GB2576078A - Utilisation of mid-infrared lasers for rapid evaporative ionisation mass spectrometry imaging and high throughput sampling - Google Patents
Utilisation of mid-infrared lasers for rapid evaporative ionisation mass spectrometry imaging and high throughput sampling Download PDFInfo
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- H01J49/049—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components with means for heating or cooling the sample with means for applying heat to desorb the sample; Evaporation
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Abstract
Pulses of laser radiation having a wavelength in the range 2700-3100 nm obtained from an optical parametric oscillator laser 1 on to a fixed and embedded tissue sample 2 and generating aerosol, smoke or vapour 4 from the tissue sample 2. The tissue sample is formalin fixed and paraffin embedded (FFPE) prior to analysis. Analyte ions are generated from the aerosol, smoke or vapour 4 and the analyte ions are then mass analysed. An ion imaging map may be obtained by translating the tissue specimen with respect to the laser beam during analysis.
Description
UTILISATION OF MID-INFRARED LASERS FOR RAPID EVAPORATIVE
IONISATION MASS SPECTROMETRY IMAGING AND HIGH THROUGHPUT
SAMPLING
CROSS-REFERENCE TO RELATED APPLICATION
This application claims priority from and the benefit of United Kingdom patent application No. 1808916.9 filed on 31 May 2018. The entire content of this application is incorporated herein by reference.
FIELD OF THE INVENTION
The present invention relates generally to methods of mass spectrometry and mass spectrometers and in particular to imaging samples using Rapid Evaporative Ionisation Mass Spectrometry (“REIMS”).
BACKGROUND
Rapid Evaporative Ionisation Mass Spectrometry (“REIMS”) is a powerful ambient ionization technology that can be used for the rapid classification and identification of complex biological matter based on their mass spectral fingerprint.
Rapid Evaporative Ionisation Mass Spectrometry (“REIMS”) utilises a number of different methods for sample mobilisation. The most common method is ablation using RF electric currents applied to a standard electrosurgical handpiece.
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 such as 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.
-2By 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 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:
directing one or more pulses of laser radiation having a wavelength in the range 2700-3100 nm from a pulsed optical parametric oscillator (ΌΡΟ”) laser on to a fixed and embedded tissue sample and generating aerosol, smoke or vapour from the tissue sample;
generating analyte ions from the aerosol, smoke or vapour; and mass analysing the analyte ions.
The method according to various embodiments is particularly suited to the analysis of fixed or embedded tissue samples and has been shown to produce high quality mass spectral data with higher signal to noise ratio data than other mid-infrared lasers and other REIMS ionisation sources.
The fixed and embedded tissue sample may be fixed in formalin and the tissue sample may be embedded in paraffin. Accordingly, the fixed and embedded tissue sample may comprise a formalin fixed paraffin-embedded (“FFPE”) tissue sample. The fixed and embedded tissue sample comprises an ex vivo tissue sample and the tissue sample may be mounted or otherwise provided on a substrate such as a microscope or other glass slide.
According to various embodiments a laser sampling system is disclosed which is capable both of sampling from well plates and also creating molecular images from ex vivo tissues or tissue slices which may be mounted on slides. In particular, the laser
- 3sampling system according to various embodiments has been found to be particularly effective at generating analyte ions from formalin fixed paraffin-embedded (“FFPE”) tissue samples thereby enabling ion maps or mass spectral imaging of formalin fixed paraffin-embedded (“FFPE”) tissue samples to be obtained.
It will be understood that 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.
The present inventors have found that a pulsed optical parametric oscillator (ΌΡΟ”) laser having a wavelength in the range 2700-3100 nm is particularly effective at accessing analyte ions or generating analyte ions from a fixed and embedded tissue sample. In particular, the present inventors have found that it is possible to obtain strong analyte ion signals of interest from a formalin fixed paraffin-embedded (“FFPE”) tissue sample using a pulsed optical parametric oscillator (ΌΡΟ”) laser having a wavelength in the range 2700-3100 nm without needing to pre-process the tissue sample and without needing to apply a reversal agent to the formalin fixed paraffinembedded (“FFPE”) tissue sample. The pulsed optical parametric oscillator (ΌΡΟ”) laser may be Q-switched and may have a pulse length of 1-10 ns, optionally 3-7 ns.
The ability to analyse formalin fixed paraffin-embedded (“FFPE”) tissue samples without needing to pre-process the tissue sample by applying a reversal agent to the tissue sample enables current and historic formalin fixed paraffin-embedded (“FFPE”) tissue samples to be analysed in a simple and effective manner which is particularly beneficial.
The method of laser sampling which is disclosed according to various embodiments avoids any need to physically contact the sampled surface. The spatial resolution of the laser beam emitted from the laser may be easily and precisely defined which is beneficial for generating an ion map of the tissue sample. Furthermore, the system enables proper or precise focusing to be achieved. Accordingly, the method which is disclosed according to various embodiments is particularly suitable as a tissue imaging tool.
Formalin fixed paraffin-embedded (“FFPE”) tissue samples which may be analysed according to various embodiments may comprise a tissue sample which has first been 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 have then been embedded in an embedding medium which may comprise paraffin. However, other embodiments are contemplated wherein the fixed tissue sample may be embedded in one or more plastic polymers, a
-4polyester wax, polyethylene glycol (“PEG”), polyfin (RTM), a tissue freezing medium (“TFM”), glycol methacrylate (“GMA”), gelatin or carboxy methyl cellulose (“SMC”).
The fixed and embedded tissue sample may result in the formation of methylene bridge cross-linkages being formed between proteins.
The fixed and embedded tissue sample may comprise a tissue sample which has been dehydrated with an alcohol.
The fixed and embedded tissue sample may comprise a tissue sample which has been infiltrated with molten paraffin.
The fixed and embedded tissue sample may be provided on a substrate which may comprise a microscope slide, a transparent slide, a glass slide, a plastic slide or an indium-tin-oxide (ITO) coated glass or other slide.
According to various embodiments the fixed and embedded tissue sample is not pre-processed prior to the step of directing one or more pulses of laser radiation on to the tissue sample. In particular, according to various embodiments the fixed and embedded tissue sample is not subjected to the application of a reversal agent prior to the step of directing one or more pulses of laser radiation on to the tissue sample. Accordingly, the method according to various embodiments is particularly beneficial.
It is known to apply a reversal agent to a formalin fixed paraffin embedded (“FFPE”) tissue sample in order to reverse cross-linked bonds of proteins present in the tissue sample. The reversal agent may, for example, comprise a formalin or formaldehyde scavenger. However, avoiding the need to use a reversal agent is beneficial.
According to various embodiments the method may further comprise adding a matrix solvent to the aerosol, smoke or vapour to form a mixture.
The matrix solvent may comprise an organic solvent such as 2-propanol (isopropanol).
The step of generating analyte ions from the aerosol, smoke or vapour may comprise causing the mixture to impact upon a heated collision surface located within a vacuum chamber of a mass spectrometer.
The addition of a matrix solvent to the aerosol, smoke or vapour together with directing the resulting mixture onto a heated collision surface maintained at subatmospheric pressure within a vacuum chamber of the mass spectrometer has been found to result in a significant enhancement in the intensity of analyte ions which are generated from the aerosol, smoke or vapour.
