining and Fig. 42G shows an example spectrum from section A.
  • a reduced solvent flow rate of 0.5 ⁇ /min (compared to 1.5 ⁇ /min) was used. Although signal intensity was reduced proportionally, it was still sufficient to receive high quality images at 50 and 20 ⁇ pixel size.
  • Fig. 42A shows desorption electrospray ionisation-MS imaging of adjacent colorectal cancer sections at different spatial resolutions.
  • the image shown in Fig. 42A was acquired using a solvent flow rate of 1.5 ⁇ /min and a gas pressure of 7 bars and corresponds with the trace shown in Fig. 42D on rhodamine B.
  • Figs. 42B and 42C were acquired using a solvent flow rate of 0.5 ⁇ /min and a gas pressure of 6 bars and correspond with the trace shown in Fig. 42E.
  • Fig. 42F shows an optical image of the section shown in Fig. 42A after hematoxylin and eosin staining.
  • Fig. 42G shows an example spectrum from the section shown in Fig. 42A.
  • Fig. 43 shows another embodiment and comprises a Desorption Electrospray Ionisation ("DESI") sprayer 300 in which a solvent capillary 302 may be arranged to direct electrically charged droplets 304 of solvent at a sample surface 310.
  • a sample 311 may be located on the sample surface 310, which may comprise analyte particles.
  • the charging of the solvent droplets may be achieved through the use of a high-voltage power supply 306 that contacts the capillary 302.
  • the high-voltage power supply 306 may comprise an electrode 307 which may contact any portion of the capillary 302 so that it is operable to charge the solvent droplets as they leave an outlet end 303 of the capillary 302.
  • the outlet end 303 of the capillary may be directed towards the sample surface 310.
  • a sheath gas 308 (e.g., nitrogen) may be arranged to surround the capillary 302 so as to nebulise the solvent as it emerges from the capillary 302 and direct the electrically charged solvent droplets 304 towards the surface 310.
  • the sheath gas may be introduced through a tube 312 that may be coaxial to the solvent capillary 302, having an inlet 314 at an end distal to the sample surface 310 and an outlet 316 at an end facing the sample surface 310.
  • the outlet 316 of the sheath gas tube 312 may be concentric to the outlet end 303 of the capillary, which can facilitate in nebulising the solvent as it emerges from the capillary 302.
  • the solvent emerging from the outlet end 303 of the solvent capillary 302 may be nebulised by the sheath gas 308.
  • a connector 318 may connect the tube 312 to a source of gas suitable to use as a sheath gas.
  • the sheath gas 308 may comprise nitrogen or standard medical air, and the source of sheath gas may be a source of nitrogen gas or standard medical air.
  • analyte particles on the sample can desorb and the charged droplets and analyte mixture 320 may be transferred into a transfer capillary or transfer device 330 that may lead to an ion analyser, mass analyser or mass spectrometer 340.
  • the charged droplet and analyte mixture may be transferred through an inlet 332 of the transfer capillary or transfer device 330. This may be achieved by placing the opposite end 333 of the transfer capillary or transfer device 330 in a low pressure region 352, for example a vacuum stage of the mass analyser or mass spectrometer 340.
  • the charged droplet and analyte mixture may be transferred by ion optics 352 to an analysis region of the ion analyser or mass
  • the ion optics 352 may comprise an ion guide, for example a Stepwave (RTM) ion guide.
  • RTM Stepwave
  • the analyte ions may be guided to the analysis region by applying voltages to the ion optics 352.
  • the analyte ions may then be analysed by the mass analyser or mass spectrometer 340.
  • the ion analyser, mass analyser or mass spectrometer 340 may comprise an ion mobility spectrometer.
  • the ion analyser, mass analyser or mass spectrometer 340 may comprise the combination of an ion mobility spectrometer and a mass spectrometer.
  • One or more heaters may be provided to heat the various parts of the apparatus shown in Fig. 43.
  • a heater may be provided to heat one or more of the solvent capillary 302, the sheath gas tube 312, the sample surface 310 and the transfer or inlet capillary 330.
  • the one or more heaters may comprise a wire heater (e.g., a tungsten wrap) and/or may be configured to heat the respective part to at least 50 °C, 100 °C, 200 °C, 300 °C, 400 °C, 500 °C, 600 °C, 700 °C or 800 °C.
  • a wire heater e.g., a tungsten wrap
  • any type of heater may be used that has the function of heating the respective part, for example a blower or an inductive heater.
  • Fig. 43 shows a first heater 342 that may be arranged and adapted to heat the transfer or inlet capillary 330, such that the solvent and analyte mixture 320 may be heated before being passed onward, for example to the mass analyser or mass spectrometer 340.
  • the first heater 342 may be located anywhere along the solvent capillary 330, for example adjacent to or at the inlet 341 of the ion analyser, mass analyser or mass spectrometer. Alternatively, the first heater 342 may be located adjacent to or at the inlet 332 of the solvent capillary or transfer device 330.
  • the first heater 342 may comprise a wire heater (e.g., a tungsten wrap) and/or may be configured to heat the inlet capillary to at least 50 °C, 100 °C, 200 °C, 300 °C, 400 °C, 500 °C, 600 °C, 700 °C or 800 °C.
  • a second heater 344 may be arranged and adapted to heat the sheath gas tube
  • the second heater 344 may be located at the end of the tube 312 nearest the sample surface 310, such that the solvent and/or sheath gas may be heated before being directed at the sample surface 310.
  • the second heater 344 may comprise a wire heater (e.g., a tungsten wrap) and/or may be configured to heat the tube 312 and/or the solvent and/or the sheath gas to at least 50°C, 100°C, 200°C, 300°C, 400°C, 500°C, 600 °C, 700 °C or 800 °C.
  • a third heater 346 may be arranged and adapted to heat the solvent capillary 302, such that the solvent may be heated.
  • the third heater 346 may be located anywhere along the solvent capillary 302, for example nearest the end 305 located away from the sample surface 310, such that the solvent may be heated before it is surrounded by the sheath gas tube 312.
  • the third heater 346 may comprise a wire heater (e.g., a tungsten wrap) and/or may be configured to heat the solvent capillary 302 and/or the solvent to at least 50 °C, 100 °C, 200 °C, 300 °C, 400 °C, 500 °C, 600 °C, 700 °C or 800 °C.
  • a fourth heater 348 may be arranged and adapted to heat the sample surface 310, such that the sample 31 1 and/or the sample surface 310 may be heated.
  • the fourth heater 348 may be located beneath a portion of the sample surface 310 arranged and adapted to hold or contain the sample 311.
  • the fourth heater 348 may comprise a wire heater (e.g., a tungsten wrap) and/or may be configured to heat the sample 311 and/or sample surface 310 and/or the solvent to at least 50 °C, 100 °C, 200 °C, 300 °C, 400 °C, 500 °C, 600 °C, 700 °C or 800 °C.
  • any one or more or all of the heaters may comprise an inductive or eddy current heater.
  • the ion transfer capillary or transfer device 330 was heated using a nickel wire heater in a range from 100 to 490 °C. Pork liver sections were used and the intensities for selected fatty acids and phospholipids were compared. Inlet capillary heating was found to have some impact on fatty acid intensities using a Xevo (RTM) mass spectrometer and no impact using a Synapt (RTM) mass spectrometer. Intensities for the monitored
  • Figs. 44A-D show the impact of inlet capillary heating on absolute intensity.
  • Figs. 44A and 44C relate to a Waters Synapt G2-Si (RTM) mass spectrometer and
  • Figs. 44B and 44D relate to a Waters Xevo G2-XS (RTM) mass spectrometer.
  • Average intensities for selected fatty acids (FA), phosphatidyl ethanolamines (PE) and the most abundant phosphatidylinositol (PI) from pork liver sections are shown.
  • the sample surface 310 forms the surface of a swab.
  • the swab itself may be heated so as to heat the sample 31 1 that is located on the swab.
  • the fourth heater 348 may be a wire heater that is located within the swab, and may be arranged and adapted to heat the end of the swab configured to hold and/or retain biologic samples for analysis.
  • Methods of analysis e.g., methods of medical treatment, surgery and diagnosis and nonmedical methods
  • tissue may comprise human or non-human animal or plant tissue.
  • target may comprise biological matter or organic matter (including a plastic).
  • the target comprises one or more bacterial colonies or one or more fungal colonies.
  • analyte ions generated by an ambient ionisation ion source are then subjected either to: (i) mass analysis by a mass analyser or filter such as a quadrupole mass analyser or a Time of Flight mass analyser; (ii) ion mobility analysis (IMS) and/or differential ion mobility analysis (DMA) and/or Field Asymmetric Ion Mobility Spectrometry (FAIMS) analysis; and/or (iii) a combination of firstly (or vice versa) ion mobility analysis (IMS) and/or differential ion mobility analysis (DMA) and/or Field Asymmetric Ion Mobility Spectrometry (FAIMS) analysis followed by secondly (or vice versa) mass analysis by a mass analyser or filter such as a quadrupole mass analyser or a Time of Flight mass analyser.
  • Various embodiments also relate to an ion mobility spectrometer and/or mass analyser and a method of ion mobility spectrome
  • analyte ions may be subjected to a combination of both ion mobility analysis and mass analysis i.e. that both (a) the ion mobility, differential ion mobility, collision cross section or interaction cross section of analyte ions together with (b) the mass to charge of analyte ions is determined.
  • hybrid ion mobility-mass spectrometry (IMS-MS) and mass spectrometry-ion mobility (MS-IMS) embodiments are contemplated wherein both the ion mobility and mass to charge ratio of analyte ions generated e.g. by an ambient ionisation ion source are determined. Ion mobility analysis may be performed prior to mass to charge ratio analysis or vice versa.
  • references to mass spectrometric data and databases comprising mass spectrometric data should also be understood as
  • ion mobility data and differential ion mobility data etc. encompassing ion mobility data and differential ion mobility data etc. and databases comprising ion mobility data and differential ion mobility data etc. (either in isolation or in combination with mass spectrometric data).

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Abstract

A method is disclosed comprising applying a reversal agent, such as urea, to a formalin fixed tissue sample in order to reverse the effects of the fixation process. An ambient ionisation ion source such as a desorption electrospray ionisation ("DESI") ion source is used to generate analyte or analyte ions from multiple regions of the modified target so that an ion image of the modified target may be generated.

Description

DESORPTION ELECTROSPRAY IONISATION ("DESI") IMAGING CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority from and the benefit of United Kingdom patent application No. 1503876.3 filed on 6 March 2015, United Kingdom patent application No. 1503864.9 filed on 6 March 2015, United Kingdom patent application No. 1518369.2 filed on 16 October 2015, United Kingdom patent application No. 1503877.1 filed on 6 March 2015, United Kingdom patent application No. 1503867.2 filed on 6 March 2015, United Kingdom patent application No. 1503863.1 filed on 6 March 2015, United Kingdom patent application No. 1503878.9 filed on 6 March 2015, United Kingdom patent application No. 1503879.7 filed on 6 March 2015 and United Kingdom patent application No. 1516003.9 filed on 9 September 2015. The entire contents of these applications are incorporated herein by reference.
FIELD OF THE INVENTION The present invention relates generally to the analysis of a target (which may, for example, comprise ex vivo tissue) by ambient ionisation techniques such as desorption electrospray ionisation ("DESI"), methods of analysis and diagnosis and apparatus for analysing a target using an ambient ionisation ion source. Various embodiments are contemplated wherein analyte ions generated by an ambient ionisation ion source are then subjected either to: (i) mass analysis by a mass analyser such as a quadrupole mass analyser or a Time of Flight mass analyser; (ii) ion mobility analysis (IMS) and/or differential ion mobility analysis (DMA) and/or Field Asymmetric Ion Mobility Spectrometry (FAIMS) analysis; and/or (iii) a combination of firstly ion mobility analysis (IMS) and/or differential ion mobility analysis (DMA) and/or Field Asymmetric Ion Mobility Spectrometry (FAIMS) analysis followed by secondly mass analysis by a mass analyser such as a quadrupole mass analyser or a Time of Flight mass analyser (or vice versa). Various embodiments also relate to an ion mobility spectrometer and/or mass analyser and a method of ion mobility spectrometry and/or method of mass analysis. BACKGROUND
Rapid evaporative ionisation mass spectrometry ("REIMS") is a relatively new technique that is useful for the analysis of many different samples including the
identification of tissue.
Reference is made to N. Strittmatter et al., Anal. Chem. 2014, 86, 6555-6562 which discloses an investigation into the suitability of using rapid evaporative ionisation mass spectrometry as a general identification system for bacteria and fungi.
The known approach for analysing bacterial colonies by rapid evaporative ionisation mass spectrometry involves using bipolar electrosurgical forceps and an electrosurgical RF generator. A bacterial colony is scraped from the surface of an agar layer using the bipolar electrosurgical forceps and a short burst of RF voltage from the electrosurgical RF generator is applied between the bipolar electrosurgical forceps. For example, it is known to apply 60 W of power in a bipolar mode at a frequency of 470 kHz sinusoid. The RF voltage which is applied to the electrosurgical forceps has the result of rapidly heating the particular portion of the bacterial colony which is being analysed due to its nonzero impedance. The rapid heating of the microbial mass results in an aerosol being generated. The aerosol is transferred directly into a mass spectrometer and/or ion mobility
spectrometer and the aerosol sample may then be analysed by the mass spectrometer and/or ion mobility spectrometer. It is known to utilise multivariate statistical analysis in order to help distinguish and identify different samples.
Reference is made to Z. Takats et al., Science 2004, 306, 471-473 which discloses a related ambient ionisation technique wherein mass spectrometry sampling is performed under ambient conditions using a Desorption Electrospray Ionisation ("DESI") ion source. Various compounds were ionised including peptides and proteins present on metal, polymer and mineral surfaces. Desorption electrospray ionization ("DESI") was carried out by directing electrosprayed charged droplets and ions of solvent onto the surface to be analysed. The impact of the charged droplets on the surface produces gaseous ions of material originally present on the surface. The resulting mass spectra are similar to normal electrospray mass spectra in that they show mainly singly or multiply charged molecular ions of the analytes. The desorption electrospray ionisation phenomenon was observed both in the case of conductive and insulator surfaces and for compounds ranging from nonpolar small molecules such as lycopene, the alkaloid coniceine, and small drugs, through polar compounds such as peptides and proteins. Changes in the solution that is sprayed can be used to selectively desorb and ionize particular compounds, including those in biological matrices. In vivo analysis was also demonstrated.
Numerous diseases are diagnosed on the basis of histology i.e. microscopic analysis of tissue samples which have been resected from patients. The gold standard for histopathological analysis is based upon formalin-fixed, paraffin-embedded ("FFPE") patient samples. Formalin fixation preserves tissues by forming methylene bridge cross- linkages between proteins. The sample is then dehydrated with ethanol and is immersed in an organic solvent (e.g., xylene) in order to remove the alcohol. The tissue sample is then infiltrated with molten paraffin.
According to a protocol, a tissue sample may by fixed for 12-24 hours in 10% neutral buffered formalin and then embedded in paraffin using standardised and automated procedures. Samples may be dehydrated in graded ethanol, cleared with xylene and then embedded in molten paraffin.
By way of contrast, fresh frozen samples may be stored post-surgery in liquid nitrogen. Prior to analysis, fresh frozen samples may be dehydrated in a vacuum for 1 hour at room temperature. Whereas fresh frozen samples can only be preserved for a short period of time, formalin fixed paraffin embedded samples can be archived for many years allowing retrospective reassessment and analysis.
However, it is problematic to generate ion images from formalin fixed tissue samples since when analyte ions from a formalin fixed tissue sample are generated there is a strong ion signal due to the formalin. Furthermore, other potentially important ion signals are suppressed.
Accordingly, attempts to generate high quality ion images of a formalin fixed tissue sample using e.g. a desorption electrospray ionisation ("DESI") ion source are particularly problematic.
It is desired to provide an improved method of ambient ionisation imaging and in particular to be able to perform ion imaging on a formalin fixed tissue sample.
SUMMARY
According to an aspect there is provided a method comprising:
applying a reversal agent to a first target in order to modify said target thereby producing a modified target;
using a first device to generate analyte or analyte ions from multiple regions of the modified target; and
generating an ion image of the modified target.
Conventionally, desorption electrospray ionisation ("DESI") ion imaging of a formalin fixed tissue sample is problematic due to the fact that the formalin results in a strong signal in the resulting mass spectrum and also because weaker analyte ion signals may be suppressed.
The application of a reversal agent such as urea and optionally also subjecting the tissue sample to one or more cycles of heat treatment according to various embodiments enables the cross-linkages between proteins to be reversed and the formalin and any embedding material such as paraffin to be removed from the tissue sample.
As a result, ambient ionisation ion imaging may be performed on chemically modified targets (e.g., formalin fixed tissue samples) which have been exposed to a reversal agent (such as urea) with the result that significantly improved ion images can be obtained (compared to conventional ion images of formalin fixed tissue samples).
The first device may comprise or form part of an ambient ion or ionisation source or the first device may generate aerosol, smoke or vapour for subsequent ionisation by an ambient ion or ionisation source or other ionisation source.
The first device may comprise a desorption electrospray ionisation ("DESI") ion source or a desorption electro-flow focusing ("DEFFI") ion source.
The first device may comprise an ion source selected from the group consisting of: (i) a rapid evaporative ionisation mass spectrometry ("REIMS") ion source; (ii) a laser desorption ionisation ("LDI") ion source; (iii) a thermal desorption ion source; (iv) a laser diode thermal desorption ("LDTD") ion source; (v) a dielectric barrier discharge ("DBD") plasma ion source; (vi) an Atmospheric Solids Analysis Probe ("ASAP") ion source; (vii) an ultrasonic assisted spray ionisation ion source; (viii) an easy ambient sonic-spray ionisation ("EASI") ion source; (ix) a desorption atmospheric pressure photoionisation ("DAPPI") ion source; (x) a paperspray ("PS") ion source; (xi) a jet desorption ionisation ("JeDI") ion source; (xii) a touch spray ("TS") ion source; (xiii) a nano-desorption electrospray ionisation ("nano-DESI") ion source; (xiv) a laser ablation electrospray ("LAESI") ion source; (xv) a direct analysis in real time ("DART") ion source; (xvi) a probe electrospray ionisation ("PESI") ion source; (xvii) a solid-probe assisted electrospray ionisation ("SPA-ESI") ion source; (xviii) a cavitron ultrasonic surgical aspirator ("CUSA") device; (xix) a focussed or unfocussed ultrasonic ablation device; (xx) a microwave resonance device; and (xxi) a pulsed plasma RF dissection device.
The first target may comprise a chemically modified target.
The first target may comprise a fixed tissue sample.
The fixed tissue sample may be fixed with either: (i) formalin or a formaldehyde solution; or (ii) formalin or a formaldehyde solution with glycerol or another chemical agent.
The fixed tissue sample may comprise a formalin fixed, paraffin-embedded
("FFPE") tissue sample.
The fixed tissue sample may be further embedded in an embedding medium selected from the group consisting of: (i) glycerol; (ii) optimum cutting temperature ("OCT") (RTM) medium; (iii) paraffin; (iv) one or more plastic polymers; (v) polyester wax; (vi) polyethylene glycol ("PEG"); (vii) polyfin (RTM); (viii) a tissue freezing medium ("TFM"); (ix) glycol methacrylate ("GMA"); (x) gelatin; and (xi) carboxy methyl cellulose ("SMC").
The first target may comprise a tissue sample wherein methylene bridge cross- linkages are formed between proteins.
The first target may comprise a tissue sample which has been dehydrated with an alcohol.
The first target may comprise a tissue sample which has been infiltrated with molten paraffin.
The first target and/or the modified target may be provided on a substrate.
The substrate may be selected from the group consisting of: (i) a microscope slide;
(ii) a transparent slide; (iii) a glass slide; (iv) a plastic slide; and (v) an indium-tin-oxide (ITO) coated glass or other slide.
The step of applying the reversal agent to the first target may reverse cross-linked bonds of proteins present in the first target.
The reversal agent may be arranged to reverse cross-linked bonds of formalin fixed tissue proteins or formalin fixed paraffin embedded ("FFPE") tissue proteins.
The reversal agent may comprise a formalin or formaldehyde scavenger.
The reversal agent may comprise water.
The reversal agent may comprise sodium metabisulfate or sodium metabisulfite (Na2S205).
The reversal agent may comprise urea. The reversal agent may be selected from the group consisting of: (i) at least one dithioxamide; (ii) at least one carbodiimide; (iii) at least one monomeric, oligomeric or polymeric carbodiimide; (iv) at least one aminoplastic, thermoplastic, binder, polymeric compound or copolymeric compound which are able to cleave or eliminate formalin or formaldehyde; (v) pyrogallol; (vi) 1 ,2 hexanediol; (vii) one or more polyhydric alcohols, such as diethylene glycol and sorbitol; (viii) a nitrogen containing compound such as urea, melamine, diazine, triazine and amine compounds; (ix) a nitrogenous compound such as urea, ethyleneurea or carbohydrazide; (x) Dimethyl-1 ,3-acetonedicarboxylate, diethyl malonate or ethylacetoacetate; (xi) a water-soluble active methylene compound; (xii) hydrazine; (xiii) polyalkylene glycol diester; and (xiv) ammonium bisulfite.
