EP1639622A2 - Source d'ions de desorption laser - Google Patents

Source d'ions de desorption laser

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Publication number
EP1639622A2
EP1639622A2 EP04754443A EP04754443A EP1639622A2 EP 1639622 A2 EP1639622 A2 EP 1639622A2 EP 04754443 A EP04754443 A EP 04754443A EP 04754443 A EP04754443 A EP 04754443A EP 1639622 A2 EP1639622 A2 EP 1639622A2
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EP
European Patent Office
Prior art keywords
sample
ions
ion
gas phase
desorption
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Granted
Application number
EP04754443A
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German (de)
English (en)
Other versions
EP1639622A4 (fr
EP1639622B1 (fr
Inventor
Ross C. Willoughby
Edward W. Sheehan
Craig M. c/o Analytica of Branford WHITEHOUSE
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Publication of EP1639622A4 publication Critical patent/EP1639622A4/fr
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Publication of EP1639622B1 publication Critical patent/EP1639622B1/fr
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Classifications

    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/10—Ion sources; Ion guns
    • H01J49/16—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission
    • H01J49/161—Ion sources; Ion guns using surface ionisation, e.g. field-, thermionic- or photo-emission using photoionisation, e.g. by laser
    • H01J49/164—Laser desorption/ionisation, e.g. matrix-assisted laser desorption/ionisation [MALDI]
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/0027—Methods for using particle spectrometers
    • H01J49/0031—Step by step routines describing the use of the apparatus
    • H—ELECTRICITY
    • H01—ELECTRIC ELEMENTS
    • H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
    • H01J49/00—Particle spectrometers or separator tubes
    • H01J49/02—Details
    • H01J49/06—Electron- or ion-optical arrangements

Definitions

  • This invention relates to the generation of gas-phase ions or charged particles from condensed phase sample (e.g. liquid or solid) using laser desorption ionization and related techniques, primarily for analysis of chemical species with mass spectrometers or ion mobility spectrometers.
  • MALDI Matrix-assisted laser desorption/ionization
  • MALDI samples are generally mixtures of matrix and analyte, whereby the light energy from the laser is absorbed primarily by the matrix, facilitating both ionization and desorption of analyte.
  • Gas-phase anayte ions are generally analyzed by time-of-flight mass spectrometers; however, any number of gas-phase ion analyzers have been considered and employed for MALDI analysis.
  • Vacuum MALDI has developed into a widely used commercial technology for analysis of proteins and other macromolecules.
  • the present invention relates to the application of MALDI to desorption and ionization in vacuum and at intermediate and higher pressures, including atmospheric pressure.
  • Franzen and Koster US 5,663,561 first described atmospheric pressure MALDI in reference to their atmospheric pressure desorption/ionization technique by stating, "In contrast to MALDI, at atmospheric pressure, the related molecules of the decomposed matrix material are not needed to ionize the macromolecules. The selection of matrix molecules is solely dependent upon their ability to release the large molecules.”
  • the concept of atmospheric pressure MALDI (or AP-MALDI) was clearly first described by Franzen and Koster.
  • Laiko and Burlingame (US 5,965,884) distinguish their AP-MALDI from Franzen and Koster by arguing simplicity and non-destructive matrices. This patent dismisses the key arguments made by Franzen and Koster that AP-MALDI is inefficient.
  • the Laikd patent teaches AP-MALDI with the requirement of close coupling of a sample target to the conductance aperture into vacuum.
  • the lack of efficient atmospheric pressure optics with this device requires precise alignment and positioning of sample and the laser beam relative to the vacuum inlet.
  • Laiko provides for a sweep gas to assist in transport of the ions from the target surface to the vacuum inlet. The transmission of this device is low.
  • the lack of time-sequenced optics with the laser pulse limit ion extraction and transmission efficiency.
  • Sheehan and Willoughby describe separation of the ionization process [and sample target posision] from the conductance aperture using atmospheric pressure optics. They describe efficient atmospheric pressure transport and compression optics that allow relative independence of sample location from the position of the vacuum inlet. Components of this invention are included by reference into the present invention.
  • Sheehan and Willoughby describe further improvement of transmission of MALDI generated ions at atmospherip pressure by laminating high transmission elements and incorporating a "back-well" geometry whereby MALDI samples can be placed facing away from the conductance aperture.
  • This geometry facilitates easier access of the laser beam to the sample targets compared to close-coupled designs.
  • the back-well geometry also provides a simplification of sample insertion and easier access to the ionization chamber. Components of this invention are also included by reference into the present invention.
  • Willoughby and Sheehan also describe improvements in transmission of ions from atmospheric pressure sources [including AP-MALDI]. These improvements are accomplished by precisely controlling the electric field through the entire conductance pathway from atmospheric pressure into vacuum. Components of this invention are included by reference into the present invention. Willoughby and Sheehan (US PPA 60/476,582) also teach that conductance arrays and patterned optics can further enhance the transmission of ions from atmospheric pressure sources and improve the transmission of MALDI ions from either intermediate of higher-pressure sources. Components of this invention are included by reference into the present invention.
  • Whitehouse (US 20020175278) describes the use of a variety of RF multipole devices and DC funnel devices to focus and entrain the flow of ions from atmospheric and intermediate pressure MALDI targets to detection. Components of this invention are included by reference into the present invention.
  • Truche et al. (US 6,707,039 B1) describe a wide variety of alternatives for close- coupling the sample target to the conductance aperture. This technology places high tolerance on sample position and laser position.
  • mirrored reflective surfaces close to the plume of the MALDI target would tend to become contaminated and degraded in their optical performance.
  • sampling of ions from an electric field between the target and aperture into the field-free region of the vacuum inlet tube would cause rim losses from field penetration and degrade the transport efficiency.
  • Makarov and Bondarenko (US 6,707,036 B2) teach of a positionally optimized sample target device with a close-coupled conductance opening for atmospheric pressure and intermediate pressure MALDI. This device is still subordinate to alignment of laser, target, and lacks spatial or temporal optics to facilitate efficient ion transmission to the mass analyzer. The lack of time-sequenced optics with the laser pulse limit ion extraction and transmission efficiency.
  • Dispersive sources of ions at or near atmospheric pressure such as, atmospheric pressure discharge ionization, chemical ionization, photoionization, or matraix assisted laser desorption ionization, and electrospray ionization generally have low sampling efficiency through conductance or transmission apertures, where less than 1 % [often less than 1 ion in 10,000] of the ion current emanating from the ion source make it into the lower pressure regions of the present commercial interfaces for mass spectrometry.
  • associated methods of sample charging, laser desorption and sample ionization are intended to improve the collection efficiency and ionization efficiency of atmospheric pressure, intermediate pressure and vacuum laser desorption ionization.
  • Two advantages of the current device should be emphasized. First, precisely timing the sequence of laser pulse with ion extraction under high voltage followed by reduction of the electric field in the extraction and focusing region before losing ions to surfaces. The field in the extraction and focusing region is reduced so that the ions are efficiently focused and transmitted through a conductance aperture into a lower pressure region on the path to a mass analyzer.
  • the second important advantage is the ability to populate the sample surface with ions of the sample polarity as the analyte ions to be extracted. This condition drives the equilibrium toward product with an excess of reagent ions compared to conventional MALDI and increases the efficiency of ionization of analyte.
  • One aspect of the current invention is to precharge a sample prior to laser desorption to enhance the yield of ions from a given sample.
  • Another object of this patent is to incorporate precision precharging of a sample to predetermined spots on a sample (e.g. biopsy of suspected cancer tissue) in order to facilitate enhance yield of ions from a given spot.
  • Optical imaging can be used to determine the precise position of sample precharging and laser pulse impingement (e.g. dye markers or fluorescent tags visualized by microscopes with video recording).
  • An object of this invention is to use specialized target surfaces with shaped needles or electrodes behind the sample in order to control the electric field experienced by the sample during and after laser pulse. By varying voltage in space and time, optimum sample precharging, ion generation and extraction of ions can be achieved.