The tissue sample may be translated relative to the laser multiple times. Mass spectral data may be obtained relating to different regions of the tissue sample and an
-5ion map or mass spectral image may be generated of at least a portion of the tissue sample.
The generation of an ion map or mass spectral image enables a human or software to determine regions of interest of the tissue sample. For example, regions of the tissue sample which relate to healthy or normal tissue can be identified and regions of the tissue sample which relate to diseased or tumorous tissue can also be identified.
According to various embodiments an ion map or mass spectral image may be generated of at least a portion of the tissue sample over a range 600-800 m/z corresponding to phospholipids.
This region is of particular interest for identifying ion peaks which may be indicative of tumorous tissue. For example, ion peaks having a m/z of 744 may be indicative of tumorous tissue.
According to various embodiments an ion map or mass spectral image may be generated of at least a portion of the tissue sample over a range 890-900 m/z corresponding to triglycerides.
This region is of particular interest for identifying ion peaks which may be indicative of normal or healthy tissue. For example, ion peaks having a m/z of 893 may be indicative of normal (fatty) tissue.
According to various embodiments the one or more pulses of laser radiation may have a pulse width or pulse length in the range 1-10 ns, optionally 3-7 ns.
According to various embodiments the pulsed laser may comprise a 2940 nm Q-switched optical parametric oscillator laser. Other embodiments are also contemplated wherein the pulsed laser may comprise a Q-switched optical parametric oscillator laser having a wavelength more generally in the range 2700-3100 nm.
According to another aspect there is provided a method of ion mapping or mass spectral imaging comprising a method as described above.
According to another aspect there is provided a method of mass spectrometry comprising a method as described above.
According to another aspect there is provided a mass spectrometer comprising: a pulsed optical parametric oscillator (ΌΡΟ”) laser arranged and adapted to emit, in use, one or more pulses of laser radiation having a wavelength in the range 2700-3100 nm which are directed on to a fixed and embedded tissue sample so as to generate, in use, aerosol, smoke or vapour from the tissue sample;
a translation device for translating the tissue sample relative to the laser;
a device arranged and adapted to generate analyte ions from the aerosol, smoke or vapour; and a mass analyser for mass analysing the analyte ions.
-6The mass spectrometer according to various embodiments is particularly suited to the analysis of fixed or embedded tissue samples and has been shown to produce high quality mass spectral data with higher signal to noise ratio data than other midinfrared lasers or other REIMS ionisation sources.
The fixed and embedded tissue sample may comprise a formalin fixed paraffinembedded (“FFPE”) tissue sample. According to various embodiments the fixed and embedded tissue sample is not pre-processed prior to directing one or more pulses of laser radiation on to the tissue sample. In particular, the fixed and embedded tissue sample is not subjected to the application of a reversal agent prior to directing one or more pulses of laser radiation on to the tissue sample. Accordingly, the mass spectrometer according to various embodiments is particularly beneficial.
The mass spectrometer may further comprise a device arranged and adapted to add a matrix solvent to the aerosol, smoke or vapour to form a mixture.
The matrix solvent may comprise an organic solvent such as 2-propanol (isopropanol).
The device arranged and adapted to generate analyte ions from the aerosol, smoke or vapour may comprise a heated collision surface located within a vacuum chamber of the mass spectrometer and wherein, in use, the mixture is caused to impact the heated collision surface.
The addition of a matrix solvent to the aerosol, smoke or vapour together with directing the resulting mixture onto a heated collision surface maintained at subatmospheric pressure within a vacuum chamber of the mass spectrometer has been found to result in a significant enhancement in the generation of analyte ions.
The translation device may be arranged and adapted to translate the tissue sample relative to the laser multiple times. The mass spectrometer may be arranged and adapted to obtain mass spectral data relating to different regions of the tissue sample and the mass spectrometer may be arranged and adapted to generate an ion map or mass spectral image of at least a portion of the tissue sample.
The generation of an ion map or mass spectral image enables a human or software to determine regions of interest of the tissue sample. For example, regions of the tissue sample which relate to healthy or normal tissue can be identified and regions of the tissue sample which relate to diseased or tumorous tissue can also be identified.
According to various embodiments the one or more pulses of laser radiation may have a pulse width or pulse length in the range 1-10 ns, optionally 3-7 ns.
The mass spectrometer may comprise one or more ion guides comprising a plurality of electrodes.
The mass spectrometer may comprise a device arranged and adapted to supply
- 7an AC or RF voltage to the electrodes. The AC or RF voltage optionally has an amplitude selected from the group consisting of: (i) < 50 V peak to peak; (ii) 50-100 V peak to peak; (iii) 100-150 V peak to peak; (iv) 150-200 V peak to peak; (v) 200-250 V peak to peak; (vi) 250-300 V peak to peak; (vii) 300-350 V peak to peak; (viii) 350-400 V peak to peak; (ix) 400-450 V peak to peak; (x) 450-500 V peak to peak; and (xi) > 500 V peak to peak.
The AC or RF voltage may have a frequency selected from the group consisting of: (i) < 100 kHz; (ii) 100-200 kHz; (iii) 200-300 kHz; (iv) 300-400 kHz; (v) 400-500 kHz; (vi) 0.5-1.0 MHz; (vii) 1.0-1.5 MHz; (viii) 1.5-2.0 MHz; (ix) 2.0-2.5 MHz; (x) 2.5-3.0 MHz; (xi) 3.0-3.5 MHz; (xii) 3.5-4.0 MHz; (xiii) 4.0-4.5 MHz; (xiv) 4.5-5.0 MHz; (xv) 5.0-5.5 MHz; (xvi) 5.5-6.0 MHz; (xvii) 6.0-6.5 MHz; (xviii) 6.5-7.0 MHz; (xix) 7.0-7.5 MHz; (xx) 7.5-8.0 MHz; (xxi) 8.0-8.5 MHz; (xxii) 8.5-9.0 MHz; (xxiii) 9.0-9.5 MHz; (xxiv) 9.5-10.0 MHz; and (xxv) > 10.0 MHz.
The mass spectrometer may comprise one or more ion mobility separation devices and/or one or more Field Asymmetric Ion Mobility Spectrometer devices.
The mass spectrometer may comprise one or more ion traps or one or more ion trapping regions.