The reversal agent may comprise an organic solvent.
The reversal agent may comprise an organic compound having at least one active methylene hydrogen and a pKa (logarithmic acid dissociation constant) in the range 5-13.
The step of applying the reversal agent to the first target may comprise applying the reversal agent to the first target at a concentration of: (i) 0-5 % w/v; (ii) 5-10 % w/v; (iii) 10- 15 % w/v; (iv) 15-20% w/v; (v) 20-25% w/v; (vi) 25-30 % w/v; (vii) 30-35% w/v; (viii) 35-40 % w/v; (ix) 40-45 % w/v; (x) 45-50 % w/v; (xi) 50-55% % w/v; (xii) 55-60% w/v; (xiii) 60-65% w/v; (xiv) 65-70% w/v; (xv) 70-75%; and (xvi) > 75 % w/v.
The step of applying the reversal agent to the first target may comprise applying the reversal agent to the first target at a concentration of: (i) 0-1 M; (ii) 1-2 M; (iii) 2-3 M; (iv) 3-4 M; (v) 4-5 M; (vi) 5-6 M; (vii) 6-7 M; (viii) 7-8 M; (ix) 8-9 M; (x) 9-10 M; (xi) 10-11 M; (xii) 11- 12 M; (xiii) 12-13 M; (xiv) 13-14 M; (xv) 14-15 M; and (xvi) > 15 M.
The method may further comprise subjecting the first target or the modified target to one or more heat treatment cycles.
A or each heat treatment cycle may comprise heating the first target or the modified target at a temperature selected from the group consisting of: (i) 20-30 °C; (ii) 30-40 °C; (iii) 40-50 °C; (iv) 50-60 °C; (v) 60-70 °C; (vi) 70-80 °C; (vii) 80-90 °C; (viii) 90-100 °C; (ix) 100- 1 10 °C; (x) 110-120 °C; and (xi) > 120 °C.
A or each heat treatment cycle may comprise heating the first target or the modified target for a period of time selected from the group consisting of: (i) 0-1 minutes; (ii) 1-2 minutes; (iii) 2-3 minutes; (iv) 3-4 minutes; (v) 4-5 minutes; (vi) 5-6 minutes; (vii) 6-7 minutes; (viii) 7-8 minutes; (ix) 8-9 minutes; (x) 9-10 minutes; and (xi) > 10 minutes.
The reversal agent may be applied by the first device.
According to another aspect there is provided a method of ambient ionisation comprising a method as disclosed above.
According to another aspect there is provided a method of desorption electrospray ionisation ("DESI") imaging comprising a method as disclosed above.
According to another aspect there is provided a method of desorption electroflow focusing ionisation ("DEFFI") imaging comprising a method as disclosed above.
According to another aspect there is provided a method of ion imaging comprising a method as disclosed above. According to another aspect there is provided a method of analysis comprising a method as disclosed above.
According to another aspect there is provided a method of surgery, diagnosis, therapy or medical treatment comprising a method as disclosed above.
According to another aspect there is provided a non-surgical, non-therapeutic method of mass spectrometry and/or ion mobility spectrometry comprising a method as disclosed above.
According to another aspect there is provided a method of mass spectrometry and/or ion mobility spectrometry comprising a method as disclosed above.
Various embodiments are contemplated wherein analyte ions generated by the ambient ionisation ion source are then subjected either to: (i) mass analysis by a mass analyser or filter such as a quadrupole mass analyser or a Time of Flight mass analyser; (ii) ion mobility analysis (IMS) and/or differential ion mobility analysis (DMA) and/or Field Asymmetric Ion Mobility Spectrometry (FAIMS) analysis; and/or (iii) a combination of firstly ion mobility analysis (IMS) and/or differential ion mobility analysis (DMA) and/or Field Asymmetric Ion Mobility Spectrometry (FAIMS) analysis followed by secondly mass analysis by a mass analyser or filter such as a quadrupole mass analyser or a Time of Flight mass analyser (or vice versa). Various embodiments also relate to an ion mobility spectrometer and/or mass analyser and a method of ion mobility spectrometry and/or method of mass analysis.
According to another aspect there is provided apparatus comprising:
a first device arranged and adapted to generate analyte or analyte ions from multiple regions of a modified target, wherein the modified target comprises a first target to which a reversal agent has been applied; and
a device for generating an ion image of the modified target.
The first device may comprise or form part of an ambient ion or ionisation source or the first device may generate aerosol, smoke or vapour for subsequent ionisation by an ambient ion or ionisation source or other ionisation source.
The first device may comprise a desorption electrospray ionisation ("DESI") ion source or a desorption electro-flow focusing ("DEFFI") ion source.
The first device may comprise an ion source selected from the group consisting of: (i) a rapid evaporative ionisation mass spectrometry ("REIMS") ion source; (ii) a laser desorption ionisation ("LDI") ion source; (iii) a thermal desorption ion source; (iv) a laser diode thermal desorption ("LDTD") ion source; (v) a dielectric barrier discharge ("DBD") plasma ion source; (vi) an Atmospheric Solids Analysis Probe ("ASAP") ion source; (vii) an ultrasonic assisted spray ionisation ion source; (viii) an easy ambient sonic-spray ionisation ("EASI") ion source; (ix) a desorption atmospheric pressure photoionisation ("DAPPI") ion source; (x) a paperspray ("PS") ion source; (xi) a jet desorption ionisation ("JeDI") ion source; (xii) a touch spray ("TS") ion source; (xiii) a nano-desorption electrospray ionisation ("nano-DESI") ion source; (xiv) a laser ablation electrospray ("LAESI") ion source; (xv) a direct analysis in real time ("DART") ion source; (xvi) a probe electrospray ionisation ("PESI") ion source; (xvii) a solid-probe assisted electrospray ionisation ("SPA-ESI") ion source; (xviii) a cavitron ultrasonic surgical aspirator ("CUSA") device; (xix) a focussed or unfocussed ultrasonic ablation device; (xx) a microwave resonance device; and (xxi) a pulsed plasma RF dissection device.
The first target may comprise a chemically modified target.
The first target may comprise a fixed tissue sample.
The fixed tissue sample may be fixed with either: (i) formalin or a formaldehyde solution; or (ii) formalin or a formaldehyde solution with glycerol or another chemical agent.
The fixed tissue sample may comprise a formalin fixed, paraffin-embedded
("FFPE") tissue sample.
The fixed tissue sample may be further embedded in an embedding medium selected from the group consisting of: (i) glycerol; (ii) optimum cutting temperature ("OCT") (RTM) medium; (iii) paraffin; (iv) one or more plastic polymers; (v) polyester wax; (vi) polyethylene glycol ("PEG"); (vii) polyfin (RTM); (viii) a tissue freezing medium ("TFM"); (ix) glycol methacrylate ("GMA"); (x) gelatin; and (xi) carboxy methyl cellulose ("SMC").
The first target may comprise a tissue sample wherein methylene bridge cross- linkages are formed between proteins.
The first target may comprise a tissue sample which has been dehydrated with an alcohol.
The first target may comprise a tissue sample which has been infiltrated with molten paraffin.
The first target and/or the modified target may be provided on a substrate.
The substrate may be selected from the group consisting of: (i) a microscope slide; (ii) a transparent slide; (iii) a glass slide; (iv) a plastic slide; and (v) an indium-tin-oxide (ITO) coated glass or other slide.
The reversal agent may be applied to the first target in order to reverse cross-linked bonds of proteins present in the first target.
The reversal agent may be arranged to reverse cross-linked bonds of formalin fixed tissue proteins or formalin fixed paraffin embedded ("FFPE") tissue proteins.
The reversal agent may comprise a formalin or formaldehyde scavenger.
The reversal agent may comprise water.
The reversal agent may comprise sodium metabisulfate or sodium metabisulfite (Na2S205).
The reversal agent may comprise urea.
The reversal agent may be selected from the group consisting of: (i) at least one dithioxamide; (ii) at least one carbodiimide; (iii) at least one monomeric, oligomeric or polymeric carbodiimide; (iv) at least one aminoplastic, thermoplastic, binder, polymeric compound or copolymeric compound which may be able to cleave or eliminate formalin or formaldehyde; (v) pyrogallol; (vi) 1 ,2 hexanediol; (vii) one or more polyhydric alcohol, such as diethylene glycol and sorbitol; (viii) a nitrogen containing compound such as urea, melamine, diazine, triazine and amine compounds; (ix) a nitrogenous compound such as urea, ethyleneurea or carbohydrazide; (x) Dimethyl-1 ,3-acetonedicarboxylate, diethyl malonate or ethylacetoacetate; (xi) a water-soluble active methylene compound; (xii) hydrazine; (xiii) polyalkylene glycol diester; and (xiv) ammonium bisulfite.
The reversal agent may comprise an organic solvent.
The reversal agent may comprise an organic compound having at least one active methylene hydrogen and a pKa (logarithmic acid dissociation constant) in the range 5-13.
The reversal agent may be applied to the first target at a concentration of: (i) 0-5 % w/v; (ii) 5-10 % w/v; (iii) 10-15 % w/v; (iv) 15-20% w/v; (v) 20-25% w/v; (vi) 25-30 % w/v; (vii) 30-35% w/v; (viii) 35-40 % w/v; (ix) 40-45 % w/v; (x) 45-50 % w/v; (xi) 50-55% % w/v; (xii) 55-60% w/v; (xiii) 60-65% w/v; (xiv) 65-70% w/v; (xv) 70-75%; and (xvi) > 75 % w/v.
The reversal agent may be applied to the first target at a concentration of: (i) 0-1 M;
(ii) 1-2 M; (iii) 2-3 M; (iv) 3-4 M; (v) 4-5 M; (vi) 5-6 M; (vii) 6-7 M; (viii) 7-8 M; (ix) 8-9 M; (x) 9-10 M; (xi) 10-11 M; (xii) 11-12 M; (xiii) 12-13 M; (xiv) 13-14 M; (xv) 14-15 M; and (xvi) > 15 M.
The first target or the modified target may be subjected to one or more heat treatment cycles.
A or each heat treatment cycle may comprise heating the first target or the modified target at a temperature selected from the group consisting of: (i) 20-30 °C; (ii) 30-40 °C; (iii) 40-50 °C; (iv) 50-60 °C; (v) 60-70 °C; (vi) 70-80 °C; (vii) 80-90 °C; (viii) 90-100 °C; (ix) 100- 1 10 °C; (x) 110-120 °C; and (xi) > 120 °C.
A or each heat treatment cycle may comprise heating the first target or the modified target for a period of time selected from the group consisting of: (i) 0-1 minutes; (ii) 1-2 minutes; (iii) 2-3 minutes; (iv) 3-4 minutes; (v) 4-5 minutes; (vi) 5-6 minutes; (vii) 6-7 minutes; (viii) 7-8 minutes; (ix) 8-9 minutes; (x) 9-10 minutes; and (xi) > 10 minutes.
The reversal agent may be applied by the first device.
According to another aspect there is provided an ambient ionisation ion source comprising apparatus as disclosed above.
According to another aspect there is provided a desorption electrospray ionisation ("DESI") imaging system comprising apparatus as disclosed above.
According to another aspect there is provided a desorption electroflow focusing ionisation ("DEFFI") imaging system comprising apparatus as disclosed above.
According to another aspect there is provided an ion imager comprising apparatus as disclosed above.
According to another aspect there is provided analysis apparatus comprising apparatus as disclosed above.
According to another aspect there is provided a mass and/or ion mobility spectrometer comprising apparatus as disclosed above.
Various embodiments are contemplated wherein analyte ions generated by the ambient ionisation ion source are then subjected either to: (i) mass analysis by a mass analyser or filter such as a quadrupole mass analyser or a Time of Flight mass analyser; (ii) ion mobility analysis (IMS) and/or differential ion mobility analysis (DMA) and/or Field Asymmetric Ion Mobility Spectrometry (FAIMS) analysis; and/or (iii) a combination of firstly ion mobility analysis (IMS) and/or differential ion mobility analysis (DMA) and/or Field Asymmetric Ion Mobility Spectrometry (FAIMS) analysis followed by secondly mass analysis by a mass analyser or filter such as a quadrupole mass analyser or a Time of Flight mass analyser (or vice versa). Various embodiments also relate to an ion mobility spectrometer and/or mass analyser and a method of ion mobility spectrometry and/or method of mass analysis.
According to another aspect there is provided apparatus comprising:
an ion source comprising a nozzle having an aperture;
a solvent emitter which extends through the aperture; and
a centering device which is arranged and adapted to locate or position the solvent emitter substantially centrally within the aperture.
The ion source may comprise a Desorption Electrospray lonisation ("DESI") ion source or a desorption electro-flow focusing ("DEFFI") ion source.
The apparatus may further comprise a solvent emitter guide.
The solvent emitter may be located or positioned within the solvent emitter guide. The centering device may be arranged and adapted to locate or position the solvent emitter guide substantially centrally within an internal volume of the nozzle.
The solvent emitter guide may be electrically conductive.
The solvent emitter guide may comprise metal.
The solvent emitter guide may comprise stainless steel.
The nozzle may be electrically conductive.
The nozzle may comprise metal.
The nozzle may comprise stainless steel.
The centering device may comprise a disc.
The centering device may comprise or may be coated with an insulator.
The centering device may comprise or may be coated with a ceramic.
The centering device may comprise or may be coated with a polymer.
The solvent emitter guide may be electrically insulating.
The solvent emitter guide may comprise a ceramic.
The solvent emitter guide may comprise a polymer.
The apparatus may further comprise a device for supplying a solvent to the solvent emitter.
The apparatus may further comprise a device for supplying a nebulising gas within the nozzle so that, in use, the nebulising gas exits the nozzle via the aperture.
According to another aspect there is provided an ambient ionisation ion source comprising apparatus as disclosed above.
According to another aspect there is provided a desorption electrospray ionisation ("DESI") imaging system comprising apparatus as disclosed above.
According to another aspect there is provided a desorption electroflow focusing ionisation ("DEFFI") imaging system comprising apparatus as disclosed above.
According to another aspect there is provided an ion imager comprising apparatus as disclosed above. According to another aspect there is provided analysis apparatus comprising apparatus as disclosed above.
According to another aspect there is provided a mass and/or ion mobility spectrometer comprising apparatus as disclosed above.
According to another aspect there is provided a method comprising:
providing an ion source comprising a nozzle having an aperture;
providing a solvent emitter which extends through the aperture; and
using a centering device to locate or position the solvent emitter substantially centrally within the aperture.
According to another aspect there is provided a method of mass spectrometry and/or ion mobility spectrometry comprising a method as disclosed above.
According to another aspect there is provided apparatus comprising:
a first device arranged and adapted to emit a stream of electrically charged droplets towards a target in use;
a transfer capillary or transfer device arranged and adapted to transfer ions generated from the target towards an ion analyser or mass and/or ion mobility
spectrometer; and
a heating device arranged and adapted to heat either: (i) a capillary of the first device; (ii) the stream of electrically charged droplets emitted from the first device; (iii) the target; or (iv) the transfer capillary or transfer device.
The first device may comprise a Desorption Electrospray lonisation ("DESI") ion source or a desorption electroflow focusing ionisation ("DEFFI") ion source.
The heating device may comprise a heater.
The heater may comprise a wire heater.
The heater may comprise an inductive or eddy current heater.
The heating device may be arranged and adapted to heat the capillary of the first device, the stream of electrically charged droplets emitted from the first device, the target or the transfer capillary or transfer device to a temperature of at least 50 °C, 100 °C, 200 °C, 300 °C, 400 °C, 500 °C, 600 °C, 700 °C or 800 °C.
According to another aspect there is provided a mass and/or ion mobility spectrometer comprising apparatus as disclosed above.
According to another aspect there is provided a method comprising:
emitting a stream of electrically charged droplets from a first device towards a target;
providing a transfer capillary or transfer device for transferring ions generated from the target towards an ion analyser or mass and/or ion mobility spectrometer; and
heating either: (i) a capillary of the first device; (ii) the stream of electrically charged droplets emitted from the first device; (iii) the target; or (iv) the transfer capillary or transfer device.
The first device may comprise a Desorption Electrospray lonisation ("DESI") ion source or a desorption electroflow focusing ionisation ("DEFFI") ion source.
The heating device may comprise a heater. The heater may comprise a wire heater.
The heater may comprise an inductive or eddy current heater.
According to an embodiment the method may comprise heating the capillary of the first device, the stream of electrically charged droplets emitted from the first device, the target or the transfer capillary or transfer device to a temperature of at least 50 °C, 100 °C, 200 °C, 300 °C, 400 °C, 500 °C, 600 °C, 700 °C or 800 °C.
According to another aspect there is provided a method of mass spectrometry and/or ion mobility spectrometry comprising a method as disclosed above.
According to another aspect there is provided apparatus comprising:
an ion source comprising a nozzle having an aperture;
a solvent emitter which extends through the aperture; and
a fixing device which is arranged and adapted to fix the solvent emitter relative to the aperture.
The fixing device may comprise a centering device which is arranged and adapted to locate or position the solvent emitter substantially centrally within the aperture.
The fixing device comprises a device which is arranged and adapted to locate or position the solvent emitter substantially non-centrally within the aperture.
The fixing device may comprise a device which is arranged and adapted to locate or position the solvent emitter substantially non-centrally within the aperture, such that the solvent emitter is substantially closer to and/or oriented towards an inlet of a mass and/or ion mobility spectrometer.
The fixing device may be arranged and adapted such that gas may flow through the nozzle and/or aperture relatively unimpeded by the fixing device.
According to an aspect there is provided apparatus comprising:
an ion source comprising a nozzle having an aperture;
a solvent emitter which extends through the aperture; and
a device which is arranged and adapted to monitor the solvent emitter position relative to the aperture.
The apparatus may comprise a device arranged and adapted to calibrate and/or correct the ion source and/or mass and/or ion mobility spectrometric data acquired using the ion source based on the monitoring.
According to an aspect there is provided a mass and/or ion mobility spectrometer comprising apparatus as described above.
According to an aspect there is provided a method comprising:
providing an ion source comprising a nozzle having an aperture;
providing a solvent emitter which extends through the aperture; and
using a fixing device to fix the solvent emitter relative to the aperture.
The method may comprise using the fixing device to locate or position the solvent emitter substantially centrally within the aperture.
The method may comprise using the fixing device to locate or position the solvent emitter substantially non-centrally within the aperture. The method may comprise using the fixing device to locate or position the solvent emitter substantially non-centrally within the aperture, such that the solvent emitter is substantially closer to and/or oriented towards an inlet of a mass and/or ion mobility spectrometer.
The method may comprise passing gas through the nozzle and/or aperture such that the gas flows relatively unimpeded by the fixing device.
According to an aspect there is provided a method comprising:
providing an ion source comprising a nozzle having an aperture;
providing a solvent emitter which extends through the aperture; and
monitoring the solvent emitter position relative to the aperture.
The method may comprise calibrating and/or correct the ion source and/or mass and/or ion mobility spectrometric data acquired using the ion source based on the monitoring.
According to an aspect there is provided a method of mass spectrometry and/or ion mobility spectrometry comprising a method as described above.
The mass and/or ion mobility spectrometer may obtain data in negative ion mode only, positive ion mode only, or in both positive and negative ion modes. Positive ion mode spectrometric data may be combined or concatenated with negative ion mode
spectrometric data.
Ion mobility spectrometric data may be obtained using different ion mobility drift gases and/or dopants. This data may then be combined or concatenated.
Various embodiments are contemplated which relate to generating smoke, aerosol or vapour from a target (details of which are provided elsewhere herein) using an ambient ionisation ion source. The aerosol, smoke or vapour may then be mixed with a matrix and aspirated into a vacuum chamber of a mass spectrometer and/or ion mobility spectrometer. The mixture may be caused to impact upon a collision surface causing the aerosol, smoke or vapour to be ionised by impact ionization which results in the generation of analyte ions. The resulting analyte ions (or fragment or product ions derived from the analyte ions) may then be mass analysed and/or ion mobility analysed and the resulting mass spectrometric data and/or ion mobility spectrometric data may be subjected to multivariate analysis or other mathematical treatment in order to determine one or more properties of the target in real time.
According to an embodiment the first device for generating aerosol, smoke or vapour from the target may comprise a tool which utilises an RF voltage, such as a continuous RF waveform.