  • atmospheric pressure, intermediate pressure and vacuum laser desorption ion sources comprise ionization chambers and transmission devices encompassing targets for holding samples, lasers to illuminate said targets resulting in desorption and ionization of the samples, time-sequenced electrostatic potentials to foster efficient extraction, focusing, and selecting of resulting gas-phase ions.
  • Laser desorption ion sources in accordance with the invention also comprise a means to accumulate charge on a sample prior to laser desorption of the sample and a means to conduct gas phase ionization of laser desorbed neutral sample molecules to increase the ionization efficiency of a sample during and after a desorption laser pulse.
  • FIG. 1 is a diagram of an atmospheric pressure Laser Desorption Ionization source, incorporating surface charging, interfaced to a mass spectrometer.
  • FIG. 2A is a diagram of the atmospheric pressure Laser Desorption Ionization source shown in Figure 1 during the operating step of charge accumulation on the sample surface.
  • FIG. 2B is a diagram of the atmospheric pressure Laser Desorption Ionization source shown in Figure 1 during the operating step of laser firing and charge release from the sample.
  • FIG. 2C is a diagram of the atmospheric pressure Laser Desorption Ionization source shown in Figure 1 during the operating step of focusing the ion population produced into the orifice to vacuum.
  • FIG. 2D is a diagram of the atmospheric pressure Laser Desorption Ionization source shown in Figure with the view turned 90 degrees showing a imaging apparatus with magnification.
  • FIG. 3 is a diagram of one embodiment of the electric fields applied during surface charging and ion release and focusing operation in the atmospheric pressure Laser Desorption Ionization source shown in Figure 1.
  • FIG. 4A is a timing diagram of one operating sequence embodiment used in the atmospheric pressure Laser Desorption Ionization source shown in Figure 1.
  • FIG. 4B is a timing diagram of a second operating sequence embodiment used in the atmospheric pressure Laser Desorption Ionization source shown in Figure 1.
  • FIG. 5 is a diagram of an atmospheric pressure Laser Desorption Ionization source, incorporating surface charging, with the target surface configured in proximity to the orifice into vacuum.
  • FIG. 6 is a timing diagram of the of one operating sequence embodiment used in the atmospheric pressure Laser Desorption Ionization source shown in Figure 5.
  • FIG. 7 is diagram of an intermediate pressure Laser Desorption Ionization source, incorporating surface charging; interfaced to a mass spectrometer.
  • FIG. 8A is a diagram of the one embodiment of a Laser Desorption target surface configured with an insulated charging electrode.
  • FIG. 8B is a diagram of an alternative embodiment of a Laser Desorption target surface configured with an insulated and shielded charging electrode.
  • FIG. 8C is a diagram of an alternative embodiment of a Laser Desorption target surface configured with an array of insulated and shielded charging electrodes.
  • FIG. 9A is a diagram of one embodiment of a Laser Desorption target surface.
  • FIG. 9B is a diagram of an alternative embodiment of a Laser Desorption target surface comprising an array of charging electrodes with integral fiber optics for applying a laser pulse to the back side of the sample.
  • FIG. 9C is a diagram of a renewable liquid Laser Desorption target surface with liquid sample delivered to the target surface through a liquid flow channel.
  • FIG. 9D is a diagram of a renewable liquid Laser Desorption target surface with integral fiber optics for applying a laser pulse to the back side of the sample.
  • FIG. 1OA is a diagram of an atmospheric Laser Desorption Ionization source comprising surface charging and a annular ion focusing lens embodiment interfaced to a mass spectrometer during the operating step of surface charging.
  • FIG 1OB is a diagram of the Laser Desorption Ionization source shown in Figure 1OA during the operating step of laser firing and charge release from the sample surface.
  • FIG 1OC is a diagram of the Laser Desorption Ionization source shown in Figure 1OA during the operating step of focusing the ion population produced into the orifice to vacuum.
  • FIG 11 is a diagram of an atmospheric Laser Desorption Ionization source comprising surface charging, a reversing annular ion focusing lens and surface imaging.
  • FIG 12A is a diagram of a vacuum Laser Desorption Ionization source configured with surface charging and a near surface potential trap configured in the pulsing region of a Time-Of-Flight mass spectrometer during surface charging operation.
  • FIG 12B is a diagram of the vacuum Laser Desorption Ionization source shown in Figure 12A during the operating step of laser firing and charge release from the sample surface.
  • FIG 12C is a diagram of the vacuum Laser Desorption Ionization source shown in Figure 12A during the operating step of trapping the ion population produced on the dynamic field trapping surface.
  • FIG 12D is a diagram of the vacuum Laser Desorption Ionization source shown in Figure 12A during the operating step of pulsing the ion population produced into the Time- OF-Flight mass spectrometer flight tube.
  • a preferred embodiment of the invention comprising an atmospheric pressure Laser Desorption Ionization source with sample surface charging is diagrammed in Figure 1. Operating details for Laser Desorption Ionization source 1 are diagrammed in Figures 2A through 2D.
  • Laser Desorption Ionization (LDI) source 1 interfaced to vacuum system 2 comprising ion transfer optics and mass to charge analyzer with detector 3, produces ions from sample 4 on target plate 5.
  • a portion of the laser desorption ion population produced is focused into bore 10 of capillary 11. Ions exit capillary bore 10 at capillary exit end 12 into vacuum and are accelerated in a free jet expansion of neutral background gas flowing through capillary bore 10 from atmospheric pressure ion source 1.
  • Capillary 11 may comprise a dielectric capillary with conductive electrodes on the entrance and exit faces, a heated electrically conductive capillary, a nozzle, an orifice or an array of orifices into vacuum. Ions pass through skimmer 13 orifice 14 and into ion guide 15 where their translational energies are damped through collisions with background gas. Ions exiting ion guide 15 pass through exit lens 17 and are mass to charge analyzed in mass to charge analyzer and detector 3. Ion guide 15 may comprise a multipole ion guide, a segmented multipole ion guide, a sequential disk RF ion guide, an ion funnel or other ion guides known in the art.
  • Ion guide 15 may extend continuously into one or more vacuum pumping stages or may begin and end in one vacuum stage.
  • Mass analyzer and detector 3 may comprise a quadrupole, triple quadrupole, three dimensional ion trap, linear ion trap, Time-Of-Flight (TOF), magnetic sector, Fourier Transform Ion-Cyclotron Resonance (FTICR), Orbitrap or other mass to charge analyzer known in the art.
  • Vacuum system 2 comprises vacuum stages 18, 19 and 20.
  • embodiments of the invention may comprise vacuum systems with more or less vacuum stages depending on the requirements of the vacuum ion optics and mass to charge analyzer.
  • Atmospheric pressure ion source 1 produces ions from a sample deposited on or part of a surface. As will be described below, the sample may comprise a solid or liquid.
  • Sample 4 on target plate 5 is positioned in target plate chamber 22.
  • Gas or gas containing ions 23 enters target surface chamber 22 through target gas controller 24.
  • Target gas controller 24 comprises a gas heater and an ion source to generate reagent ions from a gas and/or liquid input 25.
  • Target gas controller 25 may comprise a pneumatic nebulization charge droplet sprayer followed by a vaporizer producing a heated carrier gas containing reagent ions formed from the evaporating charged droplets.
  • target gas controller 25 may comprise a photoionization source, a glow discharge ionizer, a corona discharge ionizer configured in an atmospheric pressure chemical ionization (APCI) source or other type of gas or liquid sample ion source.
  • APCI atmospheric pressure chemical ionization
  • target gas controller 24 can be configured and operated to deliver unheated neutral gas, heated neutral gas or an ion and gas mixture into target plate chamber 22 during laser desorption ion source operation.
  • Reagent ion containing gas flow 23 passes between target plate 5 and target plate counter electrode 28 exiting target plate chamber 22 at opening 27 in target plate counter electrode lens 28.
  • Electrode 28 is electrically insulated from target plate chamber 22 by insulators 29.
  • reagent ions entrained gas flow 23 may be selectively deposited on sample 4, directed through opening 27 or discharged on target lens 28 during laser desorption ion source operation.
  • Target plate 5 can be moved manually or by software control in the x and y directions using x-y translator 26.