The mass spectrometer may comprise one or more collision, fragmentation or reaction cells selected from the group consisting of: (i) a Collisional Induced Dissociation (CID) fragmentation device; (ii) a Surface Induced Dissociation (SID) fragmentation device; (iii) an Electron Transfer Dissociation (ETD) fragmentation device; (iv) an Electron Capture Dissociation (ECD) fragmentation device; (v) an Electron Collision or Impact Dissociation fragmentation device; (vi) a Photo Induced Dissociation (PID) fragmentation device; (vii) a Laser Induced Dissociation fragmentation device; (viii) an infrared radiation induced dissociation device; (ix) an ultraviolet radiation induced dissociation device; (x) a nozzle-skimmer interface fragmentation device; (xi) an in-source fragmentation device; (xii) an in-source Collision Induced Dissociation fragmentation device; (xiii) a thermal or temperature source fragmentation device; (xiv) an electric field induced fragmentation device; (xv) a magnetic field induced fragmentation device; (xvi) an enzyme digestion or enzyme degradation fragmentation device; (xvii) an ion-ion reaction fragmentation device; (xviii) an ion-molecule reaction fragmentation device; (xix) an ion-atom reaction fragmentation device; (xx) an ion-metastable ion reaction fragmentation device; (xxi) an ion-metastable molecule reaction fragmentation device; (xxii) an ion-metastable atom reaction fragmentation device; (xxiii) an ion-ion reaction device for reacting ions to form adduct or product ions; (xxiv) an ion-molecule reaction device for reacting ions to form adduct or product ions; (xxv) an ion-atom reaction device for reacting ions to form adduct or
- 8product ions; (xxvi) an ion-metastable ion reaction device for reacting ions to form adduct or product ions; (xxvii) an ion-metastable molecule reaction device for reacting ions to form adduct or product ions; (xxviii) an ion-metastable atom reaction device for reacting ions to form adduct or product ions; and (xxix) an Electron Ionisation
Dissociation (“EID”) fragmentation device.
The ion-molecule reaction device may be configured to perform ozonolysis for the location of olefinic (double) bonds in lipids.
The mass spectrometer may comprise a mass analyser selected from the group consisting of: (i) a quadrupole mass analyser; (ii) a 2D or linear quadrupole mass analyser; (iii) a Paul or 3D quadrupole mass analyser; (iv) a Penning trap mass analyser; (v) an ion trap mass analyser; (vi) a magnetic sector mass analyser; (vii) Ion Cyclotron Resonance (ICR) mass analyser; (viii) a Fourier Transform Ion Cyclotron Resonance (FTICR) mass analyser; (ix) an electrostatic mass analyser arranged to generate an electrostatic field having a quadro-logarithmic potential distribution; (x) a Fourier Transform electrostatic mass analyser; (xi) a Fourier Transform mass analyser; (xii) a Time of Flight mass analyser; (xiii) an orthogonal acceleration Time of Flight mass analyser; and (xiv) a linear acceleration Time of Flight mass analyser.
The mass spectrometer may comprise one or more energy analysers or electrostatic energy analysers.
The mass spectrometer may comprise one or more ion detectors.
The mass spectrometer may comprise one or more mass filters selected from the group consisting of: (i) a quadrupole mass filter; (ii) a 2D or linear quadrupole ion trap; (iii) a Paul or 3D quadrupole ion trap; (iv) a Penning ion trap; (v) an ion trap; (vi) a magnetic sector mass filter; (vii) a Time of Flight mass filter; and (viii) a Wien filter.
The ion guide may be maintained at a pressure selected from the group consisting of: (i) < 0.0001 mbar; (ii) 0.0001-0.001 mbar; (iii) 0.001-0.01 mbar; (iv) 0.010.1 mbar; (v) 0.1-1 mbar; (vi) 1-10 mbar; (vii) 10-100 mbar; (viii) 100-1000 mbar; and (ix) > 1000 mbar.
Analyte ions may be subjected to Electron Transfer Dissociation (“ETD”) fragmentation in an Electron Transfer Dissociation fragmentation device. Analyte ions may be caused to interact with ETD reagent ions within an ion guide or fragmentation device.
The mass spectrometer may be operated in various modes of operation including a mass spectrometry (MS) mode of operation; a tandem mass spectrometry (MS/MS) mode of operation; a mode of operation in which parent or precursor ions are alternatively fragmented or reacted so as to produce fragment or product ions, and not fragmented or reacted or fragmented or reacted to a lesser degree; a Multiple Reaction
- 9Monitoring (MRM) mode of operation; a Data Dependent Analysis (DDA) mode of operation; a Data Independent Analysis (DIA) mode of operation; a Quantification mode of operation; or an Ion Mobility Spectrometry (IMS) mode of operation.
The electrodes of the ion guide may comprise electrodes which are formed on a printed circuit board, printed wiring board or an etched wiring board. For example, according to various embodiments the electrodes may comprise a plurality of traces applied or laminated onto a non-conductive substrate. The electrodes may be provided as a plurality of copper or metallic electrodes arranged on a substrate. The electrodes may be screen printed, photoengraved, etched or milled onto a printed circuit board or equivalent. According to an embodiment the electrodes may comprise electrodes arranged on a paper substrate impregnated with phenolic resin or a plurality of electrodes arranged on a fibreglass mat impregnated within an epoxy resin. More generally, the electrodes may comprise one or more electrodes arranged on a nonconducting substrate, an insulating substrate or a plastic substrate. According to embodiments the plurality of electrodes may be arranged on a substrate.
A plurality of insulator layers may be interspersed or interleaved between an array of electrodes. The plurality of electrodes may be arranged on or deposited on one or more insulator layers.
BRIEF DESCRIPTION OF THE DRAWINGS
Various embodiments together will other arrangements given for illustrative purposes will now be described, by way of example only, and with reference to the accompanying drawings in which:
Fig. 1 shows a laser REIMS sampling method according to various embodiments wherein laser radiation from a pulsed laser source is directed on to a tissue sample provided on a translation stage;
Fig. 2 shows an illustrative arrangement wherein an optical parametric oscillator (ΌΡΟ”) laser was used to generate aerosol, smoke or vapour from sample wells of a 96-well sample plate and wherein a translation stage was used to translate the 96-well sample plate relative to the laser;
Fig. 3 shows mass spectra relating to different tissue samples obtained using a first midrange infrared laser comprising an optical parametric oscillator (OPO) according to various embodiments and mass spectra relating to different tissue samples obtained using a second midrange infrared laser comprising an Er:YAG laser, wherein both lasers were pulsed but had different pulse lengths;
Fig. 4 shows mass spectra obtained using a third midrange infrared laser
- 10comprising a CO2 laser having a wavelength of 10600 nm and wherein the results may be compared with mass spectra obtained using a first midrange laser comprising an optical parametric oscillator (ΌΡΟ”) laser according to various embodiments;
Fig. 5 shows a visual image together with two 200 pm resolution mass spectral images of a porcine kidney slice wherein the two mass spectral images were obtained using the second midrange infrared laser comprising an Er:YAG laser having a wavelength of 2940 nm and wherein the first mass spectral image shows the spatial distribution of analyte ions having a mass to charge ratio of 722.55 m/z and the second mass spectral image shows the spatial distribution of analyte ions having a mass to charge ratio of 893.75 m/z which corresponds with fatty tissues comprising triglycerides;
Fig. 6 shows two 250 pm resolution ion images of a human breast tissue sample which comprises a region of healthy tissue and a region of tumorous tissue which were obtained using the first midrange infrared laser comprising an optical parametric oscillator (ΌΡΟ”) laser according to various embodiments, wherein an image of an H&E (haematoxylin and eosin) stained slide is also shown, wherein the left hand side of the tissue sample comprises healthy tissue as confirmed by the first ion map image at 893.73 m/z and wherein the right hand side of the tissue sample comprises tumorous breast tissue as confirmed by the second ion map image at 744.55 m/z wherein analyte ions having a mass to charge ratio of 744.45 are characteristic of tumorous breast tissue;
Fig. 7 shows as a comparative example less pronounced mass spectral data which was obtained using the third midrange infrared laser comprising a CO2 laser which utilised a 200 pm I.D. hollow core fibre which enabled the laser to be focused to a smaller spot size, wherein a first 50 pm resolution mass spectral ion image at 744.40 m/z of another human breast tissue sample is shown together with an associated first mass spectrum, wherein the human breast tissue sample comprises both a region of healthy or normal tissue and a region of tumorous tissue and wherein a major ion peak at 744.40 m/z is apparent in the first mass spectrum and wherein a second 50 pm resolution mass spectral ion image at 893.45 m/z is shown together with an associated second mass spectrum, wherein the second ion image relates to normal or healthy tissue as evidenced by the presence of a major ion peak at 893.45 m/z in the second mass spectrum which is indicative of normal or healthy (fatty) tissue;
Fig. 8A shows an image of a formalin-fixed paraffin embedded colorectal tissue sample which was analysed according to various embodiments using the first midrange infrared laser comprising an optical parametric oscillator (OPO) laser, Fig. 8B shows a molecular image or ion map at 701.51 m/z of the formalin-fixed paraffin embedded colorectal tissue sample which was acquired using the first midrange infrared laser
- 11 comprising an optical parametric oscillator (OPO) laser according to various embodiments and Fig. 8C shows a molecular image or ion map at 374.11 m/z of the formalin-fixed paraffin embedded colorectal tissue sample which was acquired using the first midrange infrared laser comprising an optical parametric oscillator (OPO) laser according to various embodiments; and
Fig. 9A shows for illustrative purposes a 96-well sample well containing samples of yeast and yoghurt cultures which were used to obtain cell line sampling data, Fig. 9B shows a total ion current (TIC”) of the measurements which were obtained as a function of time wherein 96 data points were acquired within seven minutes and Fig. 9C shows a PCA model of the binary yeast-yoghurt sample, wherein it is apparent that there is a clear separation between the two spectral groups.