Other embodiments are contemplated wherein the first device for generating aerosol, smoke or vapour from the target may comprise an argon plasma coagulation ("APC") device. An argon plasma coagulation device involves the use of a jet of ionised argon gas (plasma) that is directed through a probe. The probe may be passed through an endoscope. Argon plasma coagulation is essentially a non-contact process as the probe is placed at some distance from the target. Argon gas is emitted from the probe and is then ionized by a high voltage discharge (e.g., 6 kV). High-frequency electric current is then conducted through the jet of gas, resulting in coagulation of the target on the other end of the jet. The depth of coagulation is usually only a few millimetres.
The first device, surgical or electrosurgical tool, device or probe or other sampling device or probe disclosed in any of the aspects or embodiments herein may comprise a non-contact surgical device, such as one or more of a hydrosurgical device, a surgical water jet device, an argon plasma coagulation device, a hybrid argon plasma coagulation device, a water jet device and a laser device.
A non-contact surgical device may be defined as a surgical device arranged and adapted to dissect, fragment, liquefy, aspirate, fulgurate or otherwise disrupt biologic tissue without physically contacting the tissue. Examples include laser devices, hydrosurgical devices, argon plasma coagulation devices and hybrid argon plasma coagulation devices.
As the non-contact device may not make physical contact with the tissue, the procedure may be seen as relatively safe and can be used to treat delicate tissue having low intracellular bonds, such as skin or fat.
According to various embodiments the mass spectrometer and/or ion mobility spectrometer may obtain data in negative ion mode only, positive ion mode only, or in both positive and negative ion modes. Positive ion mode spectrometric data may be combined or concatenated with negative ion mode spectrometric data. Negative ion mode can provide particularly useful spectra for classifying aerosol, smoke or vapour samples, such as aerosol, smoke or vapour samples from targets comprising lipids.
Ion mobility spectrometric data may be obtained using different ion mobility drift gases, or dopants may be added to the drift gas to induce a change in drift time of one or more species. This data may then be combined or concatenated.
It will be apparent that the requirement to add a matrix or a reagent directly to a sample may prevent the ability to perform in vivo analysis of tissue and also, more generally, prevents the ability to provide a rapid simple analysis of target material.
According to other embodiments the ambient ionisation ion source may comprise an ultrasonic ablation ion source or a hybrid electrosurgical -ultrasonic ablation source that generates a liquid sample which is then aspirated as an aerosol. The ultrasonic ablation ion source may comprise a focused or unfocussed ultrasound.
Optionally, the first device comprises or forms part of an ion source selected from the group consisting of: (i) a rapid evaporative ionisation mass spectrometry ("REIMS") ion source; (ii) a desorption electrospray ionisation ("DESI") ion source; (iii) a laser desorption ionisation ("LDI") ion source; (iv) a thermal desorption ion source; (v) a laser diode thermal desorption ("LDTD") ion source; (vi) a desorption electro-flow focusing ("DEFFI") ion source; (vii) a dielectric barrier discharge ("DBD") plasma ion source; (viii) an Atmospheric Solids Analysis Probe ("ASAP") ion source; (ix) an ultrasonic assisted spray ionisation ion source; (x) an easy ambient sonic-spray ionisation ("EASI") ion source; (xi) a desorption atmospheric pressure photoionisation ("DAPPI") ion source; (xii) a paperspray ("PS") ion source; (xiii) a jet desorption ionisation ("JeDI") ion source; (xiv) a touch spray ("TS") ion source; (xv) a nano-DESI ion source; (xvi) a laser ablation electrospray ("LAESI") ion source; (xvii) a direct analysis in real time ("DART") ion source; (xviii) a probe electrospray ionisation ("PESI") ion source; (xix) a solid-probe assisted electrospray ionisation ("SPA- ESI") ion source; (xx) a cavitron ultrasonic surgical aspirator ("CUSA") device; (xxi) a hybrid CUSA-diathermy device; (xxii) a focussed or unfocussed ultrasonic ablation device; (xxiii) a hybrid focussed or unfocussed ultrasonic ablation and diathermy device; (xxiv) a microwave resonance device; (xxv) a pulsed plasma RF dissection device; (xxvi) an argon plasma coagulation device; (xxvi) a hybrid pulsed plasma RF dissection and argon plasma coagulation device; (xxvii) a hybrid pulsed plasma RF dissection and JeDI device; (xxviii) a surgical water/saline jet device; (xxix) a hybrid electrosurgery and argon plasma
coagulation device; and (xxx) a hybrid argon plasma coagulation and water/saline jet device.
BRIEF DESCRIPTION OF THE DRAWINGS
Various embodiments will now be described, by way of example only, and with reference to the accompanying drawings in which:
Fig. 1A shows a desorption electrospray ionisation ion image for counts in the range 0-250 at m/z 726.5 obtained in negative ion mode from fresh pork liver embedded in a cryoprotective embedding medium OCT, Fig. 1 B shows a desorption electrospray ionisation image for counts in the range 0-12000 at m/z 766.5 obtained in negative ion mode from fresh pork liver embedded in a cryoprotective embedding medium OCT and Fig. 1 C shows a desorption electrospray ionisation image for counts in the range 0-1400 at m/z 740.5 obtained in negative ion mode from fresh pork liver embedded in a cryoprotective embedding medium OCT;
Fig. 2A shows a desorption electrospray ionisation mass spectrometry imaging (MSI) ion image obtained in negative ion mode of formalin fixed pork liver embedded in OCT and Fig. 2B shows a mass spectrum obtained in negative ion mode from formalin fixed pork liver embedded in OCT;
Fig. 3A shows a spectrum intensity in negative ion mode from frozen fresh pork liver at m/z 885.5 (characteristic of phosphatidylinositol (Pl(18:0/20:4)) and Fig. 3B shows a corresponding spectrum intensity from formalin fixed pork liver washed in LC-MS grade water at m/z 885.5 (characteristic of phosphatidylinositol (Pl(18:0/20:4));
Fig. 4A shows a desorption electrospray ionisation mass spectrum obtained in negative ion mode from fresh frozen pork liver and Fig. 4B shows a desorption electrospray ionisation mass spectrum from formalin fixed pork liver washed in LC-MS grade water showing a lower signal intensity;
Fig. 5A shows a spectrum intensity in negative ion mode from fresh frozen pork liver, Fig. 5B shows a spectrum intensity from formalin fixed pork liver and Fig. 5C shows a spectrum intensity from heat-treated formalin fixed pork liver;
Fig. 6A shows a desorption electrospray ionisation mass spectrometry imaging (MSI) ion image obtained in negative ion mode from frozen fresh pork liver, Fig. 6B shows a desorption electrospray ionisation mass spectrometry imaging (MSI) ion image obtained from formalin fixed pork liver and Fig. 6C shows a desorption electrospray ionisation mass spectrometry imaging (MSI) ion image obtained from heat-treated formalin fixed pork liver;
Fig. 7 A shows spectra intensity obtained in negative ion mode from formalin fixed pork liver heat-treated with 15% (w/v) sodium metabisulfate and Fig. 7B shows a corresponding ion image for m/z 794.5;
Fig. 8A shows a microscope image from formalin fixed pork liver heat-treated with sodium metabisulfate prior to desorption electrospray ionisation mass spectrometry imaging (MSI) analysis and Fig. 8B shows a corresponding microscope image post- desorption electrospray ionisation mass spectrometry imaging (MSI) analysis;
Fig. 9A shows a spectral intensity obtained in negative ion mode of formalin fixed pork liver using sodium metabisulfate 15% (w/v) and one heat-treatment cycle as formalin scavengers, Fig. 9B shows a spectral intensity obtained in negative ion mode of formalin fixed pork liver using sodium metabisulfate 15% (w/v) and three heat-treatment cycles as formalin scavengers and Fig. 9C shows a spectral intensity obtained in negative ion mode of formalin fixed pork liver using sodium metabisulfate 40% (w/v) and one heat-treatment cycle as formalin scavengers;
Fig. 10A shows a desorption electrospray ionisation mass spectrometry imaging (MSI) ion image at m/z 794.53 from formalin fixed pork liver, heat-treated for 3 cycles with 15% (w/v) sodium metabisulfate after desorption electrospray ionisation mass spectrometry imaging (MSI) analysis in negative ion mode ion image and Fig. 10B shows a
corresponding microscope image of the tissue after desorption electrospray ionisation mass spectrometry imaging (MSI) (square);
Fig. 11 shows a comparison of spectra intensity obtained in negative ion mode from formalin fixed pork liver using either 1 M or 5M of urea and either 3 or 5 heat-treatment cycles as formalin scavengers;
Fig. 12A shows a microscope image of formalin fixed pork liver post-desorption electrospray ionisation mass spectrometry imaging (MSI) analysis after being subjected to 5M urea and 3 heat-treatment cycles using a SuperFrost slide, Fig. 12B shows a microscope image of formalin fixed pork liver post-desorption electrospray ionisation mass spectrometry imaging (MSI) analysis after being subjected to 5M urea and 3 heat-treatment cycles using a SuperFrostPlus slide and Fig. 12C shows a microscope image of formalin fixed pork liver post-desorption electrospray ionisation mass spectrometry imaging (MSI) analysis after being subjected to 5M urea and 5 heat-treatment cycles on a Poly-Lysine slide;
Fig. 13A shows an ion image of formalin fixed pork liver heat-treated for 3 cycles with 5M urea after desorption electrospray ionisation mass spectrometry imaging (MSI) analysis in negative ion mode with ion image (m/z 794.53) using a SuperFrost slide and Fig. 13B shows an ion image (m/z 794.53) using a SuperFrostPlus slide;
Fig. 14 shows a desorption electrospray ionisation mass spectrometry imaging (MSI) image of a human lung biopsy after EVLP treatment wherein the image comprises a RGB image of PCs 1 , 2 and 5 for a human lung tissue sample (EXT8-T2-S1);
Fig. 15 shows a method of analysis that comprises building a classification model according to various embodiments;
Fig. 16 shows a set of reference sample mass spectra obtained from two classes of known reference samples;
Fig. 17 shows a multivariate space having three dimensions defined by intensity axes, wherein the multivariate space comprises plural reference points, each reference point corresponding to a set of three peak intensity values derived from a reference sample spectrum;
Fig. 18 shows a general relationship between cumulative variance and number of components of a PCA model;
Fig. 19 shows a PCA space having two dimensions defined by principal component axes, wherein the PCA space comprises plural transformed reference points or scores, each transformed reference point corresponding to a reference point of Fig. 17;
Fig. 20 shows a PCA-LDA space having a single dimension or axis, wherein the
LDA is performed based on the PCA space of Fig. 19, the PCA-LDA space comprising plural further transformed reference points or class scores, each further transformed reference point or class score corresponding to a transformed reference point or score of
Fig. 19;
Fig. 21 shows a method of analysis that comprises using a classification model according to various embodiments;
Fig. 22 shows a sample spectrum obtained from an unknown sample;
Fig. 23 shows the PCA-LDA space of Fig. 20, wherein the PCA-LDA space further comprises a PCA-LDA projected sample point derived from the peak intensity values of the sample spectrum of Fig. 22;
Fig. 24 shows a method of analysis that comprises building a classification library according to various embodiments;
Fig. 25 shows a method of analysis that comprises using a classification library according to various embodiments;
Fig. 26A shows RGB images of PCA components 1 , 2 and 3 in negative ion mode along with a respective histological image of a Grade II Invasive Ductal Carcinoma (IDC) and Fig. 26B shows RGB images of PCA components 1 , 2 and 3 in positive ion mode along with a respective histological image of a Grade II Invasive Ductal Carcinoma (IDC);
Fig. 27A shows PCA analysis of a Grade II Invasive Ductal Carcinoma in negative ion mode and Fig. 27B shows MMC analysis of a Grade II Invasive Ductal Carcinoma in negative ion mode;
Fig. 28A shows PCA analysis of a Grade II Invasive Ductal Carcinoma in positive ion mode and Fig. 28B shows MMC analysis of a Grade II Invasive Ductal Carcinoma in positive ion mode;
Fig. 29A shows leave one out cross validation of different tissue types in a Grade II Invasive Ductal Carcinoma in negative ion mode and Fig. 29B shows leave one out cross validation of different tissue types in a Grade II Invasive Ductal Carcinoma in positive ion mode; Fig. 30 shows histological images with annotated regions of interest by a histopathologist together with PCA analysis of these assigned regions and MMC
supervised analysis components from samples analysed in the negative ion mode;
Fig. 31 A shows analysis of a combined dataset from multiple samples (negative ion mode) of PCA of identified regions, Fig. 31 B shows MMC supervised analysis, Fig. 31 C shows MMC analysis excluding the samples with outliers identified in Fig. 31 B and Fig. 31 D shows respective leave-one-region-per-patient-out cross validation;
Fig. 32A shows PCA of identified regions, Fig. 32B shows MMC supervised analysis and Fig. 32C shows leave-one-region-per-patient-out cross validation for the positive ion mode data;
Fig. 33A shows supervised MMC analysis of healthy ovary, borderline tumours and carcinomas and Fig. 33B shows corresponding leave one patient out cross validation for negative ion mode;
Fig. 34A shows supervised MMC analysis of healthy ovary and different epithelial carcinomas (endometrioid and serous) and Fig. 34B shows corresponding leave one patient out cross validation for negative ion mode;
Fig. 35A shows a sample with unknown histology used to predict different tissue types and Fig. 35B shows a cross validation of the prediction based on the histological annotation;
Fig. 36 shows a schematic of a desorption electroflow focusing ionisation ("DEFFI") setup according to an embodiment;
Fig. 37 shows a total ion count for desorption electroflow focusing ionisation ("DEFFI") on a pork liver at different applied voltages in negative ion mode;
Fig. 38A shows a desorption electroflow focusing ionisation ("DEFFI") mass spectrometric image of human breast cancer tissue and shows RGB images of the first three principal components from principal component analysis of all pixels using the full acquired m/z range from 150 to 1500, Fig. 38B shows base peak intensity (BPI) normalised example spectra for breast tissue for desorption electroflow focusing ionisation ("DEFFI"), Fig. 38C shows desorption traces in line-scanning mode on rhodamine B-coated glass slides for desorption electroflow focusing ionisation ("DEFFI"), Fig. 38D shows a desorption electrospray ionisation mass spectrometric image of human breast cancer tissue and shows RGB images of the first three principal components from principal component analysis of all pixels using the full acquired m/z range from 150 to 1500, Fig. 38E shows base peak intensity (BPI) normalised example spectra for breast tissue for desorption electrospray ionisation and Fig. 38F shows desorption traces in line-scanning mode on rhodamine B-coated glass slides for desorption electrospray ionisation;
Fig. 39A shows a RGB image for pork liver of the first three principal components of principal component analysis across the full m/z range for desorption electroflow focusing ionisation ("DEFFI"), Fig. 39B shows a RGB image for pork liver of the first three principal components of principal component analysis across the full m/z range for desorption electrospray ionisation, Fig. 39C shows representative spectra from pork liver in negative ion mode for desorption electroflow focusing ionisation ("DEFFI"), Fig. 39D shows representative spectra from pork liver in negative ion mode for desorption electrospray ionisation and Fig. 39E show desorption traces on rhodamine B for both methods for three different solvent flow rates;
Fig. 40 shows normalised base peak intensity for pork liver in negative ion mode for desorption electroflow focusing ionisation ("DEFFI") and desorption electrospray ionisation at three different solvent flow rates;
Fig. 41 shows a diagram of a sprayer nozzle according to an embodiment wherein a stainless steel emitter guide and gas nozzle are electrically insulated from each other to avoid charging of the gas nozzle and wherein this is achieved by using a ceramic-coated centering disc;
Fig. 42A shows desorption electrospray ionisation-MS imaging of adjacent colorectal cancer sections at different spatial resolutions wherein RGB images for the first three components of principal component analysis of images were acquired at 100 μηι, Fig. 42B shows RGB images for the first three components of principal component analysis of images which were acquired at 50 μηι, Fig. 42C shows RGB images for the first three components of principal component analysis of images which were acquired at 20 μηι pixel size/ scan speed per second, Fig. 42D shows a trace on rhodamine B corresponding to the experimental conditions of Fig. 42A, Fig. 42E shows a trace on rhodamine B corresponding to the experimental conditions of Figs. 42B and 42C, Fig. 42F shows an optical image of section A after hematoxylin and eosin staining and Fig. 42G shows an example spectrum from section A;
Fig. 43 shows a Desorption Electrospray Ionisation ("DESI") device according to another embodiment; and
Fig. 44A shows a graph of intensity versus inlet capillary temperature for analysis of fatty acids using a Waters Synapt (RTM) mass spectrometer, Fig. 44B shows a graph of intensity versus inlet capillary temperature for analysis of fatty acids using a Waters Xevo (RTM) mass spectrometer, Fig. 44C shows a graph of intensity versus inlet capillary temperature for analysis of phospholipids using a Waters Synapt (RTM) mass
spectrometer and Fig. 44D shows a graph of intensity versus inlet capillary temperature for analysis of phospholipids using a Waters Xevo (RTM) mass spectrometer.
DETAILED DESCRIPTION
Various embodiments will now be described in more detail below which in general relate to obtaining an ion image of a target (e.g., ex vivo tissue) using an ambient ionisation ion source such as a desorption electrospray ionisation ("DESI") ion source.
Ambient ionisation ion sources According to various embodiments a device is used to generate analyte ions from one or more regions of a target (e.g., ex vivo tissue). The device may comprise an ambient ionisation ion source which is characterised by the ability to generate analyte aerosol, smoke or vapour from a native or unmodified target. For example, other types of ionisation ion sources such as Matrix Assisted Laser Desorption lonisation ("MALDI") ion sources require a matrix or reagent to be added to the sample prior to ionisation.
It will be apparent that the requirement to add a matrix or a reagent to a sample prevents the ability to perform in vivo analysis of tissue and also, more generally, prevents the ability to provide a rapid simple analysis of target material.
In contrast, therefore, ambient ionisation techniques are particularly advantageous since firstly they do not require the addition of a matrix or a reagent (and hence are suitable for the analysis of in vivo tissue) and since secondly they enable a rapid simple analysis of target material to be performed.
A number of different ambient ionisation techniques are known and are intended to fall within the scope of the present invention. As a matter of historical record, Desorption Electrospray lonisation ("DESI") was the first ambient ionisation technique to be developed and was disclosed in 2004. Since 2004, a number of other ambient ionisation techniques have been developed. These ambient ionisation techniques differ in their precise ionisation method but they share the same general capability of generating gas-phase ions directly from native (i.e. untreated or unmodified) samples. A particular advantage of the various ambient ionisation techniques which are intended to fall within the scope of the present invention is that the various ambient ionisation techniques do not require any prior sample preparation. As a result, the various ambient ionisation techniques enable both in vivo tissue and ex vivo tissue samples to be analysed without necessitating the time and expense of adding a matrix or reagent to the tissue sample or other target material.
A list of ambient ionisation techniques which are intended to fall within the scope of the present invention are given in the following table:
Acronym lonisation technique
Desorption electrospray ionization
DeSSI Desorption sonic spray ionization
Desorption atmospheric pressure
DAPPI
photoionization
EASI Easy ambient sonic-spray ionization
Jet desorption electrospray ionization
Transmission mode desorption electrospray
TM-DESI
ionization
LMJ-SSP Liquid microjunction-surface sampling probe
DICE Desorption ionization by charge exchange
Nano-DESI Nanospray desorption electrospray ionization
Electrode-assisted desorption electrospray
EADESI
ionization
Atmospheric pressure thermal desorption
APTDCI
chemical ionization
V-EASI Venturi easy ambient sonic-spray ionization AFAI Air flow-assisted ionization
LESA Liquid extraction surface analysis
PTC-ESI Pipette tip column electrospray ionization
Air flow-assisted desorption electrospray
AFADESI
ionization
DEFFI Desorption electro-flow focusing ionization
ESTASI Electrostatic spray ionization
Plasma-based ambient sampling ionization
PASIT
transmission
Desorption atmospheric pressure chemical
DAPCI
ionization
DART Direct analysis in real time
ASAP Atmospheric pressure solid analysis probe
Atmospheric pressure thermal desorption
APTDI
ionization
PADI Plasma assisted desorption ionization
DBDI Dielectric barrier discharge ionization
FA PA Flowing atmospheric pressure afterglow
Helium atmospheric pressure glow discharge
HAPGDI
ionization
Atmospheric pressure glow discharge
APGDDI
desorption ionization
LTP Low temperature plasma
Liquid sampling-atmospheric pressure glow
LS-APGD
discharge
Microwave induced plasma desorption
MIPDI
ionization
MFGDP Microfabricated glow discharge plasma
RoPPI Robotic plasma probe ionization
PLASI Plasma spray ionization
Matrix assisted laser desorption electrospray
MALDESI
ionization
ELDI Electrospray laser desorption ionization
LDTD Laser diode thermal desorption
LAESI Laser ablation electrospray ionization
CALDI Charge assisted laser desorption ionization
Laser ablation flowing atmospheric pressure
LA-FAPA
afterglow
Laser assisted desorption electrospray
LADESI
ionization
LDESI Laser desorption electrospray ionization LEMS Laser electrospray mass spectrometry
LSI Laser spray ionization
Infrared laser ablation metastable induced
IR-LAMICI
chemical ionization
LDSPI Laser desorption spray post-ionization
Plasma assisted multiwavelength laser
PAMLDI
desorption ionization
High voltage-assisted laser desorption
HALDI
ionization
PALDI Plasma assisted laser desorption ionization
ESSI Extractive electrospray ionization
PESI Probe electrospray ionization
Neutral desorption extractive electrospray
ND-ESSI
ionization
PS Paper spray
Direct inlet probe-atmospheric pressure chemical ionization
TS Touch spray
Wooden-tip Wooden-tip electrospray
Coated blade spray solid phase
CBS-SPME
microextraction
TSI Tissue spray ionization
RADIO Radiofrequency acoustic desorption ionization
Laser induced acoustic desorption
LIAD-ESI
electrospray ionization
SAWN Surface acoustic wave nebulization
UASI Ultrasonication-assisted spray ionization
Solid probe assisted nanoelectrospray
SPA-nanoESI
ionization
PAUSI Paper assisted ultrasonic spray ionization
DPESI Direct probe electrospray ionization
ESA-Py Electrospray assisted pyrolysis ionization
APPIS Ambient pressure pyroelectric ion source
Remote analyte sampling transport and
RA TI R
ionization relay
SACI Surface activated chemical ionization
Desorption electrospray metastable-induced
DFMI
ionization
Rapid evaporative ionization mass
RFIM
spectrometry
SPAM Single particle aerosol mass spectrometry Thermal desorption-based ambient mass
TDAMS
spectrometry
MAN Matrix assisted inlet ionization
SAN Solvent assisted inlet ionization
SwiFERR Switched ferroelectric plasma ionizer
Leidenfrost phenomenon assisted thermal
LPTD
desorption
According to an embodiment the ambient ionisation ion source may comprise a rapid evaporative ionisation mass spectrometry ("REIMS") ion source wherein a RF voltage is applied to one or more electrodes in order to generate an aerosol or plume of surgical smoke by Joule heating.