  • Charging electrode assembly 8 remains fixed in position while target plate 5 slides over it.
  • a more detailed diagram of charging electrode assembly 8 is shown in Figures 2A through 2D and 8B.
  • Charging electrode assembly 8 comprises charging electrode 30 and shielding electrode 32 forming an electrically conductive cylinder around charging electrode 30.
  • Charging electrode 30 and shielding electrode 32 are embedded in dielectric block 31 to allow the application of high voltage to charging electrode 30 without the onset of gas phase corona discharge or arcing. Voltages are applied to electrodes 30 and 32 through power supplies 34 and 35 respectively.
  • Laser 7 is configured to deliver laser pulse 40 through lens or window 38 and reflected off mirror 39 to impinge on sample 4 as shown in Figure 2B.
  • Countercurrent gas 45 passes through gas heater 42 and exits through opening 43 of endplate electrode 44 forming countercurrent gas flow 41 in LDI source 1.
  • Gas 53 and desorbed ions pass through opening 52 in electrode 47 and capillary entrance electrode 48 into capillary 10 bore 11.
  • Voltages applied to electrodes 28, 44, 47 and 48 through power supplies 56, 49, 50 and 51 respectively are set to maximize focusing and ion transmission into capillary bore 11 as will be described below.
  • Charged droplet sprayer 58 comprises liquid inlet 59, nebulization gas inlet 60, sprayer tip 61 and ring electrode 63 as shown in Figure 2A. Voltages are applied to charged droplet sprayer 58 and ring electrode 63 through power supplies 65 and 64 respectively.
  • charged droplet sprayer 58 is configured to produce a spray of charged droplets oriented orthogonal to ion source centerline 68.
  • Charged droplets are produced through conventional Electrospray or pneumatic nebulization in the presence of an electric field. Heated countercurrent drying gas 41 and target plate gas 74 aid in evaporating the charged droplets in spray 62.
  • voltages applied to electrodes 30, 28 44, 47 and 48 are set to direct ions generated from evaporating the charged droplets in spray 62 into capillary bore 11.
  • ions produced from sample bearing solution 59 are directed into vacuum and mass to charge analyzed.
  • Ion source 1 can be operated in Electrospray or atmospheric pressure Laser Desorption ionization mode individually or both ionization modes can be run simultaneously. Rapid switching between Electrospray and Laser Desorption ionization can by achieved using the ion source embodiment shown in Figure 1.
  • charged droplet sprayer 58 is replaced by an Atmospheric Pressure Chemical Ionization source comprising a pneumatic nebulizer, vaporizer and corona discharge needle.
  • a glow discharge, photoionization or other type of ion source can be configured to produce ion species in region 73 between electrodes 28 and 44.
  • LDI source 1 can be configured with multiple ion generation sources delivering ions individually or simultaneously into region 73.
  • the voltages applied to electrodes 30, 28, 44, 47, 48, 63 and charged droplet sprayer 58 are set to direct ions 75 generated from charged droplet sprayer 58 to accumulate on the surface sample 4 on target plate 5 prior to desorbing sample 4 by laser pulse 40.
  • Ion or charged species 75 generated from charged droplet sprayer 58 and ion species 71 entrained in target plate gas flow 23 are directed to the surface of sample 4 prior to desorbing sample 4 with laser pulse 40 as shown in Figure 2A.
  • the accumulation and subsequent laser desorption of positive polarity ions is illustrated in Figures 2A through 2C but the same sequence of steps can be applied for negative ion accumulation and laser desorption with the reversal of voltage polarities applied to electrodes.
  • FIG 2A appropriate voltages are applied to charged droplet sprayer 58, ring electrode 63 and electrodes 28 and 44 to produce positive polarity charged droplet spray 62.
  • the potentials applied to charged droplet sprayer tip 61 , ring electrode 63, electrode 28 and electrode 44 may be set to +4KV, +OV, -1KV and + 1KV respectively.
  • the voltage applied to electrically insulated charging electrode 30 through power supply 34 may by set to - 10 to - 20 KV with the shielding electrode voltage set close to -1 KV through power supply 35.
  • the electric field formed at the sharp tip of charging electrode 30 penetrates dielectric target plate 5 and extends through opening 27 of electrode 28 into region 73 between electrodes 28 and 44 as shown in Figure 2A.
  • Heated target gas 74 aids in drying charged droplets produced by charged droplet sprayer 58.
  • Ions 75 generated from evaporating droplets produced from charged droplet spray 62 follow electric field lines 72 and are directed to the surface of sample 4 on dielectric target plate 5.
  • charged species 71 entrained in target plate gas flow 23 pass between target plate 5 and electrode 28 and are attracted to the surface of sample 4 by the same attractive electric field formed by the electrical potential applied to charging electrode 30.
  • Charge 70 accumulates on the surface of sample 4 until the space charge limit is reached. When the space charge limit is reached additional positive polarity ions turned away from the surface of sample 4 and neutralized on electrode 28.
  • Image charge 73 in this case electrons, are drawn to the tip of charging electrode 73 as positive ions accumulate on the surface of sample 4.
  • Charging electrode 30 and sample 4 form a capacitor with a charge capacity in part determined by the electric field strength maintained between the surface of sample 4 and the tip of charging electrode 30.
  • the tip sharpness of insulated charging electrode 30, the proximity of this tip to the surface of sample 4, the voltage applied to charging electrode 30 relative to the voltage applied to electrodes 28 and 44 and the dielectric constant of target plate 5 and insulation 31 will effect the electric field strength at the surface of sample 4.
  • Charge may accumulate on the surface of sample 4 until the electric field is locally reduced and ultimately neutralized preventing additional ions of the same polarity from further accumulating on the surface of sample 4.
  • Minimum charge migration or neutralization occurs on the surface of dielectric target plate 5.
  • a single ion species or a mixture of ion species can be accumulated on surface 4 depending on the requirements of an analytical application. For example, if sample 4 comprises a mixture of proteins with a matrix such as Sinapinic acid typically used in Matrix Assisted Laser Desorption Ionization (MALDI), protons may be an optimal choice of charged species to accumulate on the surface of sample 4.
  • MALDI Matrix Assisted Laser Desorption Ionization
  • Protons can be directed to the surface as protonated water or protonated methanol ions generated from charged droplet sprayer 58 or a charged droplet sprayer or APCI ion generator configured in target gas controller 24. Proteins form ions generally as protonated species so the protons accumulated on the surface of sample 4 will supply a source of protons to increase ionization efficiency during laser desorption of sample 4. Alternatively, metal ions such as sodium can be accumulated on the surface of sample 4 if carbohydrate analysis is required to enhance ionization efficiency. If sample 4 comprises a liquid such as water or a low volatility surface such as glycerol, accumulating ions can react with or attach to sample species in solution prior to laser desorption.
  • Infrared lasers can be used to desorb aqueous sample solutions at atmospheric pressure.
  • Sample 4 may include no matrix and laser desorption may occur directly from the sample as is used with Direct Ionization Off Surfaces (DIOS) techniques.
  • DIOS Direct Ionization Off Surfaces
  • Accumulating charged charged species may be in direct contact with sample molecules when no matrix is used on target plate 5. This direct charge species and sample species association can improve ionization efficiency for select sample types when compared with charge accumulation in the case where the sample is associated with a matrix.
  • Different ion species may be supplied by charged droplet sprayer 58 and target gas controller 24. Ions species may be generated from charged droplet sprayer 58 and target gas controller 24 simultaneously or individually. Charged species production by either device may be rapidly switched off or on, if required during laser desorption ionization operation.
  • Charged droplet sprayer 58 can be rapidly turned off and on by adjusting the relative potentials applied sprayer tip 61 and ring electrode 63.
  • laser pulse 40 is applied to the surface of sample 4 from laser 7 to desorb sample from target plate 5.
  • the voltage applied to charging electrode 30 is rapidly reversed just prior to, during or just after laser pulse 40 to release the charge from the surface of sample 4. This effectively reverses the potential across the capacitor formed by the charge accumulated on the surface of sample 4 and the image charge accumulated near the tip of charging electrode 30.