DETAILED DESCRIPTION
A laser Rapid Evaporative Ionisation Mass Spectrometry (“REIMS”) sampling system which was utilised according to various embodiments will now be described in more detail with reference to Fig. 1.
Fig. 1 shows an overall laser REIMS system according to various embodiments and comprises a mid-infrared laser 1. In particular, according to various embodiments the mid-infrared laser 1 comprises an optical parametric oscillator (“OPO”) laser having a wavelength in the range of 2700-3100 nm. The wavelength of laser radiation emitted by the optical parametric oscillator (“OPO”) laser according to various embodiments may be tuned or other selected within the wavelength range of 2700-3100 nm.
As shown in Fig. 1, the pulsed laser 1 may, for illustrative purposes only, be considered to be operated with a frequency of 10 Hz and at a laser output of 200 mW. However, other embodiments are contemplated wherein the frequency of operation may be higher or lower than 10 Hz. Furthermore, according to other embodiments the intensity of the laser output may be set higher or lower than 200 mW.
According to various embodiments the frequency of the optical parametric oscillator (“OPO”) laser 1 may be set at < 1 Hz, 1-10 Hz, 10-100 Hz or > 100 Hz. The intensity of the laser output of the optical parametric oscillator (“OPO”) laser 1 may be set at an intensity <10 mW, 10-50 mW, 50-100 mW, 100-150 mW, 150-200 mW, 200250 mW or > 250 mW.
According to various embodiments the pulsed laser 1 may comprise a 2940 nm Q-switched optical parametric oscillator laser. Other embodiments are contemplated wherein the pulsed laser 1 may comprise a Q-switched optical parametric oscillator laser having a wavelength more generally in the range 2700-3100 nm.
- 12As will be discussed in more detail below, according to various embodiments the pulsed laser 1 may comprise an optical parametric oscillator (ΌΡΟ”) laser 1 having an maximum energy output < 200 mW, optionally <100 mW. According to an embodiment the output power may be in the range of 35-90 mW. The optical parametric oscillator (ΌΡΟ”) laser 1 according to various embodiments may have a pulse width or pulse length in the range 1-10 ns, particularly 3-7 ns and may have a pulse frequency of, for example, 20 Hz. Other embodiments are contemplated wherein the optical parametric oscillator (ΌΡΟ”) laser 1 has a frequency higher or lower than 20 Hz. The laser beam output by the optical parametric oscillator (ΌΡΟ”) laser 1 may have a beam diameter of approximately 4 mm.
The laser 1 may be in optical communication with a laser fibre optic or other optical transmission device 1a which may be arranged to transmit light or laser radiation emitted from the laser 1 towards a sample 2. The sample 2 may comprise a tissue sample which may be provided on a substrate such as a microscope or glass slide. The sample and optional substrate may be located on or mounted upon a translation stage 3. The translation stage 3 may comprise an XY horizontal translation stage 3 which may be translatable in two orthogonal horizontal directions.
According to various embodiments the sample 2 may comprise a fixed and embedded ex vivo tissue sample. For example, the tissue sample may be fixed in formalin and the tissue sample may further be embedded in paraffin. Accordingly, the fixed and embedded tissue sample 2 may comprise a formalin fixed paraffin-embedded (“FFPE”) tissue sample.
The sample or tissue sample 2 may be moved or translated under or relative to a laser spot or laser beam which emerges either direct from the laser 1 or from a laser fibre or fibre optic 1a which is arranged to transmit the laser beam and to direct pulses of laser radiation on to the surface of the sample or tissue sample 2. The laser fibre or fibre optic 1a may alter the diameter of the laser beam which is emitted direct from the laser 1. For example, the laser fibre or fibre optic 1a may be arranged to reduce the diameter of the laser beam which impinges upon the sample or tissue sample 2 to approximately 250 pm or smaller. A focusing lens or other optical component may be provided to focus the laser radiation to a focal point and small spot size. The sample or tissue sample 2 may be translated or otherwise moved by the XY translation stage 3 thereby allowing sampling of different regions of interest of the sample or tissue sample 2. Accordingly, an ion map or mass spectral image of at least a portion or substantially the whole of the tissue sample 2 may be obtained.
- 13It will be understood that other embodiments are also contemplated wherein the sample or tissue sample 2 may remain essentially static and instead the laser radiation may be moved or scanned across the sample or tissue sample 2.
Pulsed laser radiation from the laser 1 is directed onto the sample or tissue sample 2 with the result that when laser radiation impinges upon the sample or tissue sample 2 then aerosol, smoke or vapour 4 will be generated by the laser 1 as material is ablated from the surface of the sample 2.
The resulting aerosol, smoke or vapour 4 may be aspirated through or via a PTFE tube, tubing or other transfer device 5 into a REIMS atmospheric interface 6. As shown in more detail in Fig. 1, the PTFE tube, tubing or other transfer device 5 may be coupled to or otherwise comprise an aerosol transfer capillary 7. Aerosol, smoke or vapour 4 which is transmitted through the PTFE tube, tubing or other transfer device 5 and/or through the aerosol transfer capillary 7 is then mixed with a matrix solvent such as 2-propanol (isopropanol).
According to an embodiment the aerosol, smoke or vapour 4 may be mixed with matrix solvent via a T-connection or other mixing device 9. Matrix solvent such as 2propanol (isopropanol) may be supplied to the T-connection or other mixing device 9 via a matrix transfer capillary or matrix transfer device 8.