However, it will be appreciated that other ambient ion sources including those referred to above may also be utilised. For example, according to another embodiment the ambient ionisation ion source may comprise a laser ionisation ion source. According to an embodiment the laser ionisation ion source may comprise a mid-1 R laser ablation ion source. For example, there are several lasers which emit radiation close to or at 2.94 μηι which corresponds with the peak in the water absorption spectrum. According to various embodiments the ambient ionisation ion source may comprise a laser ablation ion source having a wavelength close to 2.94 μηι on the basis of the high absorption coefficient of water at 2.94 μηι. According to an embodiment the laser ablation ion source may comprise a Er:YAG laser which emits radiation at 2.94 μηι.
Other embodiments are contemplated wherein a mid-infrared optical parametric oscillator ("OPO") may be used to produce a laser ablation ion source having a longer wavelength than 2.94 μηι. For example, an Er:YAG pumped ZGP-OPO may be used to produce laser radiation having a wavelength of e.g. 6.1 μηι, 6.45 μηι or 6.73 μηι. In some situations it may be advantageous to use a laser ablation ion source having a shorter or longer wavelength than 2.94 μηι since only the surface layers will be ablated and less thermal damage may result. According to an embodiment a Co:MgF2 laser may be used as a laser ablation ion source wherein the laser may be tuned from 1.75-2.5 μηι.
According to another embodiment an optical parametric oscillator ("OPO") system pumped by a Nd:YAG laser may be used to produce a laser ablation ion source having a wavelength between 2.9-3.1 μηι. According to another embodiment a C02 laser having a wavelength of 10.6 μηι may be used to generate the aerosol, smoke or vapour.
According to other embodiments the ambient ionisation ion source may comprise an ultrasonic ablation ion source which generates a liquid sample which is then aspirated as an aerosol. The ultrasonic ablation ion source may comprise a focused or unfocussed source.
According to another embodiment the first device may comprise a tool or probe which utilises a continuous RF waveform. According to other embodiments a
radiofrequency tissue dissection tool or probe may be used which is arranged to supply pulsed plasma RF energy to the tool or probe. The tool or probe may comprise, for example, a PlasmaBlade (RTM). Pulsed plasma RF tools operate at lower temperatures than conventional electrosurgical tools (e.g. 40-170 °C c.f. 200-350 °C) thereby reducing thermal injury depth. Pulsed waveforms and duty cycles may be used for both cut and coagulation modes of operation by inducing electrical plasma along the cutting edge(s) of a thin insulated electrode.
Histologically friendly desorption electrospray ionisation
Numerous diseases are diagnosed on the basis of histology i.e. microscopic analysis of tissue samples which have been resected from patients. The gold standard for histopathological analysis is based upon formalin-fixed, paraffin-embedded ("FFPE") patient samples. Formalin fixation preserves tissues by forming methylene bridge cross- linkages between proteins. The sample is then dehydrated with ethanol and is immersed in an organic solvent (e.g., xylene) in order to remove the alcohol. The tissue sample is then infiltrated with molten paraffin.
According to a protocol, a tissue sample may by fixed for 12-24 hours in 10% neutral buffered formalin and then embedded in paraffin using standardized and automated procedures. Samples may be dehydrated in graded ethanol, cleared with xylene and then embedded in molten paraffin.
By way of contrast, fresh frozen samples may be stored post-surgery in liquid nitrogen. Prior to analysis, fresh frozen samples may be dehydrated in a vacuum for 1 hour at room temperature.
Whereas fresh frozen samples can only be preserved for a short period of time, formalin fixed paraffin embedded samples can be archived for many years allowing retrospective reassessment and analysis.
According to various embodiments a method is disclosed which involves applying a reversal agent to a chemically modified target (e.g., a FFPE tissue sample) in order to produce a modified target (e.g., a tissue sample which is no longer formalin fixed and/or embedded with paraffin). The modified target (e.g., unfixed tissue) may then be subjected to ion imaging using an ambient ionisation ion source such as a Desorption Electrospray Ionisation ("DESI") ion source.
A number of experiments were performed using a cryoprotectant glycerol at two different concentrations (0.1 % and 5%) and two embedding media were tested namely gelatin (10%) and optimum cutting temperature ("OCT") (RTM) medium using pork liver samples.
Fresh pork liver was embedded in OCT, without any previous sample treatment, and was sectioned and imaged in negative ion mode.
Figs. 1A-C show a comparison of desorption electrospray ionisation mass spectrometry imaging (MSI) in negative ion mode for morphological structures for different signal intensities from fresh pork liver embedded in OCT.
Fig. 1A shows a desorption electrospray ionisation image for counts in the range 0- 250 at m/z 726.5, Fig. 1 B shows a desorption electrospray ionisation image for counts in the range 0-12000 at m/z 766.5 and Fig. 1 C shows a desorption electrospray ionisation image for counts in the range 0-1400 at m/z 740.5.
Clear morphological structures were observed including for ions with signal intensities as low as 250 counts as shown in Fig. 1A.
After this, the fresh pork liver sample was fixed either in formalin, or in formalin with
0.1 % glycerol, or formalin with 5% glycerol for 72 hours at room temperature.
The fixed tissue sample were then embedded in 10% gelatin and in OCT, and were then sectioned onto glass slides.
Fig. 2A shows a desorption electrospray ionisation mass spectrometry imaging (MSI) in negative ion mode from formalin fixed pork liver embedded in OCT and Fig. 2B shows the signal intensity in negative ion mode from formalin fixed pork liver embedded in OCT.
It will be apparent from Fig. 2A that when the tissue slide was analysed by desorption electrospray ionisation mass spectrometry imaging (MSI) in negative ion mode, morphological structures were barely discernible, despite a relatively high signal intensity.
Further experiments were conducted with formalin fixed pork tissue using glycerol and OCT as embedding material.
Fresh frozen pork liver and formalin fixed pork liver were washed in LC-MS grade water. In the case of formalin fixed pork liver the step of washing the tissue sample in water helped to remove any excess of formalin present in the tissue.
The tissue sample were then analysed by desorption electrospray ionisation mass spectrometry imaging (MSI).
Fig. 3A shows a comparison of spectra intensity in negative ion mode from frozen fresh pork liver and Fig. 3B shows from formalin fixed pork liver washed in LC-MS grade water.
Fig. 4A shows a comparison of desorption electrospray ionisation mass
spectrometry imaging (MSI) in negative ion mode from frozen fresh pork liver and Fig. 4B shows from formalin fixed pork liver washed in LC-MS grade water.
It is apparent from Figs. 4A and 4B that the signal intensity was higher (1.19x104) for the fresh frozen pork liver than for the formalin fixed pork liver (7.23x103). Furthermore, different phospholipid species were observed in each sample treatment.
Despite the relatively high signal intensity for the formalin fixed pork liver, no morphological features could be discerned (Fig. 3B).
Experiments were performed using heat treatment and formalin scavengers (e.g. urea) in order to reverse formalin action. Formalin fixed pork liver was heat-treated at 100 °C for 4 minutes in water or in 10M urea solution and then sectioned onto glass slides for desorption electrospray ionisation mass spectrometry imaging (MSI) analysis in negative ion mode.
Fig. 5A shows the spectrum intensity in negative ion mode of fresh frozen pork liver, Fig. 5B shows the spectrum intensity of formalin fixed pork liver and Fig. 5C shows the spectrum intensity of heat-treated formalin fixed pork liver. Fig. 6A shows a comparison of desorption electrospray ionisation mass
spectrometry imaging (MSI) in negative ion mode from frozen fresh pork liver, Fig. 6B shows a mass spectrum from formalin fixed pork liver and Fig. 6C shows a mass spectrum for heat-treated formalin fixed pork liver.
It may be observed from Figs. 6C and 6A that heat-treated formalin fixed pork liver sample in water has a higher signal for fatty acids (1.24x105) by two orders of magnitude when compared to fresh frozen pork liver. This effect was also observed when comparing formalin fixed pork liver and heat-treated formalin fixed pork liver in water, where again an increase by an order of magnitude was detected, while morphological features were beginning to be discernible.
Further experiments were performed regarding removal of formalin from formalin- fixed tissues. Sodium metabisulfate (Na2S205) was tested as a formalin removal reagent in formalin pork liver and two concentrations were tested (15 and 40 % (w/v)). Formalin fixed pork liver was heat-treated with sodium metabisulfate aqueous solution at either 15 or 40 % at 95°C. After this, the tissue block was embedded in OCT, sectioned and analysed by desorption electrospray ionisation mass spectrometry imaging (MSI) in negative ion mode. It was observed that the signal intensity for the phospholipid mass range for heat-treated samples with sodium metabisulfide was high while the fatty acid:phospholipid ratio was low, as observed in other heat treatment experiments. No morphological features were detected.
Fig. 7A shows spectra intensity in negative ion mode from formalin fixed pork liver heat-treated with 15% (w/v) sodium metabisulfate. Fig. 7B shows a corresponding ion image for m/z 794.5. This phenomenon is surprising as before analyses, the large morphological features are visible to the naked eye. However, in the desorption electrospray ionisation-MS image, no morphological features are detected.
The tissue sections were examined under the microscope. It was observed that prior to analysis morphological features could be detected in the tissue section, but after analysis these seem to have been destroyed.
Fig. 8A shows a microscope image of formalin fixed pork liver heat-treated with sodium metabisulfate prior to desorption electrospray ionisation mass spectrometry imaging (MSI) analysis and Fig. 8B shows a corresponding microscope image after desorption electrospray ionisation mass spectrometry imaging (MSI) analysis.
In order to overcome this, thicker tissue sections (20 μηι) were analysed by desorption electrospray ionisation mass spectrometry imaging (MSI). However, the same effect was observed. The effect of the gas flow (7 bar gas pressure) was also examined by analyzing a sample with no solvent flow, and it was noted that morphological features were still visible after the analysis time.
One hypothesis for the tissue not adhering as firmly to the glass slide as frozen tissue is that it may be due to the fact that formalin cross-linking of proteins was not reversed to a sufficient extent to promote enough ionic interactions between the proteins and the glass slide. In order to improve these ionic interactions, the time for sample heat- treatment was increased (heat-treatment cycles), urea was used as a formalin scavenger and microscope adhesion slides were also tested.
The table below shows various formalin scavengers which were used and heat- treatment times used from formalin fixed pork liver tissue in order to reverse of formalin protein cross-linking bonds:
Figure imgf000027_0001
Figs. 9A-C shows a comparison of spectral intensity in negative ion mode of formalin fixed pork liver using different sodium metabisulfate concentrations (15% and 40% (w/v)) and different heat-treatment cycles as formalin scavengers. Fig. 9A shows the spectral intensity in negative ion mode of formalin fixed pork liver using 15% (w/v) sodium metabisulfate and one heat-treatment cycle, Fig. 9B shows the spectral intensity in negative ion mode of formalin fixed pork liver using 15% (w/v) sodium metabisulfate and three heat-treatment cycles and Fig. 9C shows the spectral intensity in negative ion mode of formalin fixed pork liver using 40% (w/v) sodium metabisulfate and one heat-treatment cycle.
Fig. 10A shows an ion image of formalin fixed pork liver, heat-treated for three cycles with 15% (w/v) sodium metabisulfate after desorption electrospray ionisation mass spectrometry imaging (MSI) analysis in negative ion mode with ion image m/z 794.53 being shown and Fig. 10B shows a microscope image of tissue after desorption electrospray ionisation mass spectrometry imaging (MSI) (square).
It was observed that when the number of heat-treatment cycles was increased for the treatment of formalin fixed pork liver, the spectra showed a difference in the ratio of fatty acids:phospholipids (Figs. 9A-9C). However, no morphological features were detected and the tissue section post desorption electrospray ionisation mass spectrometry imaging (MSI) analysis was destroyed even when using adhesion slides SuperFrost Plus (Figs. 10A-B).
As no morphological features were detected, urea was tested as a formalin scavenger. Different concentrations were tested (1M, 5M and 10M). Different heat- treatment cycles and different microscope adhesion slides were also tested. It was observed that urea has greater effective formalin reversal properties than sodium metabisulfate as the fatty acid:phospholipid ratio was improved with all urea concentrations tested. Fig. 11 shows a comparison of spectra intensity in negative ion mode of formalin fixed pork liver using different concentrations of urea and different numbers of heat- treatment cycles as formalin scavengers.
Three, four and five cycles of heat treatment were found to be particularly effective in terms of reversing the cross-link bonds of formalin fixed tissue proteins.
The comparison between common histology slides (SuperFrost) and adhesion slides (SuperFrostPlus, Poly-Lysine, SuperFrostPlus Ultra and SuperFrost Gold) showed no improvement in the adhesion of the formalin fixed pork liver tissue.
Fig. 12A shows a microscope image of formalin fixed pork liver post-desorption electrospray ionisation mass spectrometry imaging (MSI) analysis after being subjected to 5M urea and 3 heat-treatment cycles using a SuperFrost slide.
Fig. 12B shows a microscope image of formalin fixed pork liver post-desorption electrospray ionisation mass spectrometry imaging (MSI) analysis after being subjected to 5M urea and 3 heat-treatment cycles using a SuperFrostPlus slide.
Fig. 12C shows a microscope image of formalin fixed pork liver post-desorption electrospray ionisation mass spectrometry imaging (MSI) analysis after being subjected to 5M urea and 5 heat-treatment cycles using a Poly-Lysine slide.
Fig. 13A shows an ion image from formalin fixed pork liver heat-treated for 3 cycles with 5M Urea after desorption electrospray ionisation mass spectrometry imaging (MSI) analysis in negative ion mode with ion image m/z 794.53 using a SuperFrost slide and Fig. 13B shows an ion image m/z 794.53 using a SuperFrostPlus slide.
Morphological features were most discernible when using urea at 5M concentration together with three heat-treatment cycles (Fig. 13A and 13B). Data Collection
Data acquisition of biological samples was performed using human lung samples. These samples were part of a comparision study of lungs before and after they were subjected to ex vivo lung perfusion (EVLP) in order to access their quality for
transplantation.
Desorption electrospray ionisation mass spectrometry imaging (MSI) was carried out in these samples using a modified sprayer and a heated inlet capilary at 490 °C in negative ion mode.
Fig. 14 shows a desorption electrospray ionisation mass spectrometry imaging (MSI) of human lung biopsy after EVLP treatment. The RGB image is of PCs 1 , 2 and 5 for a human lung tissue sample (EXT8-T2-S1).
Formaldehyde scavengers Any formaldehyde scavenger known in the art may potentially be used in the reversal agent in accordance with various embodiments. The action of the reversal agent may be increased by raising the temperature and/or lowering the pH. Formaldehyde scavengers react with formaldehyde, thus reducing the
concentration of free formaldehyde present.
The formaldehyde scavenger may be selected from the group consisting of: (i) at least one dithiooxamide; (ii) at least one carbodiimide; (iii) at least one polyhydroxy compound; (iv) at least one nitrogen-containing compound such as an amide, amine or hydrazide compound; (v) at least one dicarbonyl compound; (vi) at least one water-soluble active methylene compound; (vii) at least one sulfur-containing compound and (viii) at least one metabisulfite salt.
Suitable dithiooxamide scavengers include those of formula:
Figure imgf000029_0001
wherein R denotes a hydroxyalkyl group having from 1-4 carbon atoms and from 1-4 hydroxy groups, a heterocyclic group or a toluidinomethylene group. Specific such compounds include N,N'-di-(2-hydroxyethyl) dithiooxamide, N,N'-di-(3-hydroxypropyl) dithiooxamide, Ν,Ν'-diglucityl dithiooxamide, Ν,Ν'-difurfuryl dithiooxamide, N,N'-di-(2- pyridylmethyl) dithiooxamide, N,N'-di-(3-pyridylmethyl) dithiooxamide, N,N'-di-(m- toluidinoethyl) dithiooxamide and N,N'-di-[2-(N"-ethyl-m-toluidino)-ethyl dithiooxamide.
Suitable metabisulfite salts include sodium metabisulfite (Na2S205), potassium metabisulfite and ammonium metabisulfite.
Suitable carbodiimide scavengers include monomeric, oligomeric and/or polymeric carbodiimides. Carbodiimides according to various embodiments include compounds according to formula (I) R2-(N=C=N-R1)n-R3 (I) wherein R1 represents an aromatic, aliphatic, cycloaliphatic or aralkylene radical which, in the case of an aromatic or an aralkylene residue, may carry no substituents or which may also carry in at least one ortho-position, or in both ortho-positions to the aromatic carbon atom which carries the carbodiimide group, aliphatic and/or cycloaliphatic substituents with at least 2 C-atoms, which may be branched or cyclic aliphatic radicals with at least 3 C-atoms, which can also contain heteroatoms, such as N. S and/or O, i.e. imidazolyl;
wherein R2 represents aryl, aralkyl, R-NCO, R-NHCONHR4, R-NHCONR4R5 R-HCOOR6, -NHCOS-R4, -COOR4, -OR4, -N(R4)2, -SR4, -OH, -NH2, NHR4, or epoxy; and wherein R3
represents -N=C=N-aryl, -N=C=N-alkyl, -N=C=N-cycloalkyl, -N=C=N-aralkyl, -NCO, -NHC ONHR4, -NHCONR4R5, -NHCOOR6, -NHCOS-R4, -COOR4, -OR4, -N(R4)2, -SR4, -OH, -NH2, NHR4 wherein, in R2 and in R3 independently of one another, R4 and R5 are the same or different and represent an alkyl, cycloalkyl or aralkyl radical, and R has one of the meanings of R4 or represents a polyester or a polyamide radical, and n represents an integer from 1 to 5,000, such as from 2 to 500.
Suitable monomeric carbodiimide scavengers include dicyclohexylcarbodiimide, diisopropylcarbodiimide, dimethylcarbodiimide, diisobutylcarbodiimide, dioctylcarbodiimide, tert-butyl-isopropylcarbodiimide, 2,6-diisopropylphenylencarbodiimide,
diphenylcarbodiimide, di-tert-butylcarbodiimide and di-β- naphthylcarbodiimide.
Suitable polymeric carbodiimide scavengers include
4,4'-dicyclohexylmethanecarbodiimide (degree of polymerization = 2 to 20),
tetramethylxylylenecarbodiimide (degree of polymerization = 2 to 20),
Ν,Ν-dimethylphenylcarbodiimide (degree of polymerization =- 2 to 20) and N,N'-di-2,6- diisopropylphenylencarbodiimide (degree of polymerization = 2 to 20).
Suitable β-dicarbonyl scavengers include compounds of formula 1a and 1 b:
Figure imgf000030_0001
wherein X! and X4 are C, N, S, or P;
X, X2, X3, X5 and X6 are selected from the group consisting of H;
- a C1-22 straight chain, branched or cyclic hydrocarbon or an aromatic moiety
selected from phenyl, phenylene, naphthalene or other polyaromatic hydrocarbons, substituted by a polar group or 1-3 halogens.
halogen;
a polar group substituted by H or a C1-22 straight chain, branched or cyclic hydrocarbon or an aromatic moiety selected from phenyl, phenylene, naphthalene or other polyaromatic hydrocarbon; and
a polar group;
or the X, X3 and X6 groups may be chemically linked to form cyclic or heterocyclic structures.