  • the laser pulse step is illustrated in Figure 2B where the attracting electric field 72 is gone and electric field 77 attracts ions desorbed from sample 4 toward entrance orifice 78 of capillary 10.
  • Figure 3 is a diagram of one set of electrical potentials that may be applied during the ion accumulation and ion desorption steps.
  • Curve 80 shows one example of the relative potentials applied to Electrodes 30, 2844, 47 and 48 during accumulation of positive charge on the surface of sample 4.
  • Curve 82 represents the relative but off axis electrical potentials applied to charged droplet sprayer tip 61 and ring electrode 63 during production of positive polarity charged droplets from sprayer tip 61 and accumulation of positive polarity ions on the surface of sample 4.
  • Curve 81 shows the reversal of voltage polarity applied to charging electrode 30 and 28 to facilitate desorption and ionization of sample components from sample 4 when laser pulse 40 is applied.
  • the voltage applied to ring electrode 63 as shown by curve 83 is set to minimize distortion of the centerline focusing electric field directing desorbed ions into capillary entrance 78.
  • Charged droplet sprayer nebulizing gas flow is switched off during the laser desorption and ion focusing steps.
  • charged droplet sprayer 58 When charged droplet sprayer 58 is operated in non nebulizing Electrospray mode, the charged droplet spray turns off when the voltages on ring electrode 83 are set approximately equal to the voltage applied to sprayer tip 61 as shown in curve 83 or Figure 3.
  • the timing diagram of the voltage transitions illustrated in Figure 3 is shown in Figure 4A.
  • the surface charging time period is followed by laser pulse 85 and a rapid change in voltage 86 applied to charging electrode 30.
  • Electrodes 30, 28 and 63 are maintained during the ion focusing period to allow time for desorbed ions from sample 4 time to reach capillary entrance 78 where they are swept through capillary bore 11 into vacuum by gas flow 53.
  • voltages applied to Electrodes 44, 47 and 48 remain constant during the sample charging, ion desorption and ion focusing steps illustrated in Figures 2A, 2B and 2C.
  • reagent ions When positive reagent ions are generated from target gas controller 24, relative voltages can be set between electrodes 30 and 28 to allow these reagent ions to pass through opening 27 in electrode 28 and mix with neutral molecules 75 and ions 88 desorbed from sample 4. Through exchange or attachment of charge from the reagent ions to desorbed neutral species, the ionization efficiency of the desorption process is improved increasing mass to charge analysis sensitivity. As diagrammed in Figures 2B and 2C 1 reagent ions 90 mix with desorbed neutral species when the appropriate voltages are applied to electrodes 30 and 28 to direct reagent ions 71 through opening 27 and along centerline 68 moving as gas phase ions 90 toward capillary entrance 78.
  • reagent ions 90 Before countercurrent gas flow 41 sweeps desorbed neutrals away from opening 43 in endplate electrode 44, reagent ions 90 have a the opportunity to collide with and exchange charge or attach to a neutral desorbed sample molecule.
  • Target plate gas flow 74 meeting countercurrent gas flow 41 in region 73 form a stagnation and mixing area in region 71 that promotes charge exchange or attachment between reagent ions 90 and desorbed neutral species 75.
  • focusing fields 77 direct the ions towards capillary entrance 78.
  • Reagent ions species may also be selected to promote desired gas phase reactions with desorbed analyte sample molecules.
  • Reagent ion flow through opening 27 in electrode 28 can be stopped during the ion focusing step by applying the appropriate relative voltages between electrodes 30 and 28 to direct reagent ions to neutralize on electrode 28 before entering opening 27.
  • FIG. 4B An alternative sequence of surface charging step 92, sample desorption, extraction and ion focusing step 93 and gas focusing step 94 is shown in timing diagram 4B.
  • the charging and desorption steps illustrated by Figures 2A and 2B are similar to the two step sequence of Figure 4A as shown in the timing diagram shown in Figure 4B.
  • the potentials applied to electrodes 30, 28, 44, 47 and 48 are set approximately equal, as shown in step 94 of timing diagram 4B, to allow gas dynamics forces to dominate ion motion, sweeping ions into and through capillary bore 11.
  • the application of steep electric fields near capillary entrance 78 serve to focus ions toward the centerline but can also drive ions into the edge of capillary entrance electrode 48 where they are neutralized. Reducing the electric field just before the ions reach capillary entrance orifice 78 allows initial ion focusing as desorbed ions traverse from sample 4 to capillary entrance orifice 78 but reduces the amount of ion impingement occurring on capillary entrance electrode 78 as the ions enter capillary bore 11. This additional gas dynamic ion focusing step improves ion transport efficiency into vacuum increasing sensitivity in mass to charge analysis.
  • the timing of the voltage switch to the gas focusing step can be optimized for any set of focusing voltages applied by using a calibration procedure in which the duration of ion desorption, extraction and focusing step 93 is varied to find the maximum mass spectrometer signal response.
  • the diagrams of timing sequences and steps shown in Figures 2A through C, Figure 3 and Figures 4A and 4B are given to illustrate examples of operating sequences, however other switching patterns or variations on switching patterns can be employed to optimize performance for different applications.
  • Voltages can be applied to maximize ionization and sampling efficiency of negative ions.
  • Variations of step sequences and additional steps may be added to sequences to maximize performance and to optimize for differences in samples, applications, and ion source lens geometries, gas composition, temperature and flow rates.
  • multiple laser shots can be conducted on the same spot or on different spots while the voltage applied to charging electrode 30 is transitioned from charge accumulation to charge rejection potentials.
  • Laser beam 40 spot can be moved or target plate 5 can be moved between each laser shot in a series.
  • Figure 2D is a diagram of laser desorption ion source 1 viewed from an angle rotated 90 degrees to the view shown in Figures 2A through 2C.
  • Charged droplet sprayer 58 is with sprayer tip 61 is pointing orthogonal to the viewing plane.
  • Configured 90 degrees rotated from charged droplet sprayer 58 and Laser 7 is optical imaging device 95 with image magnifiers 96 and mirror 97.
  • Imaging device 95 may comprise a video camera for digital imaging or a microscope for manual viewing of the sample surface. Imaging device 95 is used to provide and image sample surface 4 allowing optimization of the target plate 5 position relative to the tip of charging electrode 30 and laser pulse 40.
  • Positioning the tip of charging electrode 30 under a sample feature will maximize charge accumulation at that location.
  • Laser desoption ionization efficiency can be improved with sample mixed in MALDI matrices when a laser pulse is applied to a MALDI crystal located using optical imaging with feedback to the target plate x-y translator stage 26. Less ion yield results when a laser pulse impinges on a MALDI matrix in a location where no matrix crystals are present.
  • Imaging device 95 can be used to located the position of MALDI matrix crystals in sample 4. Based on the image information and sample coordinates provided, target plate 5 is moved to line up the tip of charging electrode 30 and laser pulse 40 with the MALDI matrix crystal position in sample 4.
  • the position of laser beam 40 hitting sample 4 can be adjusted independent of target plate 5 movement or the location of the tip of charging electrode 30.
  • Mirror 39 can be configured with a fine resolution movement device such as a galvanometer to allow rapid steering of laser beam 40 impinging on sample 4.
  • the position of charging electrode 30 can be positioned using a separate x-y translator stage to provide movement of charging electrode 30 independent of target plate 5 x-y movement. Additional illuminating devices such as lower power lasers can be incorporated into imaging device 95 to enhance the image from florescent dyes used to stain sample 4.
  • sample 4 is a tissue slice and laser desorption source 1 is used to conduct molecular imaging of stained tissue samples
  • individual cells can be optically imaged using imaging device 95 to allow laser charge accumulation on and laser desorption from selected cells in tissue sample 4.
  • Laser beam 40 can be focused down to small spot dimensions and target plate 5 can be fabricated as a very thin dielectric sheet allowing the insulated sharp tip of charging electrode 30 to rest just under but very close to an imaged and selected cell.
  • Laser desorption ionization from individual cells or from a small group of cells in a tissue can be performed with an appropriately focused laser spot and a small local charge accumulation area.
  • Imaging device 95 can also be used determine when a sample has been depleted or damaged after several laser shots.