The mixture of aerosol, smoke or vapour 4 together with matrix solvent may then be arranged to pass via an input capillary or other transfer device 10 into the REIMS atmospheric interface 6. The REIMS atmospheric interface 6 may comprise a housing which forms an initial vacuum chamber of a mass spectrometer 14. The mixture of aerosol, smoke or vapour 4 and matrix solvent may therefore be introduced into a vacuum space or first vacuum chamber of a mass spectrometer 14. In the vacuum chamber of the mass spectrometer 14 the matrix doped sample droplets may be arranged to hit or collide with a heated collision surface 11 whereupon free ions or analyte ions may be liberated and then analysed by a mass analyser provided in a downstream region of the mass spectrometer 14.
According to an embodiment ions or analyte ions which are liberated by the heated collision surface 11 may be transmitted via a StepWave (RTM) ion guide 12. The ion guide 12 may comprise an initial ion guide comprising a plurality of ring, annular or conjoined electrodes wherein ions may be transmitted under the influence of an electric field from a first ion guide path into a second adjacent ion guide path. However, background molecules or other neutral particles are not transferred from the first ion guide path into the second ion guide path and are lost to the system. Accordingly, the ion guide 12 may be provided to help separate analyte ions (which are onwardly
- 14transmitted to a mass analyser as identified by arrow 13) from unwanted neutral particles (which are lost to the system).
The ion guide 12 may be used to confine and optionally focus analyte ions. For example, according to an embodiment the ion guide 12 may comprises a plurality of ring, annular or conjoined electrodes which have a progressively smaller or decreasing internal diameter along the optic axis. Accordingly, as analyte ions pass through the plurality of ring, annular or conjoined electrodes having decreasing internal radius then the analyte ions may be focussed so as to pass through a subsequent differential pumping aperture (not shown).
Experimental data as presented below was acquired using a Waters Xevo G2XS QToF (RTM) mass spectrometer 14. All mass spectra were acquired in negative ion mode.
Two different XY translation stages 3 were tested. A first XY translation stage 3 was constructed from a Thorlabs NRT (RTM) translation stage and a second XY translation stage 3 was constructed from a Newport MFA-CC (RTM) translation stage. All of the laser optics parts were acquired from Thorlabs (RTM) or were custom built.
Three different midrange infrared red (“IR”) lasers were tested and compared.
A first midrange infrared laser was tested which comprised an Opolette HE 2731 optical parametric oscillator (ΌΡΟ”) laser. The wavelength of the first midrange infrared laser was tunable between 2700 and 3100 nm. The first midrange infrared laser had a pulse width of 3 ns and the energy output could be set in the range 35-90 mW. The pulse width or pulse length could be set more generally between 1-10 ns, and more particularly between 3-7 ns and the optical parametric oscillator (ΌΡΟ”) laser 1 was operated with a frequency of 20 Hz. However, the optical parametric oscillator (ΌΡΟ”) laser 1 may be operated at different frequencies and with different pulse lengths or pulse widths.
The laser beam or pulse output from the optical parametric oscillator (ΌΡΟ”) laser 1 had a diameter of 4 mm. The laser beam or pulse diameter could be reduced by passing the laser beam or pulse through an optical fibre 1a and optionally by using a focusing lens.
A second midrange infrared laser was tested comprising a Medley MF3003 (RTM) Er:YAG dermatological laser. The second midrange laser had a wavelength of 2940 nm and a pulse width of 200 ps. The energy output of the second midrange infrared laser was 150 mW. The second midrange infrared laser had a pulse frequency of 10 Hz.
A third midrange infrared laser was tested comprising an OmniGuide (RTM) CO2 laser. The third midrange infrared laser had a longer wavelength of 10600 nm. The
- 15third midrange infrared laser had a pulsed output with a pulse width of 20 ms. The energy output of the third midrange infrared laser was 40 mW.
As will be discussed in more detail below, the first midrange infrared laser comprising an optical parametric oscillator (ΌΡΟ”) laser having an output wavelength in the range 2700-3100 nm was found to be particularly beneficial for analysing fixed and embedded tissue samples, particularly formalin fixed paraffin-embedded (“FFPE”) ex vivo tissue samples.
Accordingly, data obtained using the second midrange infrared laser comprising an Er:YAG laser having a wavelength of 2940 nm and a pulse width of 200 ps and the third midrange infrared laser comprising a CO2 laser having a longer wavelength of 10600 nm and a pulse width of 20 ms is provided for illustrative purposes only and provides comparative data which illustrates that the highest quality mass spectral data which was obtained was obtained by using the first midrange infrared laser comprising an optical parametric oscillator (ΌΡΟ”) laser according to various embodiments.
Fig. 2 shows an XY translation stage 3 arranged below the first midrange infrared laser 1 comprising an optical parametric oscillator (ΌΡΟ”) laser 1 according to various embodiments. For illustrative purposes only, the optical parametric oscillator (ΌΡΟ”) laser 1 according to various embodiments is shown positioned so as to direct pulsed laser radiation into a 96-well deep well sample plate 15 which was arranged on the XY translation stage. It will be understood that the 96-well deep well sample plate 15 and the laser 1 can be modified to allow different experimental geometries.
The laser pulses output from the laser 1 may be reflected by a mirror and may pass through a focusing lens 1b or other beam optics so as to focus the laser pulses either into a sample plate 15 as shown in Fig. 2 or according to various embodiments onto a tissue sample 2 which may be provided on a substrate. In particular, the laser pulses output from the laser 1 may be directed so as to impinge upon one or more fixed and embedded tissue samples, particularly one or more formalin fixed paraffinembedded (“FFPE”) ex vivo tissue samples.
The effect of different laser pulse widths was studied. In particular, as will be discussed in more detail below, an optical parametric oscillator (ΌΡΟ”) laser 1 as may be used according to various embodiments was compared with the second midrange infrared laser which comprised an Er:YAG laser.
The first midrange infrared laser 1 which may be used according to various embodiments and which comprises an optical parametric oscillator (ΌΡΟ”) laser 1 and the second midrange infrared laser comprising an Er:YAG laser were both operated at the same wavelength of 2940 nm. It will be recalled that the optical parametric oscillator (ΌΡΟ”) laser 1 according to various embodiments is tunable between 2700-3100 nm.
- 16According to various embodiments the first midrange infrared laser comprising an optical parametric oscillator (ΌΡΟ”) laser 1 may be arranged to have a pulse width in the range 1-10 ns, more particularly in the range 3-7 ns.
Although the two lasers were operated at the same wavelength, the first and second midrange infrared lasers were operated so as to have different pulse widths. The first midrange infrared laser comprising an optical parametric oscillator (ΌΡΟ”) laser 1 according to various embodiments was set to have a short pulse width of 3 ns whereas the second midrange infrared laser comprising an Er:YAG laser had a longer pulse width of 200 ps.
The second midrange infrared laser comprising an Er:YAG laser was operated at its standard operating wavelength of 2940 nm and the wavelength of the first midrange infrared laser comprising an optical parametric oscillator (ΌΡΟ”) laser 1 according to various embodiments was tuned to the same wavelength as the second midrange laser namely to a wavelength of 2940 nm.