Unless otherwise defined, as used herein halogen denotes F, CI, Br or I. Unless otherwise defined, as used herein "polar group |denotes O, OH, COOH, carbonyl, amide, amine, thiol, quaternary nitrogen, ethoxy or propoxy group, or combinations thereof.
β-dicarbonyl scavengers according to various embodiments include
acetoacetamide, ethyl acetoacetate, Ν,Ν-dimethyleneacetamide, acetoacetone, dimethyl- 1 ,3-acetonedicarboxylate, 1 ,3-acetonedicarboxylic acid, malonic acid, resorcinol, 1 ,3- cyclohexadione, barbituric acid, 5,5-dimethyl-1 ,3-cyclohexanedione (dimedone), 2,2- dimethyl-1 ,3-dioxane-4,6-dione (Meldrum's acid), salicylic acid, methyl acetoacetate, ethyl- 2-methyl acetoacetate, 3-methyl-acetoacetone, dimethyl malonate, diethyl malonate, 1 ,3- dimethyl barbituric acid, resorcinol, phloroglucinol, orcinol, 2,4-dihydroxybenzoic acid, 3,5- dihydroxybenzoic acid and malonamide.
Nitrogen-containing formaldehyde scavengers include amides, amines and hydrazides. Hydrazides such as 2,4-dinitrophenzylhydrazine and adipic acid dihydrazide can react with formaldehyde to give hydrazones.
Suitable mono or di-amide scavengers include those of formula:
Figure imgf000031_0001
wherein X17 and X18 are independently selected from the group consisting of H; - a Ci-22 straight chain, branched or cyclic hydrocarbon or aromatic moiety selected from phenyl, phenylene, naphthalene or other polyaromatic hydrocarbons, substituted by a polar group or 1-3 halogens;
- halogen;
- a polar group substituted by H or a Ci-22 hydrocarbon (straight chain, branched or cyclic) or an aromatic moiety (phenyl, phenylene, naphthalene or other polyaromatic hydrocarbon); and
- a polar group.
Examples of mono- and di-amide scavengers according to various embodiments are urea, ethylene urea, propylene urea, ε-caprolactam, glycouril, hydantoin, 2- oxazolidinone, 2-pyrrolidinone, uracil, barbituric acid, thymine, uric acid, allantoin, polyamides, 4,5-dihydroxyethylene urea (DHEU), monomethylol-4-hydroxy-4-methoxy-5,5- dimethyl-propylurea, nylon 2-hydroxyethyl ethylene urea, 2-hydroxyethyl urea, L-citrulline, biotin, N-methyl urea, N-ethyl urea, N-butyl urea, N-phenyl urea, 4,5-dimethoxy ethylene urea, succinimide, 2-imidazolidinone and 1 ,3-dimethyl-2-imidazolidinone.
Suitable amine scavengers include amines which form imines by reaction with formaldehyde as represented by the following formula:
X27-NH2 wherein X27 is selected from the group consisting of H;
- a Ci-22 straight chain, branched or cyclic hydrocarbon or aromatic moiety selected from phenyl, phenylene, naphthalene or other polyaromatic hydrocarbon substituted by a polar group or 1 to 3 halogens; - halogen;
- a polar group substituted by H or a C1-22 straight chain, branched or cyclic hydrocarbon or an aromatic moiety selected from phenyl, phenylene, naphthalene and other polyaromatic hydrocarbon; and
- a polar group.
Other amine scavengers include compounds of formula:
Figure imgf000032_0001
or salt thereof, wherein:
RA is a bond or is C(R7R8);
Ri is H, OH or Ci-Ce alkyl;
R2 is H, OH or Ci-Ce alkyl;
R3, R4, R5, and R6 are independently H, or C C6 alkyl;
R7 and R8 are independently H, OH or C C6 alkyl; and
wherein alkyl in R Rs is optionally independently substituted with OH, NR9R19, C C6 alkyl, or phenyl, wherein R9 and R19 are independently H or C C6 alkyl,
and provided that if neither R7 nor R8 is OH, then at least one of and R2 is OH.
Suitable salts include hydrochloride, acetate, formate, oxalate, citrate, carbonate, sulfate, and phosphate.
Amine scavengers according to various embodiments include 2-amino-1-butanol, 2- amino-2-ethyl-1 ,3-propanediol, 2-amino-2-methyl-1-propanol, 2-amino-1-methyl-1 ,3- propanediol, tris(hydroxymethyl)aminomethane, N-isopropylhydroxylamine, ethanolamine, diethanolamine, N-methylethanolamine, N-butylethanolamine, monoisopropanolamine, diisopropanolamine, mono-sec-butanolamine, di-sec-butanolamine, and salts thereof; polyvinyl amine), arginine, lysine, asparagine, proline, tryptophan; proteins such as casein, gelatin, collagen, whey protein, soy protein, and albumin; melamine, benzoguanamine, 4- aminobenzoic acid (PABA), 3-aminobenzoic acid, 2-aminobenzoic acid (anthranilic acid), 2-aminophenol, 3-aminophenol, 4-aminophenol, creatine, 4-aminosalicylic acid, 5- aminosalicylic acid, methyl anthranilate, methoxylamine HCI, anthranilamide, 4- aminobenzamide, p-toluidine, p-anisidine, sulfanilic acid, sulfanilamide, methyl-4- aminobenzoate, ethyl-4-aminobenzoate (benzocain), beta-diethylaminoethyl-4- aminobenzoate (procain), 4-aminobenzamide, 3,5-diaminobenzoic acid; 2,4- diaminophenol; 1 ,2-phenylenediamine, 1 ,3-phenylenediamine, 1 ,4-phenylenediamine, aniline, hexamethylenediamine, bis-hexamethylenetriamine, triethylaminetriamine, poly(propyleneoxide)triamine, and poly(propyleneglycol)diamines.
Another class of formaldehyde scavengers are polyhydroxy compounds such as those represented by the following structure:
Figure imgf000033_0001
wherein X33 and X34 are independently selected from the group consisting of H;
- C1-22 straight chain, branched or cyclic hydrocarbon or aromatic moiety selected from phenyl, phenylene, naphthalene or other polyaromatic hydrocarbons, substituted by a polar group or 1-3 halogens;
- halogen;
- a polar group substituted by H or a C1-22 straight chain, branched or cyclic hydrocarbon or aromatic moiety selected from phenyl, phenylene, naphthalene or other polyaromatic hydrocarbons; and
- a polar group.
Polyhydroxy compounds according to various embodiments include
polyvinylalcohol, pyrogallol, 1 ,2-hexanediol, diethylene glycol, ascorbic acid, saccharides such as D-sorbitol, dextrose and sucrose, tannins/tannic acid, and polysaccharides such as starches, guar, xanthan, pectin, chemically-modified cellulose, chitosan, and mixtures thereof.
Sulfur containing compounds according to various embodiments include 1 ,3,5- triazine-2,4,6-trithiol, glutathione and cysteine.
Water-soluble active methylene scavengers may include compounds of formula:
Figure imgf000033_0002
wherein R and independently denote -CH3 or H(0(CH2)m)nO-;
n denotes 1-19;
m denotes 1-3; and
Y denotes -CH2- or -CH2-C(0)-CH2-.
Active methylene scavengers may include bis(2-hydroxyethoxyethyl) malonate and (2-hydroxyethoxyethyl) acetoacetate, dimethyl-1 ,3-acetonedicarboxylate, diethyl malonate and ethylacetoacetate. Analysing sample spectra
A list of analysis techniques which are intended to fall within the scope of the present invention are given in the following table:
Analysis Techniques
Univariate Analysis Multivariate Analysis
Principal Component Analysis (PCA)
Linear Discriminant Analysis (LDA)
Maximum Margin Criteria (MMC)
Library Based Analysis
Soft Independent Modelling Of Class Analogy (SIMCA)
Factor Analysis (FA)
Recursive Partitioning (Decision Trees)
Random Forests
Independent Component Analysis (ICA)
Partial Least Squares Discriminant Analysis (PLS-DA)
Orthogonal (Partial Least Squares) Projections To Latent Structures (OPLS)
OPLS Discriminant Analysis (OPLS-DA)
Support Vector Machines (SVM)
(Artificial) Neural Networks
Multilayer Perceptron
Radial Basis Function (RBF) Networks
Bayesian Analysis
Cluster Analysis
Kernelized Methods
Subspace Discriminant Analysis
K-Nearest Neighbours (KNN)
Quadratic Discriminant Analysis (QDA)
Probabilistic Principal Component Analysis (PPCA)
Non negative matrix factorisation
K-means factorisation
Fuzzy c-means factorisation
Discriminant Analysis (DA)
Combinations of the foregoing analysis approaches can also be used, such as PCA-LDA, PCA-MMC, PLS-LDA, etc.
Analysing the sample spectra can comprise unsupervised analysis for
dimensionality reduction followed by supervised analysis for classification.
By way of example, a number of different analysis techniques will now be described in more detail.
Multivariate analysis - developing a model for classification
By way of example, a method of building a classification model using multivariate analysis of plural reference sample spectra will now be described.
Fig. 15 shows a method 1500 of building a classification model using multivariate analysis. In this example, the method comprises a step 1502 of obtaining plural sets of intensity values for reference sample spectra. The method then comprises a step 1504 of unsupervised principal component analysis (PCA) followed by a step 1506 of supervised linear discriminant analysis (LDA). This approach may be referred to herein as PCA-LDA. Other multivariate analysis approaches may be used, such as PCA-MMC. The PCA-LDA model is then output, for example to storage, in step 1508.
The multivariate analysis such as this can provide a classification model that allows an aerosol, smoke or vapour sample to be classified using one or more sample spectra obtained from the aerosol, smoke or vapour sample. The multivariate analysis will now be described in more detail with reference to a simple example.
Fig. 16 shows a set of reference sample spectra obtained from two classes of known reference samples. The classes may be any one or more of the classes of target described herein. However, for simplicity, in this example the two classes will be referred as a left-hand class and a right-hand class.
Each of the reference sample spectra has been pre-processed in order to derive a set of three reference peak-intensity values for respective mass to charge ratios in that reference sample spectrum. Although only three reference peak-intensity values are shown, it will be appreciated that many more reference peak-intensity values (e.g., ~ 100 reference peak-intensity values) may be derived for a corresponding number of mass to charge ratios in each of the reference sample spectra. In other embodiments, the reference peak-intensity values may correspond to: masses; mass to charge ratios; ion mobilities (drift times); and/or operational parameters.
Fig. 17 shows a multivariate space having three dimensions defined by intensity axes. Each of the dimensions or intensity axes corresponds to the peak-intensity at a particular mass to charge ratio. Again, it will be appreciated that there may be many more dimensions or intensity axes (e.g., - 100 dimensions or intensity axes) in the multivariate space. The multivariate space comprises plural reference points, with each reference point corresponding to a reference sample spectrum, i.e., the peak-intensity values of each reference sample spectrum provide the co-ordinates for the reference points in the multivariate space.
The set of reference sample spectra may be represented by a reference matrix D having rows associated with respective reference sample spectra, columns associated with respective mass to charge ratios, and the elements of the matrix being the peak-intensity values for the respective mass to charge ratios of the respective reference sample spectra.
In many cases, the large number of dimensions in the multivariate space and matrix
D can make it difficult to group the reference sample spectra into classes. PCA may accordingly be carried out on the matrix D in order to calculate a PCA model that defines a PCA space having a reduced number of one or more dimensions defined by principal component axes. The principal components may be selected to be those that comprise or "explain" the largest variance in the matrix D and that cumulatively explain a threshold amount of the variance in the matrix D. Fig. 18 shows how the cumulative variance may increase as a function of the number n of principal components in the PCA model. The threshold amount of the variance may be selected as desired.
The PCA model may be calculated from the matrix D using a non-linear iterative partial least squares (NIPALS) algorithm or singular value decomposition, the details of which are known to the skilled person and so will not be described herein in detail. Other methods of calculating the PCA model may be used.
The resultant PCA model may be defined by a PCA scores matrix S and a PCA loadings matrix L. The PCA may also produce an error matrix E, which contains the variance not explained by the PCA model. The relationship between D, S, L and E may be:
D = SLT + E (1)
Fig. 19 shows the resultant PCA space for the reference sample spectra of Figs. 16 and 17. In this example, the PCA model has two principal components PC0 and PCi and the PCA space therefore has two dimensions defined by two principal component axes. However, a lesser or greater number of principal components may be included in the PCA model as desired. It is generally desired that the number of principal components is at least one less than the number of dimensions in the multivariate space.
The PCA space comprises plural transformed reference points or PCA scores, with each transformed reference point or PCA score corresponding to a reference sample spectrum of Fig. 16 and therefore to a reference point of Fig. 17. As is shown in Fig. 19, the reduced dimensionality of the PCA space makes it easier to group the reference sample spectra into the two classes. Any outliers may also be identified and removed from the classification model at this stage.
Further supervised multivariate analysis, such as multi-class LDA or maximum margin criteria (MMC), in the PCA space may then be performed so as to define classes and, optionally, further reduce the dimensionality.
As will be appreciated by the skilled person, multi-class LDA seeks to maximise the ratio of the variance between classes to the variance within classes (i.e., so as to give the largest possible distance between the most compact classes possible). The details of LDA are known to the skilled person and so will not be described herein in detail. The resultant PCA-LDA model may be defined by a transformation matrix U, which may be derived from the PCA scores matrix S and class assignments for each of the transformed spectra contained therein by solving a generalised eigenvalue problem.
The transformation of the scores S from the original PCA space into the new LDA space may then be given by:
Z =SU
(2) where the matrix Z contains the scores transformed into the LDA space. Fig. 20 shows a PCA-LDA space having a single dimension or axis, wherein the LDA is performed in the PCA space of Fig. 19. As is shown in Fig. 20, the LDA space comprises plural further transformed reference points or PCA-LDA scores, with each further transformed reference point corresponding to a transformed reference point or PCA score of Fig. 19.
In this example, the further reduced dimensionality of the PCA-LDA space makes it even easier to group the reference sample spectra into the two classes. Each class in the PCA-LDA model may be defined by its transformed class average and covariance matrix or one or more hyperplanes (including points, lines, planes or higher order hyperplanes) or hypersurfaces or Voronoi cells in the PCA-LDA space.
The PCA loadings matrix L, the LDA matrix U and transformed class averages and covariance matrices or hyperplanes or hypersurfaces or Voronoi cells may be output to a database for later use in classifying an aerosol, smoke or vapour sample.
The transformed covariance matrix in the LDA space V'g for class g may be given by
Figure imgf000037_0001
(3) where Vg are the class covariance matrices in the PCA space. The transformed class average position zg for class g may be given by sgU = Zg where sg is the class average position in the PCA space.
Multivariate analysis - using a model for classification
By way of example, a method of using a classification model to classify an aerosol, smoke or vapour sample will now be described.
Fig. 21 shows a method 2100 of using a classification model. In this example, the method comprises a step 2102 of obtaining a set of intensity values for a sample spectrum. The method then comprises a step 2104 of projecting the set of intensity values for the sample spectrum into PCA-LDA model space. Other classification model spaces may be used, such as PCA-MMC. The sample spectrum is then classified at step 2106 based on the project position and the classification is then output in step 2108.
Classification of an aerosol, smoke or vapour sample will now be described in more detail with reference to the simple PCA-LDA model described above.
Fig. 22 shows a sample spectrum obtained from an unknown aerosol, smoke or vapour sample. The sample spectrum has been pre-processed in order to derive a set of three sample peak-intensity values for respective mass to charge ratios. As mentioned above, although only three sample peak-intensity values are shown, it will be appreciated that many more sample peak-intensity values (e.g., - 100 sample peak-intensity values) may be derived at many more corresponding mass to charge ratios for the sample spectrum. Also, as mentioned above, in other embodiments, the sample peak-intensity values may correspond to: masses; mass to charge ratios; ion mobilities (drift times);
and/or operational parameters.
The sample spectrum may be represented by a sample vector dx, with the elements of the vector being the peak-intensity values for the respective mass to charge ratios. A transformed PCA scores vector sx for the sample spectrum can be obtained as follows: dxL = Sx (5)
Then, a transformed PCA-LDA vector zx for the sample spectrum can be obtained as follows:
SxU = Ζχ (6)
Fig. 23 again shows the PCA-LDA space of Fig. 20. However, the PCA-LDA space of Fig. 23 further comprises the projected sample point, corresponding to the transformed PCA-LDA vector zx, derived from the peak intensity values of the sample spectrum of Fig. 22.
In this example, the projected sample point is to one side of a hyperplane between the classes that relates to the right-hand class, and so the aerosol, smoke or vapour sample may be classified as belonging to the right-hand class.
Alternatively, the Mahalanobis distance from the class centres in the LDA space may be used, where the Mahalanobis distance of the point zx from the centre of class g may be given by the square root of:
(Ζχ-Ζ( ,)T (Vg)- zx-zf
(8) and the data vector dx may be assigned to the class for which this distance is smallest.
In addition, treating each class as a multivariate Gaussian, a probability of membership of the data vector to each class may be calculated.
Library based analysis - developing a library for classification
By way of example, a method of building a classification library using plural input reference sample spectra will now be described.
Fig. 24 shows a method 2400 of building a classification library. In this example, the method comprises a step 2402 of obtaining plural input reference sample spectra and a step 2404 of deriving metadata from the plural input reference sample spectra for each class of sample. The method then comprises a step 2404 of storing the metadata for each class of sample as a separate library entry. The classification library is then output, for example to electronic storage, in step 2406.
A classification library such as this allows an aerosol, smoke or vapour sample to be classified using one or more sample spectra obtained from the aerosol, smoke or vapour sample. The library based analysis will now be described in more detail with reference to an example.
In this example, each entry in the classification library is created from plural pre- processed reference sample spectra that are representative of a class. In this example, the reference sample spectra for a class are pre-processed according to the following procedure:
First, a re-binning process is performed. In this embodiment, the data are resampled onto a logarithmic grid with abscissae:
Figure imgf000039_0001
where Nchan is a selected value and | j denotes the nearest integer below x. In one example, Nchan is 212 or 4096.
Then, a background subtraction process is performed. In this embodiment, a cubic spline with k knots is then constructed such that p% of the data between each pair of knots lies below the curve. This curve is then subtracted from the data. In one example, k is 32. In one example, p is 5.
A constant value corresponding to the q% quantile of the intensity subtracted data is then subtracted from each intensity. Positive and negative values are retained. In one example, q is 45.
Then, a normalisation process is performed. In this embodiment, the data are normalised to have mean yt. In one example, yt = 1.
An entry in the library then consists of metadata in the form of a median spectrum value μι and a deviation value Ό{ for each of the Nchan points in the spectrum.
The likelihood for the i'th channel is given by:
Figure imgf000039_0002
wherein 1/2≤ C <∞ and where (C) is the gamma function.
The above equation is a generalised Cauchy distribution which reduces to a standard Cauchy distribution for C = 1 and becomes a Gaussian (normal) distribution as C→∞. The parameter Ό{ controls the width of the distribution (in the Gaussian limit Ό{ = σ, is simply the standard deviation) while the global value C controls the size of the tails.
In one example, C is 3/2, which lies between Cauchy and Gaussian, so that the likelihood becomes: 3 1 1
Pr(y , Di) = - (9)
,ί (3/2 + (.γί-μί)2/0?)
For each library entry, the parameters are set to the median of the list of values in the i'th channel of the input reference sample spectra while the deviation Dt is taken to be the interquartile range of these values divided by V2. This choice can ensure that the likelihood for the i'th channel has the same interquartile range as the input data, with the use of quantiles providing some protection against outlying data. Library based analysis - using a library for classification
By way of example, a method of using a classification library to classify an aerosol, smoke or vapour sample will now be described.
Fig. 25 shows a method 2500 of using a classification library. In this example, the method comprises a step 2502 of obtaining a set of plural sample spectra. The method then comprises a step 2504 of calculating a probability or classification score for the set of plural sample spectra for each class of sample using metadata for the class entry in the classification library. The sample spectra are then classified at step 2506 and the classification is then output in step 2508.
Classification of an aerosol, smoke or vapour sample will now be described in more detail with reference to the classification library described above.
In this example, an unknown sample spectrum y is the median spectrum of a set of plural sample spectra. Taking the median spectrum y can protect against outlying data on a channel by channel basis.
The likelihood Ls for the input data given the library entry s is then given by:
Figure imgf000040_0001
wherein and Ό{ are, respectively, the library median values and deviation values for channel i. The likelihoods Ls may be calculated as log likelihoods for numerical safety.