  • Target plate 5 and charging electrode 30 may be configured in alternative embodiments.
  • Target plate 5 may be configured as a moving dielectric belt.
  • the eluant from a liquid chromatography (LC) run can be deposited on the moving belt as a continuous track or spots with a MALDI matrix added on line.
  • a second track of calibration sample can be added along side the LC sample track.
  • Two charging electrodes can be positioned under each track or spot train to provide simultaneous charging of both LC and calibration sample?.
  • Laser beam 40 can be rastered across both tracks or spots during the desorption step to generate ions from both the LC and calibration samples as the dielectric belt target moves past opening 27 of electrode 28.
  • the charging and laser desorption steps can occur rapidly with multiple step cycles conducted per second to maximize sample throughput.
  • FIG. 5 An alternative embodiment of the invention is diagrammed in Figure 5 where electrodes 44 and 47 are removed and target plate 100 is positioned closer to the capillary bore entrance 102.
  • Charged droplet sprayer 105 produces charged droplet spray 108 as described in Figure 2A above. Evaporating charged droplets generate ions that can be directed to accumulate on the surface of sample 101 to enhance the ionization efficiency of laser desorption or directed toward capillary bore entrance 102 when conducting Electrospray or pneumatic nebulization ionization of a sample substance.
  • charged droplet sprayer 105 may be configured as an APCI, a photoionization, glow discharge, corona discharge or other ionization source to generate of charged species for charge accumulation on sample 101 prior to laser pulse 108.
  • Multiple alternative ionization probes can be configured in one ion source with laser desorption producing ions in region 113 of ion source 114 shown in Figure 5 or region 73 of ion source 1 shown in Figure 1 and 2A through 2D.
  • Different ionization methods can be separately controlled to provide ion accumulation on sample 101 and 4 prior to laser desorption or to generate ions that are directed into vacuum through capillary bore 104 and 11 for mass to charge analysis. Combinations of multiple probes can be run simultaneously or independently in one ion source without the need to change hardware.
  • the operating sequence of laser desorption ion source 114 shown in Figure 5 is analogous to that illustrated in timing diagram 4B described above.
  • a negative voltage is applied to charging electrode 112 through power supply 123 relative to the voltages applied to target plate counter electrode 111, capillary entrance electrode 115, capillary nosepiece electrode 117, charged droplet sprayer 105 and ring electrode 106 through power supplies 118, 119, 120, 122 and 121 respectively.
  • Charged species generated by charged droplet sprayer 105 and/or target gas controller 124 are directed to the surface of sample 101 on dielectric or semiconductor target plate 100. Charge is accumulated on the surface of sample 101 until the space charge limit is reached for the relative electrode voltages applied.
  • the time period 128 of this sample charging step is illustrated in the timing diagram shown in Figure 6.
  • Laser pulse 108 is fired from laser 110 to desorb material from sample 101 as the voltages on electrodes 112, 106 and 117 are changed to facilitate extraction of desorbed ions from the surface of sample 101 and focusing of the ion population produced into capillary bore entrance 102.
  • the ion desorption, extraction and focusing step 129 is shown to occur simultaneously with laser pulse 108.
  • the electrode voltage transitions can occur before or after the laser pulse and additional laser pulses can occur during or after such electrode voltage transition.
  • the relative voltages applied to electrodes 112, 111, 106, 115 and 117 are set to be approximately equal to reduce,the electric field in region 113 between target plate 100 and capillary entrance electrode 115.
  • the relative voltages of electrodes are set to be approximately equal to initiate gas focusing step 130. With a minimum electric field in region 113, the desorbed ions are swept into capillary bore by gas flow 131.
  • the reduction of the electric field in region 113 prior to the desorbed ions reaching capillary entrance electrode 115 reduces neutralization of ions on electrode 115 and improves ion transmission efficiency into vacuum through capillary bore 104.
  • the duration of the gas focusing step 130 time period is sufficient to allow the desorbed ion population to enter capillary bore 104 prior to switching the electrode potentials back to ion accumulation step 132.
  • Heated countercurrent gas flow 127 sweeps neutral species away from capillary bore entrance 102 during ion extraction and focusing step 129 and provides the carrier gas for sweeping ions into vacuum.
  • gas phase ion species may generated in target gas controller 124 and carried in target gas 133 to charge the surface of sample 101 and provide subsequent gas phase ionization of desorbed neutral molecules traversing region 113.
  • the charging, desorption and gas focusing steps can be conducted in rapid succession cycling multiple times per second to minimize sample analysis time.
  • the laser pulse 108 spot, target plate 100, and charging electrode 112 positions can be positioned independently with or without optical imaging to optimize analytical performance for a given application.
  • FIG. 7 An alternative embodiment of the invention is diagrammed in Figure 7 where target plate 140 and target plate chamber 142 are positioned in vacuum stage 160.
  • the pressure maintained in vacuum stage 160 may range from above 4 torr to below 10 "4 torr depending on the analytical application, total gas flow through target plate gas controller 143 and ion generator 147 and vacuum stage 160 pumping speed.
  • Ion or charged species generator 147 with ion focusing electrodes 148 and target gas controller 143 may comprise a chemical ionization, glow discharge, electron bombardment, photoionization or other vacuum compatible ion source to generate charged species.
  • charging of the surface of sample 141 occurs in intermediate pressure laser desorption ion source 164 prior to applying laser pulse 165 from laser 151 to desorb sample components and ions from sample 141.
  • Charged species in either positive or negative ion operating mode are accumulated on the surface of sample 141 by applying the appropriate potentials as described above to charging electrode 166, target plate counter electrode 146, skimmer electrode 149, ion generator 147 and focusing electrodes 148.
  • Ion species are supplied from target gas controller 143 and ion generator 147 individually or simultaneously during the sample charging step.
  • Electrodes 166, 146, 149 and 148 and ion generator 147 are rapidly changed while laser pulse 165 is applied to aid in desorbing, extracting and ionizing sample components from sample 141. After ion and neutral sample components have been desorbed and extracted from sample 141 , voltages applied to these electrodes are then changed to optimize transmission efficiency of the desorbed ion population through skimmer opening 150 into ion guide 154. Timing sequence similar to that shown in Figures 4A, 4B and 6, can be applied in the operation of intermediate pressure laser desorption ion source 160.
  • Ion guide 154 can be operated as an ion trap to allow additional reaction time between reagent ions supplied from target plate gas controller 143 trapped in ion guide 154 to react with desorbed neutral species flowing through skimmer opening 150 and into ion guide 154.
  • the accumulation of charge on the sample prior to desorption and addition of further gas phase ionization increases the ionization efficiency and sensitivity of intermediate pressure laser desorption ionization and allows for ion molecule reactions with sample components prior to, during or after laser desorption of sample 141.
  • Target plate gas flow 144 aids in directing reagent ions to the surface of sample 141 during the sample charging step.
  • Target plate gas flow 145 exiting target plate chamber 142 through opening 168 in electrode 146 provides a gas load in vacuum stage 160 and, passing through skimmer 149 opening 150 into vacuum stage 161 , provides a local increase in background gas pressure at the entrance of ion guide 154.
  • the flow of target plate gas 145 through electrode 146 serves to collisionally damp translational energy spread of ions generated in the desorption process.
  • the translational energy spread of the desorbed ion population continues to be reduced through collisional cooling in ion guide 154.
  • Desorbed ions can be focused in region 167 by applying the appropriate relative voltages to electrode 146 and skimmer electrode 149.
  • Ions accelerated and focused between electrode 146 and skimmer opening 150 experience collisions with background gas that may increase or decrease internal energy of the ions depending on the rate of acceleration imposed by the applied voltages. If required, ion internal energy can be increased in region 167 to decluster or fragment of ions prior to conducting mass to charge analysis in mass to charge analyzer 158.
  • Intermediate pressure laser desorption ion source mass spectrometer 157 comprises vacuum stages 160, 161 and 162. Sufficient vacuum pumping is provided in each vacuum stage to allow optimal performance of elements within each vacuum stage. Less than three or more than three vacuum stages may be configured in alternative embodiments of the invention to provide optimal performance for specific mass analyzer types.