The first midrange infrared laser was operated in mode of operation wherein a single laser pulse had a pulse length of approximately 3 ns whilst a pulse from the second midrange laser comprising an Er:YAG laser had a longer pulse length of approximately 200 ps. Both laser output powers were set to 100 mW.
Experiments were then performed on porcine kidney tissue samples and human breast tissue samples. The food grade test samples were acquired from local stores whereas the human breast tissue samples were measured at Imperial College, London. Food grade kidney tissue (cortex, medulla and fatty tissue) was sampled using the two pulsed lasers having the same wavelength but different pulse lengths and the experimental results from using the two different lasers were compared.
Fig. 3 shows a comparison of the effect of different pulse widths on the species present in mass range 600-900 m/z of samples of kidney medulla, kidney fatty tissue and kidney cortex. In this mass range of 600-900 m/z the mass spectra mostly comprises mass peaks which correspond with phospholipids and triglycerides originating from cell membranes.
The first midrange infrared laser comprising an optical parametric oscillator (ΌΡΟ”) laser 1 according to various embodiments and which had a short pulse width of 3 ns (but which more generally may have a pulse width in the range 1-10 ns or more particularly 3-7 ns) is able to mobilise phospholipid and triglyceride molecules more efficiently and with a better signal to noise ratio compared with the second midrange infrared laser comprising an Er:YAG laser having the same wavelength and power output but a longer pulse width. It is noted that the second midrange infrared laser
- 17comprising an Er:YAG laser does have good sensitivity with respect to triglycerides within fatty tissue in the mass to charge ratio range 890-900 m/z.
However, overall an optical parametric oscillator (“OPO”) laser 1 according to various embodiments had a better overall performance and a higher signal to noise ratio across the mass range 600-900 m/z.
Although the above experiments were performed on tissue samples which were neither fixed nor embedded, the above experimental results nonetheless illustrate that there are various benefits in using an optical parametric oscillator (“OPO”) laser 1 having a wavelength in the range 2700-3100 nm to analyse tissue samples compared to other midrange infrared laser sources. These same benefits were also found to apply to the analysis of fixed and embedded tissue samples according to various embodiments.
Further illustrative and comparative data is presented below in relation to Fig. 4 which illustrates the effect of using different wavelength mid-infrared lasers. Experimental data is shown in Fig. 4 relating to using the first midrange laser comprising an optical parametric oscillator (“OPO”) laser 1 having a wavelength in the range 27003100 nm according to various embodiments and the third midrange infrared laser comprising a CO2 laser having a wavelength of 10600 nm which was tested for comparative purposes.
Fig. 4 shows mass spectra which were obtained using the third midrange infrared laser comprising a CO2 laser having a wavelength of 10600 nm and the first midrange laser comprising an optical parametric oscillator (“OPO”) laser 1 according to various embodiments which was tuned to a wavelength of 2940 nm.
It is apparent from Fig. 4 that use of the third midrange infrared laser comprising a CO2 laser having the longest wavelength of 10600 nm resulted in mass spectral data having a relatively low or relatively poor signal to noise ratio.
By comparison, use of the first midrange infrared laser comprising an optical parametric oscillator (“OPO”) laser 1 having a wavelength in the range 2700-3100 nm results in mass spectral data which has a higher or otherwise improved signal to noise ratio relative to the use of a CO2 laser having a wavelength of 10600 nm. Furthermore, use of the first midrange infrared laser comprising an optical parametric oscillator (“OPO”) laser 1 having a wavelength in the range 2700-3100 nm according to various embodiments results in mass spectral data which has a higher information content particularly in the phospholipid region corresponding to ion peaks between 600-800 m/z. The infrared absorption maximum for water is at 2940 nm and hence the use an optical parametric oscillator (“OPO”) laser 1 at a wavelength in the range 2700-3100 nm according to various embodiments may be better utilised for sample mobilisation in certain contexts.
- 18Although the experiments as discussed above in relation to Fig. 4 were performed on tissue samples which were neither fixed nor embedded, the above experimental results nonetheless illustrate that there are various benefits in using an optical parametric oscillator (ΌΡΟ”) laser 1 having a wavelength in the range 27003100 nm to analyse tissue samples compared to other midrange infrared laser sources. These same benefits were also found to apply to the analysis of fixed and embedded tissue samples according to various embodiments.
Fig. 5 shows an image and two 200 pm resolution mass spectral images of a kidney slice obtained using the second midrange infrared laser comprising an Er:YAG laser having a wavelength of 2940 nm. The first mass spectral image or ion map relates to 722.55 m/z and the second mass spectral image or ion map relates to 893.75 m/z. Some fatty tissues triglyceride content is visible in the second mass spectral image or ion map at 893.75 m/z.
Beside classic optical and staining based methods, molecular tissue imaging and histopathological characterisation is getting more attention in clinical diagnostics.
Like other ambient ionization tools (DESI, MALDI), laser REIMS is also capable of being utilized as an imaging tool. All three lasers were tested using different tissues for this purpose.
However, as will be discussed in more detail, better experimental data and improved mass spectral data having a higher signal to noise ratio was obtained from analysing tissue samples, both unfixed and fixed/embedded, using an optical parametric oscillator (“OPO”) laser 1 having a wavelength in the range 2700-3100 nm according to various embodiments.
It will be understood that laser REIMS imaging is particularly beneficial compared to Matrix Assisted Laser Desorption Ionisation (“MALDI”) imaging owing to the fact that ion maps and ion images can be obtained without any need for a matrix coating to be applied on to the tissue surface.
Fig. 6 shows two 250 pm resolution mass spectral images of human breast tumour tissue obtained using the first midrange infrared laser comprising an optical parametric oscillator (“OPO”) laser 1 according to various embodiments.
The human breast tissue sample comprises a region of healthy tissue and also a region of tumour tissue.
An optical image of an H&E (haematoxylin and eosin) stained slide is also shown in Fig. 6 and it will be understood that the left hand side region of the tissue sample comprises healthy tissue. This is confirmed by the first ion map image at 893.73 m/z.
- 19The right hand side of the tissue sample comprises tumorous breast tissue and this is confirmed by the second ion map image at 744.55 m/z which shows characteristic ion peaks at 744.55 m/z which is indicative of tumorous breast tissue.
It will be appreciated that high quality ion maps and mass spectral data as shown and described in relation to Fig. 6 were obtained using an optical parametric oscillator (ΌΡΟ”) laser 1 according to various embodiments and the quality of the mass spectral data is higher than when alternative laser sources were used.
In particular, lower quality data was obtained when similar experiments were performed using the third midrange infrared laser comprising a CO2 laser.
Fig. 7 shows as a comparative example less pronounced mass spectral data which was obtained using the third midrange infrared laser comprising a CO2 laser. The CO2 laser utilised a 200 pm I.D. hollow core fibre enabling the laser to be focused to a smaller spot size.
A first 50 pm resolution mass spectral ion image at 744.40 m/z of another human breast tissue sample is shown in the upper portion of Fig. 7 together with an associated first mass spectrum. As before, the human breast tissue sample comprises both a region of healthy or normal tissue and also a region of tumorous tissue.
A major ion peak at 744.40 m/z is observed to be present in the first (upper) mass spectrum shown in Fig. 7 which is indicative of tumorous tissue.