The likelihoods Ls are then normalised over all candidate classes 's' to give probabilities, assuming a uniform prior probability over the classes. The resulting probability for the class s is given by:
Figure imgf000040_0002
The exponent (l/F) can soften the probabilities which may otherwise be too definitive. In one example, F = 100. These probabilities may be expressed as percentages, e.g., in a user interface. Alternatively, RMS classification scores Rs may be calculated using the same median sample values and derivation values from the library:
Figure imgf000041_0001
Again, the scores Rs are normalised over all candidate classes 's'.
The aerosol, smoke or vapour sample may then be classified as belonging to the class having the highest probability and/or highest RMS classification score. Prediction of tissue types in breast cancer biopsies
Manual histological evaluation of the stained biopsy tissue sections has been the gold standard method when it comes to providing a diagnosis for breast cancers.
However, the accuracy of this morphology-based tissue diagnosis is often compromised as it is dependent on the pathologists' interpretation resulting in poor prognosis for a given patient.
Desorption electrospray ionisation mass spectrometry imaging (MSI) enables to visualise spatial distribution of lipid species across tissue sections allowing direct correlation with the histological features. Therefore, breast cancer tissues were analysed with desorption electrospray ionisation mass spectrometry imaging (MSI) to obtain lipidomic data. 45 samples were analysed in positive and negative ion mode.
Fig. 26A shows RGB images of PCA components 1 , 2 and 3 in negative ion mode and Fig. 26B shows in positive ion mode with the respective histological images of a Grade II Invasive Ductal Carcinoma (IDC).
An ovarian cancer dataset with different epithelial carcinomas (endometrioid, serous and clear cell carcinomas), borderline tumours, and healthy ovary and fallopian tube has been analysed. A total of 109 samples were collected and acquired by desorption electrospray ionisation-MS in positive and negative ion mode.
Fig. 27A shows PCA analysis of Grade II IDC in negative ion mode.
Fig. 27B shows MMC analysis of Grade II IDC negative ion mode.
Fig. 28A shows PCA analysis of Grade II IDC in positive ion mode.
Fig. 28B shows MMC analysis of Grade II IDC positive ion mode.
Fig. 29A shows leave one out cross validation of different tissue types in a Grade II IDC in negative ion mode and Fig. 29B shows in positive ion mode.
Each individual breast sample was subjected to unsupervised principal component analysis (PCA) to visualize differences between different tissue types. In both positive and negative ion mode, a clear distinction could be observed between the stroma and the tumour tissue in almost all of the samples (Fig. 27A and Fig. 28A). Recursive maximum margin criterion (RMMC) analysis was used for supervised classification (Fig. 27B and Fig. 28B). Tissue types in each sample and their spatial distribution were determined by an independent histopathologist based on the H&E stained optical image. Based on this information, a small number of representative mass spectra per tissue were selected from the integrated MS ion image to build a sample-specific RMMC model which was used to classify all pixels in the different tissue types. This data was submitted to cross validation, which exceeded 95% accuracy generally for all tissue types in all samples in both negative and positive ion mode (Fig. 29A and Fig. 29B).
Development of spatially resolved shotgun lipidomic methods for histology-level cancer diagnostics using ovarian cancer dataset
The dataset was initially pre-processed and multivariate statistical analysis was performed on each individual sample's dataset in order to compile a database of histologically authentic lipidomic profiles. The morphological regions of interest were assigned by a qualified histopathologist and automatically co-registered and aligned with the MSI dataset.
Fig. 30 shows histological images with the annotated regions of interest by histopathologist. The PCA analysis of these assigned regions together with the MMC supervised analysis components from samples analysed in the negative ion mode.
Using principal component analysis (PCA) it was observed that different tissue types within the same sample show different lipid profiles. For example, normal ovary contains corpus and stroma tissue, and these are completely separated in PCA.
In the borderline and cancer samples it was possible to distinguish two different tissue types, the tumour cells and the surrounding stroma cells presenting large differences in their lipidomic profile.
When supervised maximum margin criteria (MMC) analysis was applied and a colour map according to the MMC components was applied it was possible to produce tissue maps that reflect the different tissue types identified in the histological image.
This profile database was also used to perform comparative analysis across multiple samples. PCA was used to perform unsupervised tissue segmentation based on the lipidomic profiles, without taking into account histological assignment. A supervised analysis was then performed and a respective leave-one-tissue-per-patient-out cross validation was calculated.
Fig. 31 A shows analysis of a combined dataset from multiple samples (negative ion mode using PCA of identified regions, Fig. 31 B shows corresponding MMC supervised analysis, Fig. 31 C shows MMC analysis excluding the samples with outliers identified in Fig. 31 B and Fig. 31 D shows respective leave-one-region-per-patient-out cross validation.
PCA shows some separation between normal ovary, serous carcinoma, and serous carcinoma associated stroma. The supervised MMC analysis shows good separation between all three tissue types with six outliers (circles in Fig. 31 B). All four misclassified normal samples were samples which were classified as normal ovary but were taken from an ovary with a tumour distant from the sampling area. This suggests that the biochemistry of this tissue is altered, even though this cannot be detected in a morphological
examination.
MMC analysis was repeated under exclusion of the outliers and leave-one-region- per-patient-out cross validation was performed, showing a complete separation of normal tissue and an overall accuracy of 85%.
The same analysis was performed for the positive ion mode data shown in Figs. 28A-28C.
Fig. 32A shows PCA of identified regions, Fig. 32B shows MMC supervised analysis and Fig. 32C shows leave-one-region-per-patient-out cross validation for the positive ion mode data.
In the positive ion mode, the cross validation shows lower scores then in negative ion mode, the different tissue types being classified with an average cross validation accuracy of around 80%.
The variances between different types of samples were also examined. For example, it was evaluated how well negative ion mode desorption electrospray ionisation mass spectrometry imaging (MSI) can separate cancer tissues, borderline and healthy ovary.
Fig. 33A shows supervised MMC analysis of healthy ovary, borderline tumours and carcinomas together with Fig. 33B showing leave one patient out cross validation.
More samples are being analysed to improve the model, but even with this small data set, an overall classification accuracy of 95.6% was achieved.
A further analysis performed was the comparison between different types of epithelial carcinomas in the dataset: endometrioid and serous carcinomas. Using the negative ion mode data, healthy ovary, serous carcinoma, and endometrioid carcinoma could be classified with an overall accuracy of 90% (see Fig. 34A and Fig. 34B).
Fig. 34A shows supervised MMC analysis of healthy ovary and different epithelial carcinomas (endometrioid and serous) and Fig. 34B leave one patient out cross validation.
An examination was also performed based on the models created, as to whether it was possible to predict the different tissue types of a blind sample. The number of serous carcinomas analysed provided a robust model to perform this validation using negative ion mode data.
Fig. 35A shows a sample with unknown histology used to predict the different tissue types. Cross validation of this prediction was based on the histological annotation.
The desorption electrospray ionisation data allowed an excellent prediction of the two tissue types present in the sample i.e. stroma and cancer. A cross validation as shown in Fig. 35B was performed based on histological annotation performed after this analysis and a classification accuracy of almost 100% was achieved.
Desorption Electroflow Focussing Ionisation - application to tissue imaging
Desorption electroflow focussing ionisation (DEFFI) is a recently developed ambient ionisation technique, in which an electroFlow Focusing (RTM) nebulizer is used to desorb ions from a sample surface. This nebulizer focusses the emitted electrospray through a small orifice in a grounded plate using a concentric gas flow. Unlike desorption
electrospray ionisation (DESI), which uses very high nebulising gas pressures (100 psi) and high electrospray voltages (4.5 to 5 kV), desorption electroflow focusing ionisation ("DEFFI") has so far been operated at relatively low gas pressures (10 psi) and lower voltages (500 V), as higher voltages were reported to cause droplet discharge at the orifice and corona discharge. The potential of desorption electroflow focusing ionisation ("DEFFI") for mass spectrometric imaging applied to imaging of biological tissues was examined and its performance compared to desorption electrospray ionisation.
Fig. 36 shows a schematic of Desorption Electroflow Focussing lonisation setup.
The desorption electroflow focusing ionisation ("DEFFI") was initially tested on a mass spectrometer, with the solvent being supplied by the instrument's syringe pump. Initial operating conditions were 10 psi nitrogen gas pressure and 5 μΙ/min solvent flow rate. This produced a spray with a relatively large diameter (approx. 1 mm) impact area on the sample surface, which was visualised by directing the spray at a glass slide coated with Rhodamine B.
In order to focus the spray and reduce the impact area, the gas pressure was gradually increased. 20 psi was found to reduce the impact area to about 600 μηι in diameter, while higher gas pressures caused the spray to become irregular or cut it off completely. Initially, a voltage of 500 V was used. However, higher voltages were also tested and found to improve signal intensity by more than an order of magnitude, although the signal became unstable above 3 kV (Fig. 37) due to the discharging effects. A voltage of 3 kV was therefore used for all imaging experiments.
Fig. 37 shows total ion count for desorption electroflow focusing ionisation
("DEFFI") on pork liver at different applied voltages in negative ion mode.
To compare desorption electroflow focusing ionisation ("DEFFI") and desorption electrospray ionisation for mass spectrometric imaging of tissue, one section from the same human breast cancer sample (containing cancerous tissue and surrounding stroma) was analysed with each method. Scan speeds and line spacing corresponding to pixel sizes of 100 and 150 μηι were used for desorption electrospray ionisation and desorption electroflow focusing ionisation ("DEFFI") respectively.
Figs. 38A-F show a comparison of desorption electroflow focusing ionisation ("DEFFI") and desorption electrospray ionisation for mass spectrometric imaging of human breast cancer tissue.
Figs. 38A and 38D show RGB images of the first three principal components from principal component analysis of all pixels using the full acquired m/z range from 150 to 1500.
Figs. 38B and Fig. 38E show base peak intensity (BPI) normalised example spectra for breast tissue for desorption electroflow focusing ionisation ("DEFFI") and desorption electrospray ionisation respectively. Figs. 38C and Fig. 38F show desorption traces in line-scanning mode on rhodamine B-coated glass slides for desorption electroflow focusing ionisation ("DEFFI") and desorption electrospray ionisation, respectively.
Although the width of the desorption trace for both methods, as observed on rhodamine B-coated glass slides (Figs. 38C and 38F) was comparable, image resolution was found to be considerably better for desorption electrospray ionisation, with
morphological features being more clearly defined (Figs. 38A and 38D).
A comparison of the traces on rhodamine B (Figs. 38C and 38F) suggested that under the operating conditions used, desorption electrospray ionisation produced a more focussed primary electrospray, while the desorption electroflow focusing ionisation
("DEFFI") spray was more diffuse causing a bigger overlap and blurring of pixels. Mass spectra for both methods were similar, although desorption electroflow focusing ionisation ("DEFFI") provided higher overall intensity, probably due to the higher flow rate used, and a more balanced ratio of higher to lower m/z peaks.
In an attempt to improve the spatial resolution of desorption electroflow focusing ionisation ("DEFFI"), the solvent was delivered by a nanoflow pump, allowing for a more precise control of solvent flow rate, even under high backpressure conditions. The solvent flow rate was gradually decreased from 4 to 1.5 μΙ/min, while gas pressure was gradually increased from 10 to 30 psi, and the electrospray was found to be stable throughout. The desorption trace on rhodamine was reduced to about 450 μηι in width.
Figs. 39A-E shows a comparison of desorption electroflow focusing ionisation ("DEFFI") and desorption electrospray ionisation for mass spectrometric imaging of pork liver tissue.
Figs. 39A and 39B show RGB images for pork liver of the first three principal components of principal component analysis across the full m/z range for desorption electroflow focusing ionisation ("DEFFI") and desorption electrospray ionisation, respectively.
Fig. 39C and 39D show representative spectra from pork liver in negative ion mode for desorption electroflow focusing ionisation ("DEFFI") and desorption electrospray ionisation, respectively.
Fig. 39E show desorption traces on rhodamine B for both methods for 3 different solvent flow rates.
Again, desorption electrospray ionisation and desorption electroflow focusing ionisation ("DEFFI") were compared for tissue imaging, in this case using pork liver sections and a pixel size of 100 μηι for both (Figs. 39A and 39B). Overall intensity for desorption electroflow focusing ionisation ("DEFFI") was slightly higher, but spectral composition was similar (Figs. 39C and 39D). A comparison of desorption traces on rhodamine showed a similar desorption area of approximately 450 μηι in width, although the desorption electroflow focusing ionisation ("DEFFI") trace still showed slightly diffuser edges. To further improve spatial resolution, both methods were tested on rhodamine using flow rates of 1 and 0.5 μΙ/min. While the desorption trace for desorption electrospray ionisation decreased in size proportionally to the flow rate, the desorption electroflow focusing ionisation ("DEFFI") desorption trace decreased only very little. It did, however, show a sharpening of its edges (Fig. 39E).
In desorption electrospray ionisation, the spectral intensity tends to decrease approximately proportionally with a decrease in the solvent flow rate. This was not found to be the case for desorption electroflow focusing ionisation ("DEFFI"), where the signal intensity on pork liver was comparable for flow rates of 1.5 and 1 μΙ/min, but lower for 0.5 μΙ/min (Fig. 40). This corresponds with the observation that desorption traces on rhodamine did not decrease proportionally to the flow rate.
Fig. 40 shows normalised base peak intensity for pork liver in negative ion mode for desorption electroflow focusing ionisation ("DEFFI") and desorption electrospray ionisation at three different solvent flow rates.
In conclusion, desorption electroflow focusing ionisation ("DEFFI") was shown to be highly suitable for imaging of biological tissues. Its performance in terms of spectral intensity was comparable to, or slightly better than, that of desorption electrospray ionisation. Its spatial resolution was slightly inferior to that of desorption electrospray ionisation. This may, however, be improved by altering the dimensions of the electroFlow Focusing (RTM) nebulizer.
One potential advantage of desorption electroflow focusing ionisation ("DEFFI") over desorption electrospray ionisation is that the electrospray produced is perfectly concentric, as it is focussed by the nebulising gas, whilst a desorption electrospray ionisation spray tends to be asymmetrical, depending on the position of the solvent capillary within the gas capillary. This leads to a larger number of degrees of freedom and, potentially, higher variability. Desorption electroflow focusing ionisation ("DEFFI") is potentially able to overcome this issue.
Improved sprayer geometry
In desorption electrospray ionisation, the sprayer is potentially one of the largest sources of reproducibility problems. In conventional desorption electrospray ionisation sprayers, the solvent capillary is not fixed relative to the gas capillary. This can result in reproducibility errors, e.g. due to the variable solvent capillary geometry.
According to various embodiments, an improved desorption electrospray ionisation geometry for imaging is provided.
According to various embodiments, a desorption electrospray ionisation sprayer is provided wherein the solvent capillary is fixed relative to the gas capillary.
According to various embodiments apparatus is provided comprising an ion source comprising a nozzle having an aperture, a solvent emitter which extends through the aperture, and a fixing device which is arranged and adapted to fix the solvent emitter relative to the aperture.
The fixing device may comprise a centering device which is arranged and adapted to locate or position the solvent emitter substantially centrally within the aperture. The fixing device may comprise a device which is arranged and adapted to locate or position the solvent emitter substantially non-centrally within the aperture.
The fixing device may comprise a device which is arranged and adapted to locate or position the solvent emitter substantially non-centrally within the aperture, such that the solvent emitter is substantially closer to and/or oriented towards an inlet, such as an inlet capillary, of a mass and/or ion mobility spectrometer, e.g. relative to the centre and/or central axis of the aperture.
For example, with reference to Fig. 43, the solvent capillary 302 and/or its outlet end 303 may be positioned centrally or non-centrally within sheath gas tube 312, and may be positioned such that the outlet end 303 is closer to the transfer capillary 330 relative to the centre of the tube outlet 316 and/or such that the solvent capillary 302 and/or its outlet end 303 is angled towards the transfer capillary 330 relative to the central axis of the tube 312.
The fixing device may be arranged and adapted such that gas may flow through the nozzle and/or aperture relatively unimpeded by the fixing device.
The fixing device may be arranged and adapted so as to avoid or reduce turbulence in gas flowing through the nozzle and/or aperture.
According to an embodiment apparatus is provided comprising an ion source comprising a nozzle having an aperture, a solvent emitter which extends through the aperture, and a device which is arranged and adapted to monitor the solvent emitter position relative to the aperture.
The results of the monitoring may be used to calibrate and/or correct the ion source and/or mass and/or ion mobility spectrometric data acquired using the ion source.
According to this embodiment, rather than fixing the solvent capillary relative to the gas capillary, its position is monitored and any movements are corrected for, e.g. in post processing of the data and/or by adjusting one or more parameters of the ion source (such as gas flow rate, applied voltage, solvent flow rate, position, orientation, temperature, etc.), e.g. in real-time, e.g. using a feedback loop.
Investigations with a fixed geometry sprayer according to various embodiments showed that solvent capillary position or orientation has a significant impact on lipid signal intensity.
A sprayer, in which the solvent capillary geometry was fixed by attaching it to the gas capillary, was tested to assess the impact of solvent capillary positioning on lipid signal. A modified sprayer was produced, where cyanoacrylate glue was used to attach the inner solvent capillary to the outer gas capillary, in order ensure consistent alignment.
The sprayer with the fixed solvent capillary was mounted in four different orientations. The orientations tested were: forward, directed towards the mass
spectrometer inlet; 45° left and 45° right; backwards, directed away from the mass spectrometer inlet. All orientations were tested on the same three pork liver sections with two line scans per orientation and scan direction. A large difference between the forward and backward orientations was observed. Both fatty acid and phospholipid signals were optimal in the forward direction and almost zero in the backward direction.
A solvent capillary orientation straight towards the inlet of the mass and/or ion mobility spectrometer was found to be beneficial, while sideways rotations led to signal reductions of up to 50 % and orientation away from the mass and/or ion mobility spectrometer inlet led to a loss of signal.
Accordingly, it was determined that the forward orientation is most effective.
This result also demonstrates that conventional designs where the capillary orientation is easily shifted (i.e. not fixed) can result in radical changes to the lipid spectrum by small deviations of the capillary in the forward or backward direction, with the latter decreasing the signal dramatically. This can result in high variability in performance, e.g. where a large inner diameter of the gas nozzle allows the capillary to move aside from a central starting position. Variations in capillary orientation can have a deleterious effect on the (lipid) signal.
The effect of unmounting and remounting a non-fixed conventional geometry sprayer was tested multiple times with de-installation and re-installation between measurements on a rhodamine sample. It was found that solvent capillary positioning can change between measurements, which leads to changes in the size and shape of the desorption area. This observation further underpins the conclusion that this parameter need to be closely controlled, e.g. by fixing and/or monitoring, during a desorption electrospray experiment.
Fixing the position of the solvent capillary within the gas capillary in accordance with various embodiments provides an improved, and reproducible sprayer geometry.
It was furthermore determined that fixing the solvent capillary to the gas capillary itself in such a way so as to impede the nebulising gas flow (e.g. using glue) led to a reduction in signal of almost 50%.
The obstructed gas flow due to the glue fixing was found to reduce the total ion intensity by around 50% to 65%. The phenomenon was associated with the change in the gas flow pattern introduced by fixing the solvent capillary to the gas capillary. The mounting point may block the gas flow, so that the electrospray is no longer surrounded by the nebulising gas, which can lead to a formation of larger, slower droplets and lower desolvation and desorption in that part of the primary electrospray. That is, fixing the solvent capillary directly to the gas capillary may introduce further turbulence in the proximity of the spray tip, which can contribute to poor reproducibility.
Thus, according to various embodiments, a more carefully engineered source is provided that does not disrupt the gas flow.
According to various embodiments, the solvent capillary is fixed without obstructing the nebulising gas flow. The solvent capillary may be fixed centrally or with a forward orientation for a high and less variable lipid signal.
According to an embodiment a desorption electrospray ionisation sprayer with a capillary fixing or centring device is provided, which allows fixing the solvent capillary geometry without obstructing the nebulising gas flow. This provides improved absolute intensity repeatability and improvements in spectral pattern repeatability of up to an order of magnitude. For tissue imaging, spatial resolutions of 20 μηι are readily achieved.
According to various embodiment, a sprayer with a centred solvent capillary and relatively small inner diameter, and tapered solvent capillary, is provided.
According to various embodiments a desorption electrospray ionisation sprayer may be provided wherein the emitter guide and electrically conductive (e.g. stainless steel) gas nozzle are insulated from each other in order to avoid charging of the gas nozzle and consequently, loss of signal.
Fig. 41 shows a diagram of a sprayer nozzle according to an embodiment. A stainless steel emitter guide and a gas nozzle are electrically insulated from each other to avoid charging of the gas nozzle. This may be achieved by using an insulating fixing or centering disc. The fixing or centering disc may comprise or may be coated with a ceramic and/or a polymer.
Alternatively, the emitter guide may be made to be electrically insulating, e.g. the emitter guide may comprise a ceramic and/or a polymer. In this case, the fixing or centering disc need not be insulating.