  • Ion guide 154 as shown in Figure 7 extends into multiple vacuum stages and serves as the gas conductance orifice between vacuum stages 161 and 162. Ions traversing ion guide 154 pass through exit electrode 155 into mass to charge analyzer and detector 158. Voltage applied to exit electrode 155 may be increased relative to the offset potential applied to ion guide 154 to trap ions in ion guide 154. Trapped ions can be released from ion guide 154 by lowering the voltage applied to exit electrode 155. The release of trapped ions from ion guide 154 need not be sychronized with laser pulses in ion source 160 allowing decoupling of mass spectrometer analysis timing with the pulsed production of ions in ion source 160. Ions from multiple laser desorption shots may be stored in ion guide 154 before releasing trapped ions into mass to charge analyzer 158.
  • FIG. 8A shows charging electrode 170 insulated by dielectric insulator 171 in contact with the opposite side of dielectric target plate or belt 172 from sample spots or lines 173. Voltage is applied to charging electrode 170 through Power supply 174.
  • charging electrode 170 is not surrounded by a shielding electrode. This allows the attractive electric field to extend over a broader region on target plate 172 during the charging of sample 173 prior to applying a laser pulse.
  • the additional ions collected during sample charging are available for gas phase ionization of sample molecules after the laser pulse desorption and ion extraction step improving ionization efficiency.
  • Charging electrode 170 can be fixed in position with target plate or belt 172 moving over it or both charging electrode 170 and target plate 172 can be translated independently to optimize performance.
  • Cylindrical shielding electrode 174 is added to the charging electrode assembly 179 in Figure 8B to constrain the electric field formed by charging electrode 175 during the sample charging and desorption and ion extraction steps. Shielding electrode 174 prevents ions in the target gas from being attracted to the back side of target plate or belt 176 during the sample charging step.
  • Charging electrode 175 with shielding electrode 174 insulated by dielectric insulator 177 can be fabricated with very small dimensions. A small diameter charging and shielding electrode assembly contacting a thin target plate or belt allows charging of a small sample area when desorbing sample from specific spatial regions of sample 178.
  • Target plate or belt 176 is moved along the surface of charging electrode assembly 179 while remaining in contact with dielectric material 177 or the tip of charging electrode 175. Higher relative electrical potentials can be applied to charging electrode 179 if it is entirely insulated in dielectric 177.
  • Shielding electrode 174 may be incased in or surrounding insulator 177.
  • Multiple charging electrodes 180 with common shielding electrode 181 are insulated in dielectric 182 that also serves as the sample target surface in target plate assembly 185 shown in Figure 8C.
  • charging electrode and target plate assembly 185 are translated to align laser pulse 186 with each sample spot 187, electrical contact is made with aligned charging electrode 188 and power supply 183 through spring contact 184.
  • Integrated assemblies 185 have the advantage that shorter distances and more reproducible tolerances can be maintained between sample spots 187 and the tip of charging electrodes 188. This allows more reproducible and higher charging of sample surfaces to be achieved for different sample spots and for different target plates.
  • Figure 9A shows a conventional laser desorption target plate 190 typically used for MALDI applications where laser beam 191 impinges on the front side of target plate 190 with no prior charging of sample.
  • target plate or the surface of target plate 190 comprises a conductive material to prevent the buildup of charge during laser desorption operation.
  • the invention comprises elements and configurations that provide improved performance but depart from configurations employed conventional laser desorption ion sources that utilized target plates as shown in Figure 9A.
  • Embodiments of laser desorption target plates shown in Figures 8A through 8C and 9B through 9D contain elements and configurations not employed in laser desorption ion sources found in the prior art.
  • FIG. 9B A diagram of laser desorption target plate assembly 194 comprising fiber optic bundles 195 surrounded by charging electrodes 196 configured in dielectric block 198 is shown in Figure 9B.
  • Sample 202 is deposited on the end of each fiber optic bundle 195 on target plate surface 203.
  • Laser pulse 204 from laser 200 is focused through optical lens assembly 201 and sent through a portion of fiber optic bundle 207 to impinge on the back side of sample spot 208.
  • Laser pulse 204 can be directed to different areas of sample spot 208 by sending laser pulse 204 through different areas of fiber optic bundle 207. This can be achieved by steering laser beam 204 or by moving target plate assembly 194 using x, y and z axis translation.
  • Voltages are applied to charging electrode 197 from power supply 205 through spring contact 206 to allow charging of the surface of sample spot 208 prior to applying laser pulse 204.
  • the embodiment of the invention shown in Figure 9B allows close positioning between a sample and an orifice into vacuum or an adjacent pumping stage.
  • the laser optics are simplified and the laser beam is oriented perpendicular to the sample surface allowing a smaller laser beam spot size.
  • sample spots or lines 208 may be mounted on an optically transparent plate and the plate can be slid over the exit end of fiber bundle 207. This would allow more rapid loading and running of sample plates without the need to clean the exit end of fiber optics bundle 207 between sample runs.
  • a lens may be added to the exit end of fiber optic bundle 207 or incorporated in to a glass target plate to allow tighter focusing of laser beam 204 as it exits fiber optic bundle 207.
  • a liquid sample 210 is introduce through bore 215 of dielectric element 211 of liquid surface laser desorption probe 212 diagrammed in Figure 9C.
  • Charging electrode 213 is electrically insulated from solution 210 in dielectric element 211. If solution 210 has low conductivity or is electrically floating, charge can be accumulated at surface 214 and in bore 215 when a high potential of opposite polarity is applied to insulated charging electrode 213 through power supply 218. Charge species accumulating on the surface of and in liquid 210 are delivered to liquid surface 214 prior to laser pulse 217 as described above for the solid surface laser desorption samples. Liquid 210 can flow through channel 215 or be loaded as a static sample during laser desorption ionization.
  • Desorbed ions can be formed by laser desorption of sample components from water using infared lasers.
  • Glycerol can be used as a liquid surface with low volatility in atmospheric pressure and intermediate pressure laser desorption ion sources. Precharging the liquid surface prior to applying a laser pulse can improve the ionization efficiency of such samples during laser desorption.
  • laser pulse 220 is applied to the underside of liquid sample surface 224 as diagrammed in Figure 9D.
  • Fiber optic bundle 221 passed through dielectric block 227.
  • Liquid sample 225 is introduced through annulus 228 forming sample surface 224 as it exits annulus 228.
  • Charging electrode 229 is electrically insulated in dielectric block 227 with voltage applied through power supply 230.
  • Precharging of electrically floating surface 224 and solution 225 can occur when an opposite polarity electrical potential is applied to charging electrode 226 attracting gas phase charged species to surface 224.
  • laser 222 delivers laser pulse 220 through optical focusing elements 223 and fiber optic bundle 221 to laser desorb sample liquid 225 from surface 224.
  • Liquid sample solution 225 may contain matrix components that absorb the wavelength of laser light used to enhance laser desorption efficiency.
  • Laser desorption ion source 240 comprising target plate chamber 241 with target plate 270 and charging electrode 244 is interfaced to three stage vacuum system 288 with mass to charge analyzer and detector 267.
  • Target plate chamber 241 is separated from endplate electrode 255, focusing electrode 256 and capillary entrance electrode 271 by annular electrode assembly 252.
  • noisy of sight exists between sample 245 and capillary entrance 259 reducing the transport of contamination neutrals and charged particles into vacuum minimizing contamination vacuum ion optics and decreasing chemical noise in acquired mass spectrum.
  • Ion focusing region 272 where ions are focused into vacuum orifice 259 is separated from ion generation region 251 allowing independent optimization of both functions.
  • Charge droplet sprayer 274, employing pneumatic nebulization is positioned in center section 275 of annular electrode assembly 252 with face electrode 253 serving as the ring electrode for charged droplet sprayer 274.
  • Alternative ion generation means as described above for alternative ion source embodiments, can be can be configured in laser desorption ion source 240 replacing pneumatic nebulization charged droplet sprayer 274.
  • charged droplet sprayer 274 is positioned on the centerline 285 of ion source 240 spraying toward sample 245.