A second 50 pm resolution mass spectral ion image at 893.45 m/z is shown in the lower portion of Fig. 7 together with an associated second mass spectrum.
The second ion image relates to normal or healthy tissue as evidenced by the presence of a major ion peak at 893.45 m/z in the second mass spectrum which is indicative of normal or healthy tissue comprising fatty tissue comprising triglycerides.
As with other experiments discussed above, the experiments which are described above in relation to Fig. 7 were performed on tissue samples which were neither fixed nor embedded. However, the above experimental results nonetheless illustrate that there are various benefits in using an optical parametric oscillator (ΌΡΟ”) laser 1 having a wavelength in the range 2700-3100 nm to analyse tissue samples compared to other midrange infrared laser sources. These same benefits were also found to apply to the analysis of fixed and embedded tissue samples according to various embodiments.
Various illustrative and comparative data has been presented above in relation to the analysis of tissue samples which were neither fixed nor embedded.
Various embodiments will now be described in more detail which relate to the analysis of tissue samples which are both fixed and embedded. In particular, experimental data will now be presented with reference to Figs. 8A-8C which relates to
-20the analysis of fixed and embedded tissue samples particularly formalin-fixed paraffinembedded (“FFPE”) tissue samples.
It has been demonstrated above that there are various benefits in selecting a mid-infrared pulsed laser for performing Rapid Evaporative Ionisation Mass Spectrometry (“REIMS”) on tissue samples wherein the pulsed laser comprises an optical parametric oscillator (ΌΡΟ”) laser 1 having a wavelength in the range 27003100 nm. The various benefits of utilising an optical parametric oscillator (ΌΡΟ”) laser 1 having a wavelength in the range 2700-3100 nm are particularly pronounced when seeking to analyse fixed and embedded tissue samples particularly formalin-fixed paraffin-embedded (“FFPE”) tissue samples according to various embodiments.
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 does not involve applying a reversal agent to a FFPE tissue sample. More generally, according to various embodiments no pre-processing of the sample is required.
Formalin-fixed paraffin embedding (“FFPE”) of tissues is an important step in current histopathology workflow for preserving the structure of the tissues allowing optical imaging and histology.
Molecular imaging of FFPE tissues is challenging usually requiring extensive processing of the sample.
However, significantly, the present inventors have found that it is possible to use an optical parametric oscillator (ΌΡΟ”) laser 1 having a wavelength in the range
-21 2700-3100 nm to analyse fixed and embedded tissue samples without any need to preprocess the tissue sample. In particular, there is no need to apply a reversal agent to the fixed and embedded tissue sample when using an optical parametric oscillator (“OPO”) laser 1 having a wavelength in the range 2700-3100 nm to analyse the fixed and embedded tissue sample.
Accordingly, according to various embodiments a method and apparatus is disclosed which allows fixed and embedded tissue samples to be simply and efficiently analysed and in particular for ion maps or mass spectral imaging of the tissue sample to be obtained avoiding the need for any pre-processing of the fixed and embedded tissue sample.
It will be apparent, therefore, that the approach according to various embodiments represents a significant advance in the art particularly in the context of the analysis of fixed and embedded tissue samples.
Fig. 8A shows an image of a formalin-fixed paraffin embedded colorectal tissue sample which was analysed according to various embodiments using the first midrange infrared laser comprising an optical parametric oscillator (“OPO”) laser 1.
According to various embodiments the optical parametric oscillator (“OPO”) laser 1 may comprise a Q-switched optical parametric oscillator laser 1. In the specific example shown and described with reference to Figs. 8B and 8C the laser 1 was tuned to 2940 nm. However, other embodiments are also contemplated wherein the pulsed laser 1 may comprise a Q-switched optical parametric oscillator laser 1 having a wavelength more generally in the range 2700-3100 nm.
According to various embodiments a pulsed laser 1 comprising a midrange infrared laser comprising an optical parametric oscillator (“OPO”) laser 1 may be used to analyse the fixed and embedded tissue sample. The laser 1 according to various embodiments may be arranged to have a pulse width or pulse length in the range 1-10 ns, more particularly in the range 3-7 ns.
Fig. 8B shows a molecular image or ion map at 701.51 m/z of the formalin-fixed paraffin embedded colorectal tissue sample which was acquired using the first midrange infrared laser comprising an optical parametric oscillator (OPO) laser according to various embodiments. It will be apparent that a high quality mass spectral image is obtained and the associated mass spectral data had a high signal to noise ratio. Regions of tumorous tissue may be identified.
Fig. 8C shows a molecular image or ion map at 374.11 m/z of the formalin-fixed paraffin embedded colorectal tissue sample which was acquired using the first midrange infrared laser comprising an optical parametric oscillator (“OPO”) laser according to various embodiments. It will be apparent that a high quality mass spectral image is
-22obtained and the associated mass spectral data had a high signal to noise ratio.
Regions of normal or healthy tissue may be identified.
It will be apparent from Figs. 8B and 8C that the use of an optical parametric oscillator (ΌΡΟ”) laser 1 having a wavelength in the range 2700-3100 nm when used as a Rapid Evaporative Ionisation Mass Spectrometry (“REIMS”) ion source to analyse fixed and embedded tissue samples is particularly effective and provides high quality mass spectral data with a higher signal to noise ratio compared with other mid-infrared lasers having either longer wavelengths (such as a CO2 laser having a longer wavelength of 10600 nm) or longer pulse widths (such as a Er:YAG laser having a pulse width of 200 ps). In particular, the use of an optical parametric oscillator (ΌΡΟ”) laser 1 having a wavelength in the range 2700-3100 nm and having a pulse width or pulse length in the range 1-10 ns and in particular in the range 3-7 ns has been found to be particularly beneficial for analysing fixed and embedded tissue samples. The optical parametric oscillator (ΌΡΟ”) laser 1 may be Q-switched.
In addition to being beneficial for analysing fixed and embedded tissue samples according to various embodiments, an optical parametric oscillator (ΌΡΟ”) laser 1 having a wavelength in the range 2700-3100 nm and used as a Rapid Evaporative Ionisation Mass Spectrometry (“REIMS”) ion source has also been found to be beneficial for cell line sampling.
Methods for rapid sampling and characterisation of a large quantity of biological samples is a common requirement, particularly with the increased use of cell lines and bacteria in drug and biomolecule production.
Fig. 9A shows for illustrative purposes a 96-well containing samples of yeast and yoghurt cultures which were used to obtain cell line sampling data.
Fig. 9B shows a total ion current (TIC”) of the measurements which were obtained as a function of time wherein 96 data points were acquired within 7 minutes.
REIMS is capable of rapidly acquiring spectral profiles and when coupled with suitable multivariate statistical analytical methods such as Principal Component Analysis (“PCA”) it is able to characterise cell line sample within seconds with a high throughput.
Fig. 9C shows a PCA model of the binary yeast-yoghurt sample, wherein it is apparent that there is a clear separation between the two spectral groups.
Utilising an XY translation stage 3 together with an optical parametric oscillator (ΌΡΟ”) laser 1 according to various embodiments enabled a fast system to be developed which allowed sampling and characterisation of a full 96-well plate within seven minutes.