This sprayer was tested for imaging using human colorectal cancer sections as shown in Figs. 42A-42G.
Fig. 42A shows desorption electrospray ionisation-MS imaging of adjacent colorectal cancer sections at different spatial resolutions wherein RGB images for the first three components of principal component analysis of images were acquired at 100 μηι, Fig. 42B shows RGB images for the first three components of principal component analysis of images which were acquired at 50 μηι, Fig. 42C shows RGB images for the first three components of principal component analysis of images which were acquired at 20 μηι pixel size/ scan speed per second, wherein Fig. 42A was acquired using a solvent flow rate of 1.5 μΙ/min and a gas pressure of 7 bars and corresponds with trace D on rhodamine B, wherein Figs. 42B and 42C were acquired using a solvent flow rate of 0.5 μΙ/min and a gas pressure of 6 bars and correspond with trace E and wherein Fig. 42F shows an optical image of section A after hematoxylin and eosin staining and Fig. 42G shows an example spectrum from section A. In order to achieve higher spatial resolution, a reduced solvent flow rate of 0.5 μΙ/min (compared to 1.5 μΙ/min) was used. Although signal intensity was reduced proportionally, it was still sufficient to receive high quality images at 50 and 20 μηι pixel size.
Fig. 42A shows desorption electrospray ionisation-MS imaging of adjacent colorectal cancer sections at different spatial resolutions. RGB images for the first three components of principal component analysis of images acquired at 100 μηι (Fig. 42A), 50 μηι (Fig. 42B) and 20 μηι (Fig. 42C) pixel size/ scan speed per second. The image shown in Fig. 42A was acquired using a solvent flow rate of 1.5 μΙ/min and a gas pressure of 7 bars and corresponds with the trace shown in Fig. 42D on rhodamine B.
The images shown in Figs. 42B and 42C were acquired using a solvent flow rate of 0.5 μΙ/min and a gas pressure of 6 bars and correspond with the trace shown in Fig. 42E. Fig. 42F shows an optical image of the section shown in Fig. 42A after hematoxylin and eosin staining. Fig. 42G shows an example spectrum from the section shown in Fig. 42A.
Desorption electrospray ionisation ("DESI") sprayer with heated transfer capillary
Fig. 43 shows another embodiment and comprises a Desorption Electrospray Ionisation ("DESI") sprayer 300 in which a solvent capillary 302 may be arranged to direct electrically charged droplets 304 of solvent at a sample surface 310. A sample 311 may be located on the sample surface 310, which may comprise analyte particles. The charging of the solvent droplets may be achieved through the use of a high-voltage power supply 306 that contacts the capillary 302. The high-voltage power supply 306 may comprise an electrode 307 which may contact any portion of the capillary 302 so that it is operable to charge the solvent droplets as they leave an outlet end 303 of the capillary 302. The outlet end 303 of the capillary may be directed towards the sample surface 310.
A sheath gas 308 (e.g., nitrogen) may be arranged to surround the capillary 302 so as to nebulise the solvent as it emerges from the capillary 302 and direct the electrically charged solvent droplets 304 towards the surface 310. The sheath gas may be introduced through a tube 312 that may be coaxial to the solvent capillary 302, having an inlet 314 at an end distal to the sample surface 310 and an outlet 316 at an end facing the sample surface 310.
The outlet 316 of the sheath gas tube 312 may be concentric to the outlet end 303 of the capillary, which can facilitate in nebulising the solvent as it emerges from the capillary 302. The solvent emerging from the outlet end 303 of the solvent capillary 302 may be nebulised by the sheath gas 308. A connector 318 may connect the tube 312 to a source of gas suitable to use as a sheath gas. The sheath gas 308 may comprise nitrogen or standard medical air, and the source of sheath gas may be a source of nitrogen gas or standard medical air.
As the solvent droplets 304 contact the sample, analyte particles on the sample can desorb and the charged droplets and analyte mixture 320 may be transferred into a transfer capillary or transfer device 330 that may lead to an ion analyser, mass analyser or mass spectrometer 340. The charged droplet and analyte mixture may be transferred through an inlet 332 of the transfer capillary or transfer device 330. This may be achieved by placing the opposite end 333 of the transfer capillary or transfer device 330 in a low pressure region 352, for example a vacuum stage of the mass analyser or mass spectrometer 340.
The charged droplet and analyte mixture (including e.g., analyte ions) may be transferred by ion optics 352 to an analysis region of the ion analyser or mass
spectrometer 340. The ion optics 352 may comprise an ion guide, for example a Stepwave (RTM) ion guide.
The analyte ions may be guided to the analysis region by applying voltages to the ion optics 352. The analyte ions may then be analysed by the mass analyser or mass spectrometer 340. According to an embodiment the ion analyser, mass analyser or mass spectrometer 340 may comprise an ion mobility spectrometer. According to a yet further embodiment the ion analyser, mass analyser or mass spectrometer 340 may comprise the combination of an ion mobility spectrometer and a mass spectrometer.
As a result of the analysis, chemical information about the sample 311 may be obtained.
One or more heaters may be provided to heat the various parts of the apparatus shown in Fig. 43. For example, a heater may be provided to heat one or more of the solvent capillary 302, the sheath gas tube 312, the sample surface 310 and the transfer or inlet capillary 330.
The one or more heaters may comprise a wire heater (e.g., a tungsten wrap) and/or may be configured to heat the respective part to at least 50 °C, 100 °C, 200 °C, 300 °C, 400 °C, 500 °C, 600 °C, 700 °C or 800 °C. However, any type of heater may be used that has the function of heating the respective part, for example a blower or an inductive heater.
Fig. 43 shows a first heater 342 that may be arranged and adapted to heat the transfer or inlet capillary 330, such that the solvent and analyte mixture 320 may be heated before being passed onward, for example to the mass analyser or mass spectrometer 340.
The first heater 342 may be located anywhere along the solvent capillary 330, for example adjacent to or at the inlet 341 of the ion analyser, mass analyser or mass spectrometer. Alternatively, the first heater 342 may be located adjacent to or at the inlet 332 of the solvent capillary or transfer device 330. The first heater 342 may comprise a wire heater (e.g., a tungsten wrap) and/or may be configured to heat the inlet capillary to at least 50 °C, 100 °C, 200 °C, 300 °C, 400 °C, 500 °C, 600 °C, 700 °C or 800 °C.
A second heater 344 may be arranged and adapted to heat the sheath gas tube
312, such that the solvent and/or sheath gas may be heated.
The second heater 344 may be located at the end of the tube 312 nearest the sample surface 310, such that the solvent and/or sheath gas may be heated before being directed at the sample surface 310. The second heater 344 may comprise a wire heater (e.g., a tungsten wrap) and/or may be configured to heat the tube 312 and/or the solvent and/or the sheath gas to at least 50°C, 100°C, 200°C, 300°C, 400°C, 500°C, 600 °C, 700 °C or 800 °C.
A third heater 346 may be arranged and adapted to heat the solvent capillary 302, such that the solvent may be heated.
The third heater 346 may be located anywhere along the solvent capillary 302, for example nearest the end 305 located away from the sample surface 310, such that the solvent may be heated before it is surrounded by the sheath gas tube 312. The third heater 346 may comprise a wire heater (e.g., a tungsten wrap) and/or may be configured to heat the solvent capillary 302 and/or the solvent to at least 50 °C, 100 °C, 200 °C, 300 °C, 400 °C, 500 °C, 600 °C, 700 °C or 800 °C.
A fourth heater 348 may be arranged and adapted to heat the sample surface 310, such that the sample 31 1 and/or the sample surface 310 may be heated. The fourth heater 348 may be located beneath a portion of the sample surface 310 arranged and adapted to hold or contain the sample 311. The fourth heater 348 may comprise a wire heater (e.g., a tungsten wrap) and/or may be configured to heat the sample 311 and/or sample surface 310 and/or the solvent to at least 50 °C, 100 °C, 200 °C, 300 °C, 400 °C, 500 °C, 600 °C, 700 °C or 800 °C.
Any one or more or all of the heaters may comprise an inductive or eddy current heater.
The impact of heating an ion inlet transfer capillary (such as a transfer capillary or transfer device 330 as shown in Fig. 43) was tested on a Xevo G2-XS (RTM) quadrupole Time of Flight mass spectrometer and a Synapt G2-Si (RTM) quadrupole-ion mobility-Time of Flight mass spectrometer.
The ion transfer capillary or transfer device 330 was heated using a nickel wire heater in a range from 100 to 490 °C. Pork liver sections were used and the intensities for selected fatty acids and phospholipids were compared. Inlet capillary heating was found to have some impact on fatty acid intensities using a Xevo (RTM) mass spectrometer and no impact using a Synapt (RTM) mass spectrometer. Intensities for the monitored
phospholipids, however, could be improved by almost two orders of magnitude.
Figs. 44A-D show the impact of inlet capillary heating on absolute intensity. Figs. 44A and 44C relate to a Waters Synapt G2-Si (RTM) mass spectrometer and Figs. 44B and 44D relate to a Waters Xevo G2-XS (RTM) mass spectrometer. Average intensities for selected fatty acids (FA), phosphatidyl ethanolamines (PE) and the most abundant phosphatidylinositol (PI) from pork liver sections are shown.
It is apparent from Figs. 44A-D that increasing the temperature of the ion transfer capillary or transfer device 330 can increase the observed intensity of phospholipids by nearly two orders of magnitude.
The embodiments described above in relation to Fig. 43 may be used in
applications such as medical swabs, where the sample surface 310 forms the surface of a swab. In such a case, the swab itself may be heated so as to heat the sample 31 1 that is located on the swab. For example, the fourth heater 348 may be a wire heater that is located within the swab, and may be arranged and adapted to heat the end of the swab configured to hold and/or retain biologic samples for analysis.
Methods of analysis, e.g., methods of medical treatment, surgery and diagnosis and nonmedical methods
Various different embodiments are contemplated. According to some embodiments the methods disclosed above may be performed on in vivo, ex vivo or in vitro tissue. The tissue may comprise human or non-human animal or plant tissue. Other embodiments are contemplated wherein the target may comprise biological matter or organic matter (including a plastic). Embodiments are also contemplated wherein the target comprises one or more bacterial colonies or one or more fungal colonies.
Various embodiments are contemplated wherein analyte ions generated by an ambient ionisation ion source are then subjected either to: (i) mass analysis by a mass analyser or filter such as a quadrupole mass analyser or a Time of Flight mass analyser; (ii) ion mobility analysis (IMS) and/or differential ion mobility analysis (DMA) and/or Field Asymmetric Ion Mobility Spectrometry (FAIMS) analysis; and/or (iii) a combination of firstly (or vice versa) ion mobility analysis (IMS) and/or differential ion mobility analysis (DMA) and/or Field Asymmetric Ion Mobility Spectrometry (FAIMS) analysis followed by secondly (or vice versa) mass analysis by a mass analyser or filter such as a quadrupole mass analyser or a Time of Flight mass analyser. Various embodiments also relate to an ion mobility spectrometer and/or mass analyser and a method of ion mobility spectrometry and/or method of mass analysis. Ion mobility analysis may be performed prior to mass to charge ratio analysis or vice versa.
Various references are made in the present application to mass analysis, mass analysers or filters, mass analysing, mass spectrometric data, mass spectrometers and other related terms referring to apparatus and methods for determining the mass or mass to charge of analyte ions. It should be understood that it is equally contemplated that the present invention may extend to ion mobility analysis, ion mobility analysers, ion mobility analysing, ion mobility data, ion mobility spectrometers, ion mobility separators and other related terms referring to apparatus and methods for determining the ion mobility, differential ion mobility, collision cross section or interaction cross section of analyte ions. Furthermore, it should also be understood that embodiments are contemplated wherein analyte ions may be subjected to a combination of both ion mobility analysis and mass analysis i.e. that both (a) the ion mobility, differential ion mobility, collision cross section or interaction cross section of analyte ions together with (b) the mass to charge of analyte ions is determined. Accordingly, hybrid ion mobility-mass spectrometry (IMS-MS) and mass spectrometry-ion mobility (MS-IMS) embodiments are contemplated wherein both the ion mobility and mass to charge ratio of analyte ions generated e.g. by an ambient ionisation ion source are determined. Ion mobility analysis may be performed prior to mass to charge ratio analysis or vice versa. Furthermore, it should be understood that embodiments are contemplated wherein references to mass spectrometric data and databases comprising mass spectrometric data should also be understood as
encompassing ion mobility data and differential ion mobility data etc. and databases comprising ion mobility data and differential ion mobility data etc. (either in isolation or in combination with mass spectrometric data).
Various surgical, therapeutic, medical treatment and diagnostic methods are contemplated.
However, other embodiments are contemplated which relate to non-surgical and non-therapeutic methods of mass spectrometry and/or ion mobility spectrometry which are not performed on in vivo tissue. Other related embodiments are contemplated which are performed in an extracorporeal manner such that they are performed outside of the human or animal body.
Further embodiments are contemplated wherein the methods are performed on a non-living human or animal, for example, as part of an autopsy procedure. Although the present invention has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes in form and detail may be made without departing from the scope of the invention as set forth in the accompanying claims.

Claims

Claims 1. A method comprising:
applying a reversal agent to a first target in order to modify said target thereby producing a modified target;
using a first device to generate analyte or analyte ions from multiple regions of said modified target; and
generating an ion image of said modified target.
2. A method as claimed in claim 1 , wherein said first device comprises or forms part of an ambient ion or ionisation source or wherein said first device generates aerosol, smoke or vapour for subsequent ionisation by an ambient ion or ionisation source or other ionisation source.
3. A method as claimed in claim 1 or 2, wherein said first device comprises a desorption electrospray ionisation ("DESI") ion source or a desorption electro-flow focusing ("DEFFI") ion source.
4. A method as claimed in claim 1 , 2 or 3, wherein said first device comprises an ion source selected from the group consisting of: (i) a rapid evaporative ionisation mass spectrometry ("REIMS") ion source; (ii) a laser desorption ionisation ("LDI") ion source; (iii) a thermal desorption ion source; (iv) a laser diode thermal desorption ("LDTD") ion source; (v) a dielectric barrier discharge ("DBD") plasma ion source; (vi) an Atmospheric Solids
Analysis Probe ("ASAP") ion source; (vii) an ultrasonic assisted spray ionisation ion source; (viii) an easy ambient sonic-spray ionisation ("EASI") ion source; (ix) a desorption atmospheric pressure photoionisation ("DAPPI") ion source; (x) a paperspray ("PS") ion source; (xi) a jet desorption ionisation ("JeDI") ion source; (xii) a touch spray ("TS") ion source; (xiii) a nano-desorption electrospray ionisation ("nano-DESI")jon source; (xiv) a laser ablation electrospray ("LAESI") ion source; (xv) a direct analysis in real time ("DART") ion source; (xvi) a probe electrospray ionisation ("PESI") ion source; (xvii) a solid-probe assisted electrospray ionisation ("SPA-ESI") ion source; (xviii) a cavitron ultrasonic surgical aspirator ("CUSA") device; (xix) a focussed or unfocussed ultrasonic ablation device; (xx) a microwave resonance device; and (xxi) a pulsed plasma RF dissection device.
5. A method as claimed in any preceding claim, wherein said first target comprises a chemically modified target.
6. A method as claimed in any preceding claim, wherein said first target comprises a fixed tissue sample.
7. A method as claimed in claim 6, wherein said fixed tissue sample is fixed with either: (i) formalin or a formaldehyde solution; or (ii) formalin or a formaldehyde solution with glycerol or another chemical agent.
8. A method as claimed in claim 6, wherein said fixed tissue sample comprises a formalin fixed, paraffin-embedded ("FFPE") tissue sample.
9. A method as claimed in claim 6, wherein said fixed tissue sample is embedded in an embedding medium selected from the group consisting of: (i) glycerol; (ii) optimum cutting temperature ("OCT") (RTM) medium; (iii) paraffin; (iv) one or more plastic polymers; (v) polyester wax; (vi) polyethylene glycol ("PEG"); (vii) polyfin (RTM); (viii) a tissue freezing medium ("TFM"); (ix) glycol methacrylate ("GMA"); (x) gelatin; and (xi) carboxy methyl cellulose ("SMC").
10. A method as claimed in any preceding claim, wherein said first target comprises a tissue sample wherein methylene bridge cross-linkages are formed between proteins.
1 1. A method as claimed in any preceding claim, wherein said first target comprises a tissue sample which has been dehydrated with an alcohol.
12. A method as claimed in any preceding claim, wherein said first target comprises a tissue sample which has been infiltrated with molten paraffin.
13. A method as claimed in any preceding claim, wherein said first target and/or said modified target is provided on a substrate.
14. A method as claimed in claim 13, wherein said substrate is selected from the group consisting of: (i) a microscope slide; (ii) a transparent slide; (iii) a glass slide; (iv) a plastic slide; and (v) an indium-tin-oxide (ITO) coated glass or other slide.
15. A method as claimed in any preceding claim, wherein said step of applying said reversal agent to said first target reverses cross-linked bonds of proteins present in said first target.
16. A method as claimed in any preceding claim, wherein said reversal agent is arranged to reverse cross-linked bonds of formalin fixed tissue proteins or formalin fixed paraffin embedded ("FFPE") tissue proteins.
17. A method as claimed in any preceding claim, wherein said reversal agent comprises a formalin or formaldehyde scavenger.
18. A method as claimed in any of claims 1-17, wherein said reversal agent comprises water.
19. A method as claimed in any of claims 1-17, wherein said reversal agent comprises sodium metabisulfate or sodium metabisulfite (Na2S205).
20. A method as claimed in any of claims 1-17, wherein said reversal agent comprises urea.
21. A method as claimed in any of claims 1-17, wherein said reversal agent is selected from the group consisting of: (i) at least one dithioxamide; (ii) at least one carbodiimide; (iii) at least one monomeric, oligomeric or polymeric carbodiimide; (iv) at least one
aminoplastic, thermoplastic, binder, polymeric compound or copolymeric compound which is able to cleave or eliminate formalin or formaldehyde; (v) pyrogallol; (vi)
1 ,2 hexanediol; (vii) one or more polyhydric alcohols, such as diethylene glycol and sorbitol; (viii) a nitrogen containing compound such as urea, melamine, diazine, triazine and amine compounds; (ix) a nitrogenous compound such as urea, ethyleneurea or carbohydrazide; (x) Dimethyl-1 ,3-acetonedicarboxylate, diethyl malonate or
ethylacetoacetate; (xi) a water-soluble active methylene compound; (xii) hydrazine; (xiii) polyalkylene glycol diester; and (xiv) ammonium bisulfite.
22. A method as claimed in any of claims 1-17, wherein said reversal agent comprises an organic solvent.
23. A method as claimed in any of claims 1-17, wherein said reversal agent comprises an organic compound having at least one active methylene hydrogen and a pKa
(logarithmic acid dissociation constant) in the range 5-13.
24. A method as claimed in any preceding claim, wherein said step of applying said reversal agent to said first target comprises applying said reversal agent to said first target at a concentration of: (i) 0-5 % w/v; (ii) 5-10 % w/v; (iii) 10-15 % w/v; (iv) 15-20% w/v; (v) 20-25% w/v; (vi) 25-30 % w/v; (vii) 30-35% w/v; (viii) 35-40 % w/v; (ix) 40-45 % w/v; (x) 45- 50 % w/v; (xi) 50-55% % w/v; (xii) 55-60% w/v; (xiii) 60-65% w/v; (xiv) 65-70% w/v; (xv) 70- 75%; and (xvi) > 75 % w/v.
25. A method as claimed in any preceding claim, wherein said step of applying said reversal agent to said first target comprises applying said reversal agent to said first target at a concentration of: (i) 0-1 M; (ii) 1-2 M; (iii) 2-3 M; (iv) 3-4 M; (v) 4-5 M; (vi) 5-6 M; (vii) 6- 7 M; (viii) 7-8 M; (ix) 8-9 M; (x) 9-10 M; (xi) 10-11 M; (xii) 11-12 M; (xiii) 12-13 M; (xiv) 13- 14 M; (xv) 14-15 M; and (xvi) > 15 M.
26. A method as claimed in any preceding claim, further comprising subjecting said first target or said modified target to one or more heat treatment cycles.
27. A method as claimed in claim 26, wherein a or each heat treatment cycle comprises heating said first target or said modified target at a temperature selected from the group consisting of: (i) 20-30 °C; (ii) 30-40 °C; (iii) 40-50 °C; (iv) 50-60 °C; (v) 60-70 °C; (vi) 70-80 °C; (vii) 80-90 °C; (viii) 90-100 °C; (ix) 100-1 10 °C; (x) 1 10-120 °C; and (xi) > 120 °C.
28. A method as claimed in claim 26 or 27, wherein a or each heat treatment cycle comprises heating said first target or said modified target for a period of time selected from the group consisting of: (i) 0-1 minutes; (ii) 1-2 minutes; (iii) 2-3 minutes; (iv) 3-4 minutes; (v) 4-5 minutes; (vi) 5-6 minutes; (vii) 6-7 minutes; (viii) 7-8 minutes; (ix) 8-9 minutes; (x) 9- 10 minutes; and (xi) > 10 minutes.