  • Target plate gas controller 242 supplies heated target gas 243. If required, ions 247 can be generated in target plate gas controller 242 and delivered to target plate chamber 241 entrained in target gas flow 243.
  • Target plate gas flow 243 exits target plate chamber 241 through opening 287 in target plate counter electrode 250.
  • Target ⁇ plate gas flow 288 entering region 251 directly opposes nebulization gas flow 280 from charged droplet sprayer 274 forming a gas stagnation and mixing region in region 251.
  • the relative voltages applied to charging electrode 244, target plate counter electrode 250, annular electrode 253 and charged droplet sprayer 274 are set to accumulate charge 246 on the surface of sample 245.
  • Target plate gas flow 288 facilitates drying of charged droplets produced from charged droplet sprayer 274.
  • Ions 248 generated from the evaporating charged droplets are directed toward sample 245 by the electric field applied in region 251.
  • Charged species 247 entrained in target plate gas flow 243 are also directed toward the surface of sample 245 by the applied electric field.
  • laser pulse 281 is fired at sample 245 from laser 282 through lens 283 and reflected off mirror 284 as shown in Figure 1OB.
  • relative voltages applied to electrodes 244, 250 and 253 and charged droplet sprayer 274 are changed just before, concurrent with or just after laser pulse 281 is fired to facilitate the release of charged species from sample 245.
  • the timing of the voltage change relative the laser pulse event is optimized to maximize sample ionization efficiency.
  • the voltage applied to electrode 253 remains constant during the sample charging, ion desorption and ion focusing steps.
  • Nebulization gas flow 280 from charged droplet sprayer 274 and target plate gas flow 280 remains on during the sample charging, ion desorption and extraction and ion focusing steps providing a gas phase stagnation and mixing region in region 251 during each operating step.
  • This mixing region facilitates gas phase ionization of desorbed neutral sample molecules by ions 247 entrained in target plate gas flow 288 during the desorption, extraction and ion focusing steps.
  • the relative voltages applied to electrodes 244 and 250 and charged droplet sprayer 274 are changed to optimize ion transmission and focusing into bore 258 of capillary 257 through annulus 292 of annular electrode assembly 252 as illustrated in Figure 10C.
  • Countercurrent drying gas 262 traverses gas heater 261 and flows through the center aperture of endplate,electrode 255. Heated drying gas flow 260 is directed along endplate electrode 255 and through annulus 292 of annular electrode assembly 252. Heated countercurrent gas flow 260 becoming gas flow 277, moves in the opposite direction to ion movement through annulus 292 of annular electrode assembly 252 as ions are directed from region 251 to capillary bore entrance 259 as shown in Figure 10C. Heated countercurrent gas flows 260 and 277 sweep any neutral contamination species away from annulus 292 of annular electrode assembly 252 preventing neutral contamination species from entering vacuum through capillary bore 258.
  • Voltages are applied to the electrodes in electrode assembly 252 to focus and direct ions from region 251 to region 295 and into capillary bore entrance 259. Voltages applied to electrodes 294, 254, 255, 258 and capillary entrance electrode 271 are set to direct desorbed and gas phase generated ions 290 leaving electrode assembly annulus 292 through the center opening in endplate electrode 255 and focus ions 291 into capillary bore entrance 259 as shown in Figure 1OC. Annular electrode assembly 252 decouples ion formation region 251 from the capillary entrance region allowing the performance in both regions to be optimized independently. Gas flows and gas temperatures, surface charging, ionization efficiency and the transport of ions into annular lens assembly 252 can be optimized in region 251.
  • Ion focusing into capillary bore 258 in region 291 is decoupled from variable settings and step sequences occurring in region 251 allowing optimization of ion transport and focusing separate from performance optimization in region 251.
  • Optimization of variables in focusing region 295 increases sensitivity of mass to charge analysis by increasing the efficiency of ion transport into capillary bore 258.
  • Desorbed or gas phase generated ions entering capillary bore 258 pass into vacuum, pass through skimmer 297 and ion guide 266 and are analyzed in mass to charge analyzer and detector 267.
  • Target gas flow 243, pneumatic nebulizer gas flow 280 and countercurrent gas flow 260 and 277 exit laser desorption ion source at gas outlet 298.
  • Laser desorption ion source 240 may be operated at near atmospheric pressure. Alternatively laser desorption ion source 240 can be operated at pressures above one atmosphere to prevent outside contamination from backstreaming into the ion source chamber or at pressures below one atmosphere to accommodate negative pressure venting systems.
  • Electrospray and laser desorption ion source 300 comprises annular electrode assembly 301.
  • Charged droplet sprayer 302 with or without pneumatic nebulization generates charged species that are directed to the surface of sample 303 during the charge accumulation step.
  • Sample is desorbed and ionized by laser pulse 317 fired from laser 310.
  • the ions generated are directed into and through annular electrode assembly 301 by applying the appropriate voltages to back electrode 308, charged droplet sprayer 302, charging electrode 305 and annular electrode assembly 301.
  • Ions exiting annular electrode assembly 301 are focused into bore 313 of capillary 314 moving against countercurrent drying gas 315.
  • Optical imager 309 can be used to image the surface of sample 303.
  • sample ions can be generated from charged droplet sprayer 302.
  • Target plate gas flow 307 aids in drying charged droples 307 produced by charged droplet sprayer 302.
  • Ions generated from the evaporating charged droplets produced by charged droplet sprayer 302 are directed and focused into annulus 318 of annular electrode assembly 301.
  • the charged droplet spray generated ions are directed through annulus 318 and focused into bore 313 of capillary 314.
  • charged droplet sprayer 312 positioned orthogonal to target plate 304 can generate ions for charging sample 303 prior to laser desorption or can generate sample ions directly for mass to charged analysis.
  • Annular lens assembly 301 configured in multiple ionization type ion source 300 decouples the ion production region from the ion focusing region into bore 313 of capillary 314 allowing decoupled optimization of each region and reducing mass spectrum noise from neutral contamination components entering vacuum.
  • the sensitivity of mass to charged analysis is increased by the improved focusing of ions passing though regions 319 and 320 into capillary bore 313.
  • Laser desorption of sample 304 and Electrospray ionization of a sample solution can occur simultaneously or independently in ion source 300.
  • Running Electrospray simultaneously with laser desorption ionization allows gas phase ion-molecules reactions or the addition of known internal calibration peaks during mass spectrum acquisition.
  • the charging of a sample surface prior to conducting laser desorption can improve the efficiency of ion production in vacuum.
  • Time-Of-Flight mass to charge analysis of ions generated from laser desorption or matrix assisted laser desorption in vacuum is well known in the art.
  • Charging of sample surfaces prior to laser desorption can reduce mass measurement accuracies and resolving power in conventional MALDI TOF mass to charge analysis.
  • the steps of ion desorption and acceleration into the TOF flight tube are coupled, the kinetic energy of the desorbed ion species can effect the ion flight time.
  • Charging of the ion surface can change the desorbed ion energy from laser shot to laser shot modifying the flight time of the desorbed ion species.
  • Time delay acceleration of ions into the TOF pulsing region after a laser pulse can reduce the effects of initial ion energy spread and neutral gas interference but cannot compensate entirely for shot to shot differences in surface charging.
  • Charging of a sample prior to a laser pulse in vacuum can be used in TOF mass to charge analysis if the laser desorption step and subsequent acceleration of ions into the TOF flight tube are decoupled.
  • US Patent Number US 6,683,301 B2 (US patent '301) incorporated herein by reference, describes the apparatus and method for decoupling the steps of laser desorption of a sample in vacuum and subsequent pulsing of the ions generated into a TOF flight tube ( for mass to charged analysis.
  • ions generated in the laser desorption step are directed to and trapped above a surface in near field potential wells formed by a high frequency electric field.
  • the trapped ion population is subsequently accelerated into the TOF flight tube.
  • Charging of the sample surface prior to the laser desorption step can be incorporated into such an apparatus and method to improve ionization efficiency or to conduct ion molecule reactions prior to laser desorption as diagrammed in Figures 12A through 12D.
  • FIG. 12A An alternative embodiment of the invention is diagrammed in Figure 12A through 12D mounted in vacuum chamber 340.