-23In summary, a method and system of REIMS analysis which utilises a midinfrared laser has been disclosed. According to various embodiments the mid-infrared laser comprises an optical parametric oscillator (ΌΡΟ”) laser 1 having a wavelength in the range 2700-3100 nm. The use of such a laser has been found to be particularly beneficial compared with other mid-infrared lasers for the analysis of tissue samples in general and fixed and embedded tissue samples in particular. The particular optical parametric oscillator (ΌΡΟ”) laser 1 having a wavelength in the range 2700-3100 nm according to various embodiments is particularly effective when incorporated into an automated sampling and imaging tool for Rapid Evaporative Ionization Mass Spectrometry (“REIMS”) analysis.
Three different mid-infrared lasers having different wavelengths and laser pulse widths were tested and evaluated. The testing and evaluation using biological samples and in particular both unfixed tissue samples and fixed and embedded tissue samples indicates that a laser comprising an optical parametric oscillator (ΌΡΟ”) 1 and an output wavelength in the range 2700-3100 nm is particularly versatile and effective as a Rapid Evaporative Ionization Mass Spectrometry (“REIMS”) ion source.
In particular, a 2940 nm Q-switched optical parametric oscillator laser has been shown to be particularly versatile and effective. However, other embodiments relate more generally to a Q-switched optical parametric oscillator laser 1 having a wavelength in the range 2700-3100 nm. The pulse width of the laser may be in the range 1-10 ns, optionally 3-7 ns.
Although the various mid-infrared lasers which were tested were all able to produce molecular images to some degree, the first midrange laser comprising an optical parametric oscillator (ΌΡΟ”) laser 1 was found to be particularly effective in enabling ion maps or molecular images of formalin-fixed paraffin embedded tissue samples to be obtained without any sample preparation being required.
It has also been shown that using an optical parametric oscillator (ΌΡΟ”) laser is particularly beneficial in that it provides the capability to measure a large number of samples within short period of time.
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 (22)
1. A method comprising:
directing one or more pulses of laser radiation having a wavelength in the range 2700-3100 nm from a pulsed optical parametric oscillator (ΌΡΟ”) laser on to a fixed and embedded tissue sample and generating aerosol, smoke or vapour from the tissue sample;
generating analyte ions from the aerosol, smoke or vapour; and mass analysing the analyte ions.
2. A method as claimed in claim 1, wherein the fixed and embedded tissue sample is fixed in formalin.
3. A method as claimed in claim 1 or 2, wherein the fixed and embedded tissue sample is embedded in paraffin.
4. A method as claimed in claim 1, 2 or 3, wherein the fixed and embedded tissue sample comprises a formalin fixed paraffin-embedded (“FFPE”) tissue sample.
5. A method as claimed in any preceding claim, wherein the fixed and embedded tissue sample is not pre-processed prior to the step of directing one or more pulses of laser radiation on to the tissue sample.
6. A method as claimed in any preceding claim, wherein the fixed and embedded tissue sample is not subjected to the application of a reversal agent prior to the step of directing one or more pulses of laser radiation on to the tissue sample.
7. A method as claimed in any preceding claim, further comprising adding a matrix solvent to the aerosol, smoke or vapour to form a mixture.
8. A method as claimed in claim 7, wherein the step of generating analyte ions from the aerosol, smoke or vapour comprises causing the mixture to impact upon a heated collision surface located within a vacuum chamber of a mass spectrometer.
9. A method as claimed in any preceding claim, further comprising translating the tissue sample relative to the laser multiple times, obtaining mass spectral data relating to different regions of the tissue sample and generating an ion map or mass spectral image of at least a portion of the tissue sample.
10. A method as claimed in claim 9, further comprising generating an ion map or mass spectral image of at least a portion of the tissue sample over a range 600-800 m/z corresponding to phospholipids.
11. A method as claimed in claim 9 or 10, further comprising generating an ion map or mass spectral image of at least a portion of the tissue sample over a range 890-900 m/z corresponding to triglycerides.
12. A method as claimed in any preceding claim, wherein the one or more pulses of laser radiation have a pulse width or pulse length in the range 1-10 ns, optionally 3-7 ns.
13. A method of ion mapping or mass spectral imaging comprising a method as claimed in any of claims 1-12.
14. A method of mass spectrometry comprising a method as claimed in any preceding claim.
15. A mass spectrometer comprising:
a pulsed optical parametric oscillator (ΌΡΟ”) laser arranged and adapted to emit, in use, one or more pulses of laser radiation having a wavelength in the range 2700-3100 nm which are directed on to a fixed and embedded tissue sample so as to generate, in use, aerosol, smoke or vapour from the tissue sample;
a translation device for translating the tissue sample relative to the laser;
a device arranged and adapted to generate analyte ions from the aerosol, smoke or vapour; and a mass analyser for mass analysing the analyte ions.
16. A mass spectrometer as claimed in claim 15, wherein the fixed and embedded tissue sample comprises a formalin fixed paraffin-embedded (“FFPE”) tissue sample.
17. A mass spectrometer as claimed in claim 15 or 16, wherein the fixed and embedded tissue sample is not pre-processed prior to directing one or more pulses of laser radiation on to the tissue sample.
18. A mass spectrometer as claimed in claim 15, 16 or 17, wherein the fixed and embedded tissue sample is not subjected to the application of a reversal agent prior to the directing one or more pulses of laser radiation on to the tissue sample.
19. A mass spectrometer as claimed in any of claims 15-18, further comprising a device arranged and adapted to add a matrix solvent to the aerosol, smoke or vapour to form a mixture.
20. A mass spectrometer as claimed in claim 19, wherein the device arranged and adapted to generate analyte ions from the aerosol, smoke or vapour comprises a heated collision surface located within a vacuum chamber of the mass spectrometer and wherein, in use, the mixture is caused to impact the heated collision surface.
21. A mass spectrometer as claimed in any of claims 15-20, wherein the translation device is arranged and adapted to translate the tissue sample relative to the laser multiple times, wherein the mass spectrometer is arranged and adapted to obtain mass spectral data relating to different regions of the tissue sample and wherein the mass spectrometer is arranged and adapted to generate an ion map or mass spectral image of at least a portion of the tissue sample.
22. A mass spectrometer as claimed in any of claims 15-21, wherein the one or more pulses of laser radiation have a pulse width or pulse length in the range 1-10 ns, optionally 3-7 ns.
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| WO2016142689A1 (en) * | 2015-03-06 | 2016-09-15 | Micromass Uk Limited | Tissue analysis by mass spectrometry or ion mobility spectrometry |
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| WO2016142694A1 (en) * | 2015-03-06 | 2016-09-15 | Micromass Uk Limited | Desorption electrospray ionisation ("desi") imaging |
| WO2018026910A1 (en) * | 2016-08-02 | 2018-02-08 | Fluidigm Canada Inc. | Sample imaging apparatus and method |
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| US20110215233A1 (en) * | 2007-07-20 | 2011-09-08 | Akos Vertes | Subcellular analysis by laser ablation electrospray ionization mass spectrometry |
| WO2016142689A1 (en) * | 2015-03-06 | 2016-09-15 | Micromass Uk Limited | Tissue analysis by mass spectrometry or ion mobility spectrometry |
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| GB2590175A (en) * | 2019-10-31 | 2021-06-23 | Micromass Ltd | Ion source |
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