29. A method of ambient ionisation comprising a method as claimed in any preceding claim.
30. A method of desorption electrospray ionisation ("DESI") imaging comprising a method as claimed in any of claims 1-28.
31. A method of desorption electroflow focusing ionisation ("DEFFI") imaging comprising a method as claimed in any of claims 1-28.
32. A method of ion imaging comprising a method as claimed in any of claims 1-28.
33. A method of analysis comprising a method as claimed in any of claims 1-28.
34. A method of surgery, diagnosis, therapy or medical treatment comprising a method as claimed in any of claims 1-28.
35. A non-surgical, non-therapeutic method of mass spectrometry and/or ion mobility spectrometry comprising a method as claimed in any of claims 1-28.
36. A method of mass spectrometry and/or ion mobility spectrometry comprising a method as claimed in any of claims 1-28.
37. Apparatus comprising:
a first device arranged and adapted to generate analyte or analyte ions from multiple regions of a modified target, wherein said modified target comprises a first target to which a reversal agent has been applied; and
a device for generating an ion image of said modified target.
38. Apparatus as claimed in claim 37, wherein said first device comprises or forms part of an ambient ion or ionisation source or wherein said first device generates aerosol, smoke or vapour for subsequent ionisation by an ambient ion or ionisation source or other ionisation source.
39. Apparatus as claimed in claim 37 or 38, wherein said first device comprises a desorption electrospray ionisation ("DESI") ion source or a desorption electro-flow focusing ("DEFFI") ion source.
40. Apparatus as claimed in claim 37, 38 or 39, wherein said first device comprises an ion source selected from the group consisting of: (i) a rapid evaporative ionisation mass spectrometry ("REIMS") ion source; (ii) a laser desorption ionisation ("LDI") ion source; (iii) a thermal desorption ion source; (iv) a laser diode thermal desorption ("LDTD") ion source; (v) a dielectric barrier discharge ("DBD") plasma ion source; (vi) an Atmospheric Solids Analysis Probe ("ASAP") ion source; (vii) an ultrasonic assisted spray ionisation ion source; (viii) an easy ambient sonic-spray ionisation ("EASI") ion source; (ix) a desorption atmospheric pressure photoionisation ("DAPPI") ion source; (x) a paperspray ("PS") ion source; (xi) a jet desorption ionisation ("JeDI") ion source; (xii) a touch spray ("TS") ion source; (xiii) a nano-desorption electrospray ionisation ("nano-DESI") ion source; (xiv) a laser ablation electrospray ("LAESI") ion source; (xv) a direct analysis in real time ("DART") ion source; (xvi) a probe electrospray ionisation ("PESI") ion source; (xvii) a solid-probe assisted electrospray ionisation ("SPA-ESI") ion source; (xviii) a cavitron ultrasonic surgical aspirator ("CUSA") device; (xix) a focussed or unfocussed ultrasonic ablation device; (xx) a microwave resonance device; and (xxi) a pulsed plasma RF dissection device.
41. Apparatus as claimed in any of claims 37-40, wherein said first target comprises a chemically modified target.
42. Apparatus as claimed in any of claims 37-41 , wherein said first target comprises a fixed tissue sample.
43. Apparatus as claimed in claim 42, wherein said fixed tissue sample is fixed with either: (i) formalin or a formaldehyde solution; or (ii) formalin or a formaldehyde solution with glycerol or another chemical agent.
44. Apparatus as claimed in claim 42, wherein said fixed tissue sample comprises a formalin fixed, paraffin-embedded ("FFPE") tissue sample.
45. Apparatus as claimed in claim 42, wherein said fixed tissue sample is embedded in an embedding medium selected from the group consisting of: (i) glycerol; (ii) optimum cutting temperature ("OCT") (RTM) medium; (iii) paraffin; (iv) one or more plastic polymers; (v) polyester wax; (vi) polyethylene glycol ("PEG"); (vii) polyfin (RTM); (viii) a tissue freezing medium ("TFM"); (ix) glycol methacrylate ("GMA"); (x) gelatin; and (xi) carboxy methyl cellulose ("SMC").
46. Apparatus as claimed in any of claims 37-45, wherein said first target comprises a tissue sample wherein methylene bridge cross-linkages are formed between proteins.
47. Apparatus as claimed in any of claims 37-46, wherein said first target comprises a tissue sample which has been dehydrated with an alcohol.
48. Apparatus as claimed in any of claims 37-47, wherein said first target comprises a tissue sample which has been infiltrated with molten paraffin.
49. Apparatus as claimed in any of claims 37-48, wherein said first target and/or said modified target is provided on a substrate.
50. Apparatus as claimed in claim 49, wherein said substrate is selected from the group consisting of: (i) a microscope slide; (ii) a transparent slide; (iii) a glass slide; (iv) a plastic slide; and (v) an indium-tin-oxide (ITO) coated glass or other slide.
51. Apparatus as claimed in any of claims 37-50, wherein said reversal agent is applied to said first target in order to reverse cross-linked bonds of proteins present in said first target.
52. Apparatus as claimed in any of claims 37-51 , wherein said reversal agent is arranged to reverse cross-linked bonds of formalin fixed tissue proteins or formalin fixed paraffin embedded ("FFPE") tissue proteins.
53. Apparatus as claimed in any of claims 37-52, wherein said reversal agent comprises a formalin or formaldehyde scavenger.
54. Apparatus as claimed in any of claims 37-53, wherein said reversal agent comprises water.
55. Apparatus as claimed any of claims 37-53, wherein said reversal agent comprises sodium metabisulfate or sodium metabisulfite (Na2S205).
56. Apparatus as claimed any of claims 37-53, wherein said reversal agent comprises urea.
57. Apparatus as claimed any of claims 37-53, wherein said reversal agent is selected from the group consisting of: (i) at least one dithioxamide; (ii) at least one carbodiimide; (iii) at least one monomeric, oligomeric or polymeric carbodiimide; (iv) at least one
aminoplastic, thermoplastic, binder, polymeric compound or copolymeric compound which is able to cleave or eliminate formalin or formaldehyde; (v) pyrogallol; (vi)
1 ,2 hexanediol; (vii) one or more polyhydric alcohol, such as diethylene glycol and sorbitol; (viii) a nitrogen containing compound such as urea, melamine, diazine, triazine and amine compounds; (ix) a nitrogenous compound such as urea, ethyleneurea or carbohydrazide; (x) Dimethyl-1 ,3-acetonedicarboxylate, diethyl malonate or ethylacetoacetate; (xi) a water- soluble active methylene compound; (xii) hydrazine; (xiii) polyalkylene glycol diester; and (xiv) ammonium bisulfite.
58. Apparatus as claimed any of claims 37-53, wherein said reversal agent comprises an organic solvent.
59. Apparatus as claimed any of claims 37-53, wherein said reversal agent comprises an organic compound having at least one active methylene hydrogen and a pKa
(logarithmic acid dissociation constant) in the range 5-13.
60. Apparatus as claimed any of claims 37-59, wherein said reversal agent is applied to said first target at a concentration of: (i) 0-5 % w/v; (ii) 5-10 % w/v; (iii) 10-15 % w/v; (iv) 15- 20% w/v; (v) 20-25% w/v; (vi) 25-30 % w/v; (vii) 30-35% w/v; (viii) 35-40 % w/v; (ix) 40-45 % w/v; (x) 45-50 % w/v; (xi) 50-55% % w/v; (xii) 55-60% w/v; (xiii) 60-65% w/v; (xiv) 65- 70% w/v; (xv) 70-75%; and (xvi) > 75 % w/v.
61. Apparatus as claimed any of claims 37-60, wherein said reversal agent is applied to said first target at a concentration of: (i) 0-1 M; (ii) 1-2 M; (iii) 2-3 M; (iv) 3-4 M; (v) 4-5 M; (vi) 5-6 M; (vii) 6-7 M; (viii) 7-8 M; (ix) 8-9 M; (x) 9-10 M; (xi) 10-1 1 M; (xii) 1 1-12 M; (xiii) 12-13 M; (xiv) 13-14 M; (xv) 14-15 M; and (xvi) > 15 M.
62. Apparatus as claimed any of claims 37-61 , wherein said first target or said modified target is subjected to one or more heat treatment cycles.
63. Apparatus as claimed in claim 62, wherein a or each heat treatment cycle comprises heating said first target or said modified target at a temperature selected from the group consisting of: (i) 20-30 °C; (ii) 30-40 °C; (iii) 40-50 °C; (iv) 50-60 °C; (v) 60-70 °C; (vi) 70-80 °C; (vii) 80-90 °C; (viii) 90-100 °C; (ix) 100-1 10 °C; (x) 1 10-120 °C; and (xi) > 120 °C.
64. Apparatus as claimed in claim 62 or 63, wherein a or each heat treatment cycle comprises heating said first target or said modified target for a period of time selected from the group consisting of: (i) 0-1 minutes; (ii) 1-2 minutes; (iii) 2-3 minutes; (iv) 3-4 minutes; (v) 4-5 minutes; (vi) 5-6 minutes; (vii) 6-7 minutes; (viii) 7-8 minutes; (ix) 8-9 minutes; (x) 9- 10 minutes; and (xi) > 10 minutes.
65. An ambient ionisation ion source comprising apparatus as claimed any of claims 37-64.
66. A desorption electrospray ionisation ("DESI") imaging system comprising apparatus as claimed in any of claims 37-64.
67. A desorption electroflow focusing ionisation ("DEFFI") imaging system comprising apparatus as claimed in any of claims 37-64.
68. An ion imager comprising apparatus as claimed in any of claims 37-64.
69. Analysis apparatus comprising apparatus as claimed in any of claims 37-64.
70. A mass and/or ion mobility spectrometer comprising apparatus as claimed in any of claims 37-64.
71. Apparatus comprising:
an ion source comprising a nozzle having an aperture;
a solvent emitter which extends through said aperture; and
a centering device which is arranged and adapted to locate or position said solvent emitter substantially centrally within said aperture.
72. Apparatus as claimed in claim 71 , wherein said ion source comprises a Desorption Electrospray lonisation ("DESI") ion source or a desorption electro-flow focusing ("DEFFI") ion source.
73. Apparatus as claimed in claim 71 or 72, further comprising a solvent emitter guide.
74. Apparatus as claimed in claim 73, wherein said solvent emitter is located or positioned within said solvent emitter guide.
75. Apparatus as claimed in claim 73 or 74, wherein said centering device is arranged and adapted to locate or position said solvent emitter guide substantially centrally within an internal volume of said nozzle.
76. Apparatus as claimed in any of claims 73, 74 or 75, wherein said solvent emitter guide is electrically conductive.
77. Apparatus as claimed in any of claims 73-76, wherein said solvent emitter guide comprises metal.
78. Apparatus as claimed in any of claims 73-77, wherein said solvent emitter guide comprises stainless steel.
79. Apparatus as claimed in any of claims 73, 74 or 75, wherein said solvent emitter guide is electrically insulating.
80. Apparatus as claimed in any of claims 71-79, wherein said nozzle is electrically conductive.
81. Apparatus as claimed in any of claims 71-80, wherein said nozzle comprises metal.
82. Apparatus as claimed in any of claims 71-81 , wherein said nozzle comprises stainless steel.
83. Apparatus as claimed in any of claims 71-82, wherein said centering device comprises a disc.
84. Apparatus as claimed in any of claims 71-83, wherein said centering device comprises or is coated with an insulator.
85. Apparatus as claimed in any of claims 71-84, wherein said centering device comprises or is coated with a ceramic and/or a polymer.
86. Apparatus as claimed in any of claims 71-85, further comprising a device for supplying a solvent to said solvent emitter.
87. Apparatus as claimed in any of claims 71-86, further comprising a device for supplying a nebulising gas within said nozzle so that, in use, said nebulising gas exits said nozzle via said aperture.
88. An ambient ionisation ion source comprising apparatus as claimed any of claims 71-87.
89. A desorption electrospray ionisation ("DESI") imaging system comprising apparatus as claimed in any of claims 71-87.
90. A desorption electroflow focusing ionisation ("DEFFI") imaging system comprising apparatus as claimed in any of claims 71-87.
91 An ion imager comprising apparatus as claimed in any of claims 71-87.
92. Analysis apparatus comprising apparatus as claimed in any of claims 71-87.
93. A mass and/or ion mobility spectrometer comprising apparatus as claimed in any of claims 71-87.
94. A method comprising:
providing an ion source comprising a nozzle having an aperture;
providing a solvent emitter which extends through said aperture; and
using a centering device to locate or position said solvent emitter substantially centrally within said aperture.
95. A method of mass spectrometry and/or ion mobility spectrometry comprising a method as claimed in claim 94.
96. Apparatus comprising:
a first device arranged and adapted to emit a stream of electrically charged droplets towards a target in use;
a transfer capillary or transfer device arranged and adapted to transfer ions generated from said target towards an ion analyser or mass and/or ion mobility
spectrometer; and
a heating device arranged and adapted to heat either: (i) a capillary of said first device; (ii) said stream of electrically charged droplets emitted from said first device; (iii) said target; or (iv) said transfer capillary or transfer device.
97. Apparatus as claimed in claim 96, wherein said first device comprises a Desorption Electrospray lonisation ("DESI") ion source or a desorption electroflow focusing ionisation ("DEFFI") ion source.
98. Apparatus as claimed in claim 96 or 97, wherein said heating device comprises a heater.
99. Apparatus as claimed in claim 98, wherein said heater comprises a wire heater or an inductive heater.
100. Apparatus as claimed in any of claims 96-99, wherein said heating device is arranged and adapted to heat said capillary of said first device, said stream of electrically charged droplets emitted from said first device, said target or said transfer capillary or transfer device to a temperature of at least 50 °C, 100 °C, 200 °C, 300 °C, 400 °C, 500 °C, 600 °C, 700 °C or 800 °C.
101. A mass and/or ion mobility spectrometer comprising apparatus as claimed in any of claims 96-100.
102. A method comprising:
emitting a stream of electrically charged droplets from a first device towards a target;
providing a transfer capillary or transfer device for transferring ions generated from said target towards an ion analyser or mass and/or ion mobility spectrometer; and
heating either: (i) a capillary of said first device; (ii) said stream of electrically charged droplets emitted from said first device; (iii) said target; or (iv) said transfer capillary or transfer device.
103. A method as claimed in claim 102, wherein said first device comprises a Desorption Electrospray lonisation ("DESI") ion source or a desorption electroflow focusing ionisation ("DEFFI") ion source.
104. A method as claimed in claim 102 or 103, wherein said heating device comprises a heater.
105. A method as claimed in claim 104, wherein said heater comprises a wire heater or an inductive heater.
106. A method as claimed in any of claims 102-105, further comprising heating said capillary of said first device, said stream of electrically charged droplets emitted from said first device, said target or said transfer capillary or transfer device to a temperature of at least 50 °C, 100 °C, 200 °C, 300 °C, 400 °C, 500 °C, 600 °C, 700 °C or 800 °C.
107. A method of mass spectrometry and/or ion mobility spectrometry comprising a method as claimed in any of claims 102-106.
108. Apparatus comprising:
an ion source comprising a nozzle having an aperture;
a solvent emitter which extends through the aperture; and
a fixing device which is arranged and adapted to fix the solvent emitter relative to the aperture.
109. Apparatus as claimed in claim 108, wherein the fixing device comprises a centering device which is arranged and adapted to locate or position the solvent emitter substantially centrally within the aperture.
1 10. Apparatus as claimed in claim 108, wherein the fixing device comprises a device which is arranged and adapted to locate or position the solvent emitter substantially non- centrally within the aperture.
1 11. Apparatus as claimed in claim 110, wherein the fixing device comprises a device which is arranged and adapted to locate or position the solvent emitter substantially non- centrally within the aperture, such that the solvent emitter is substantially closer to and/or oriented towards an inlet of a mass and/or ion mobility spectrometer.
1 12. Apparatus as claimed in any of claims 108-1 11 , wherein the fixing device is arranged and adapted such that gas may flow through the nozzle and/or aperture relatively unimpeded by the fixing device.
1 13. Apparatus comprising:
an ion source comprising a nozzle having an aperture;
a solvent emitter which extends through the aperture; and
a device which is arranged and adapted to monitor the solvent emitter position relative to the aperture.
1 14. Apparatus as claimed in claim 113, further comprising a device arranged and adapted to calibrate and/or correct the ion source and/or mass and/or ion mobility spectrometric data acquired using the ion source based on the monitoring.
115. A mass and/or ion mobility spectrometer comprising apparatus as claimed in any of claims 108-114.
116. A method comprising:
providing an ion source comprising a nozzle having an aperture;
providing a solvent emitter which extends through the aperture; and
using a fixing device to fix the solvent emitter relative to the aperture.
117. A method as claimed in claim 116, further comprising using the fixing device to locate or position the solvent emitter substantially centrally within the aperture.
118. A method as claimed in claim 116, further comprising using the fixing device to locate or position the solvent emitter substantially non-centrally within the aperture.
119. A method as claimed in claim 118, further comprising using the fixing device to locate or position the solvent emitter substantially non-centrally within the aperture, such that the solvent emitter is substantially closer to and/or oriented towards an inlet of a mass and/or ion mobility spectrometer.
120. A method as claimed in any of claims 116-119, further comprising passing gas through the nozzle and/or aperture such that the gas flows relatively unimpeded by the fixing device.
121. A method comprising:
providing an ion source comprising a nozzle having an aperture;
providing a solvent emitter which extends through the aperture; and
monitoring the solvent emitter position relative to the aperture.
122. A method as claimed in claim 121 , further comprising calibrating and/or correct the ion source and/or mass and/or ion mobility spectrometric data acquired using the ion source based on the monitoring.
123. A method of mass spectrometry and/or ion mobility spectrometry comprising a method as claimed in any of claims 116-122.
124. A method as claimed in any of claims 1-28, wherein said reversal agent is applied by said first device.
125. Apparatus as claimed in any of claims 37-64., wherein said first device is arranged and adapted to apply said reversal agent.
PCT/GB2016/050624 2015-03-06 2016-03-07 Desorption electrospray ionisation ("desi") imaging WO2016142694A1 (en)

Priority Applications (2)

Application Number Priority Date Filing Date Title
GB1714122.7A GB2551926B (en) 2015-03-06 2016-03-07 Desorption electrospray ionisation ("DESI") imaging
PCT/GB2016/050624 WO2016142694A1 (en) 2015-03-06 2016-03-07 Desorption electrospray ionisation ("desi") imaging

Applications Claiming Priority (19)

Application Number Priority Date Filing Date Title
GB1503877.1 2015-03-06
GB1503864.9 2015-03-06
GB1503863.1 2015-03-06
GB201503876A GB201503876D0 (en) 2015-03-06 2015-03-06 Rapid evaporative ionization mass spectrometry (REIMS) imaging platform for direct mapping from bulk tissue
GBGB1503878.9A GB201503878D0 (en) 2015-03-06 2015-03-06 Liquid separator for endoscopic electrosurgical applications
GB201503867A GB201503867D0 (en) 2015-03-06 2015-03-06 Ionisation of gaseous samles in rapid evaporative ionisation mass spectrometry (REIMS)
GB201503864A GB201503864D0 (en) 2015-03-06 2015-03-06 Heated rapid evaporative ionisation mass spectrometry ("REIMS") collision surface
GB201503863A GB201503863D0 (en) 2015-03-06 2015-03-06 Rapid evaporation ionization mass spectrometry (REIMS) imaging platform for direct mapping from bacteria growth media
GB1503876.3 2015-03-06
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GB201503877A GB201503877D0 (en) 2015-03-06 2015-03-06 In-vivo endoscopic tissue identification tool utilising rapid evaporative ionization mass spectrometry (REIMS)
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GBGB1518369.2A GB201518369D0 (en) 2015-10-16 2015-10-16 Heated rapid evaporative ionisation mass spectrometry ("REIMS") collision surface
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CN107525482A (en) * 2017-07-30 2017-12-29 东北师范大学 A kind of method for determining the hollow herbaceous plant stem wall body product of stem and density
CN107525482B (en) * 2017-07-30 2019-09-27 东北师范大学 A method for measuring stem wall volume and density of hollow herbaceous plants
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US11794200B2 (en) 2020-01-24 2023-10-24 Waters Technologies Corporation Sprayer assembly for DESI applications
CN114264539A (en) * 2020-09-16 2022-04-01 中国烟草总公司郑州烟草研究院 Solvent-assisted thermal desorption apparatus
CN112525980A (en) * 2020-11-26 2021-03-19 厦门大学 Mass spectrum detection method for melamine in dairy products
CN112525980B (en) * 2020-11-26 2022-09-06 厦门大学 Mass spectrometry detection method for melamine in dairy products

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