  • Figure 12A illustrates the step of charge accumulation on the surface of sample 341 positioned on dielectric target plate 342.
  • Charge is accumulated on the surface of sample 341 by directing ion beam 345 to the surface of sample 341 by applying the appropriate focusing and accelerating potentials to charging electrode 346, focusing electrode set 344, target plate counter electrode 347, TOF pulsing region entrance electrode 348, trapping surface 350, trapping electrode 349, and ion accelerating electrodes 351 , 352 and 353.
  • Ion beam 345 is generated by ion source 343 operating in vacuum.
  • Ion source 343 may be an electron bombardment, chemical ionization, glow discharge, or other vacuum ion source known in the art.
  • laser pulse 358 is directed to sample 341 from laser 359 through optical lens 360 and reflected off mirror 361 as shown in Figure 12B.
  • the voltages applied to electrodes 346, 347, 344, 348, 349, 351 and trapping surface 350 are changed to direct the population of desorbed ion species 362 toward trapping surface 350 and trap desorbed ions 362 above trapping surface 350 as shown in Figures 12B and 12C.
  • the reduction of kinetic energies of ions 365 trapped above dynamic electric field trapping surface 350 may be achieved by ion collisions with neutral background gas or by laser cooling of ions.
  • Sufficient neutral background gas may be locally present in TOF pulsing region 364 to reduce trapped ion kinetic energy or neutral gas may be added to TOF pulsing region 364 through a pulsed gas valve.
  • laser cooling may be applied to reduce the trapped ion kinetic energy.
  • Laser pulse or beam 358 can be redirected toward trapping surface 350 by moving the angle of mirror 361 and the laser power can be reduced by defocusing laser pulse 358 using lens 360 or reducing the power output of laser 359. After the kinetic energy spread of trapped ions 365 has been reduced, voltages are changed on trapping surface 350, electrode 349 and grid electrodes
  • Grid electrode 353 forms part of the TOF flight tube and the voltage applied to electrode 353 and remains constant during the sample charging, laser desorption, ion trapping and ion acceleration steps described above.
  • TOF flight tube may comprise a linear flight path or be configured with one or more ion reflectors to increase mass to charge analysis resolving power.
  • Multiple sample charging and laser desorption steps may be conducted for each step of accelerating ions into TOF Flight tube 355. This will increase analytical speed if the trapped ion kinetic energy cooling step is the longest step in the ion charging, desorption, extraction and analysis sequence.
  • Target plate 342 can be rotated or translated to move different samples into position or to optimize the sample position relative to the tip of charging electrode 346 and laser pulse 358. Optical imaging of the sample may be performed to direct adjustment of the sample surface for optimal performance.
  • Target plates are removed and replaced by the changing of flange 370.
  • Flange 370 may be replaced with an automatic target plate loading and pumpdown system that allows removal and loading of target plate 342 without venting TOF flight tube vacuum chamber 340.
  • the flatness tolerance, dimensional reproducibility and material selection of target plate 342 are relaxed in the embodiment of the invention shown. This reduces cost and improves selection of materials that may be more compatible with specific samples.
  • Sample charging prior to laser desorption can be configured with ion guides in atmospheric pressure, intermediate pressure and vacuum laser desorption ion sources.
  • US Patent Number US 6,707,037 B2 (US patent O37) incorporated herein by reference describes laser desorption ion sources comprising multipole ion guides configured in atmospheric pressure, intermediate pressure and vacuum regions.
  • the step of charge accumulation on or near the sample surface prior to applying a laser desorption pulse can be added to embodiments described in US patent '037.
  • Separately generated reagent ions can be i introduced axially through the ends of multipole ion guides or radially through the gaps between rods in multipole ion guides prior to applying a laser desorption pulse to a sample.
  • the added reagent ion charge can accumulate on the sample surface or be trapped in the multipole ion guides to enhance ion-molecule reaction gas phase ionization of neutral desorbed components through ion-molecule reactions.
  • Reagent ions of the opposite polarity can be added to the multipole ion guide volume to promote gas phase ion-ion reactions.
  • the addition of an electron to multiply charged positive polarity ions through ion-ion gas phase reactions may lead to positive ion fragmentation through electron capture or electron transfer fragmentation mechanisms.
  • Ions generated through laser desorption or gas phase ion-molecule reactions are directed through the ion guide to a mass to charge analyzer for mass to charge analysis employing methods and apparatus as described in US patent '037.
  • Other ion guides such as sequential disk RF ion guides or other ion guide types known in the art may be used as an alternative to the multipole ion guide embodiments.
  • Ions generated in the laser desorption ion sources described above alternatively be analyzed using ion mobility analyzers or combinations of ion mobility analyzers with mass spectrometers.
  • Configuration and operation of the embodiments of laser desorption ion source as described above provide performance improvements as described above and as listed below: a) By precisely timing and positioning the laser desorption process to coincide with a potential pulse to the sample, the sample can be desorbed and ionized from the target in optimum electric fields and flow leading to efficient extraction of ions from the target, and by subsequently cycling the electric potential to more appropriate focusing fields the ions can be more efficiently focused and transmitted to and through the conductance opening to lower pressures. b) By charging the sample surface with reagent ions or electrons prior to the laser desorption process, the ionization process can occur more efficiently.
  • higher pressures such as at atmospheric pressure
  • the device By introducing sample from a liquid stream such as capillary electrophoresis or liquid chromatography, the device can operate as a continuous interface for LC/MS or CE/MS. g) By controlling the extraction and focusing fields in a time-sequence to optimize both processes, the alignment and position of the sample relative to the conductance opening is less critical. h) By using optical alignment instead of positional alignment of sample and conductance opening, the loading of the sample into the source becomes much easier and the nature of the sample (e.g. direct tissue samples, direct 2D gels or western blots, flowing sample) can be far more diverse than conventional MALDI spots.
  • a liquid stream such as capillary electrophoresis or liquid chromatography

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Abstract

L'invention concerne des procédés d'ionisation à la pression atmosphérique, à pression intermédiaire et de désorption-ionisation laser sous vide et des sources d'ions qui sont conçus pour augmenter le rendement de l'ionisation et le rendement de transmissions d'ions à une masse afin de charger l'analyseur ou l'analyseur de mobilité des ions. Un champ électrique est appliqué sur la région d'un échantillon cible afin d'accumuler les ions générés par une sources d'ions locale sur un échantillon en phase solide ou liquide avant d'appliquer une impulsion de désorption laser. Le champ électrique est modifié juste avant ou pendant l'impulsion de désorption laser afin de stimuler la désorption de l'espèce chargée et d'améliorer le rendement d'ionisation de l'espèce de l'échantillon désorbé. Après un certain délai, le champ électrique peut être de nouveau modifié afin d'optimiser la focalisation et la transmission d'ions à un spectromètre de masse ou à un analyseur de mobilité des ions. L'espèce chargée peut être également ajoutée à la région du panache de l'échantillon désorbé afin de stimuler les réactions ions-molécules entre les ions ajoutés et l'espèce de l'échantillon neutre désorbé, renforçant ainsi le rendement d'ionisation de l'échantillon désorbé et/ou créant une espèce d'ion souhaitée. Les cycles de modifications du champ électrique sont répétés dans une séquence chronométrée à raison d'une ou plusieurs impulsion de désorption laser par cycle de modification du champ électrique. Des modes de réalisation selon l'invention font intervenir des procédés et des dispositifs d'ionisation à la pression atmosphérique, à pression intermédiaire et de désorption-ionisation laser sous pression source afin d'augmenter la flexibilité analytique et d'améliorer la sensibilité d'analyse par spectrométrie de masse.
EP04754443.2A 2003-06-07 2004-06-07 Source d'ions de desorption laser Expired - Lifetime EP1639622B1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA2800040C (fr) * 2002-10-29 2015-12-29 Target Discovery, Inc. Procede permettant d'accroitre l'efficacite d'ionisation en spectroscopie de masse
DE102004002729B4 (de) 2004-01-20 2008-11-27 Bruker Daltonik Gmbh Ionisierung desorbierter Analytmoleküle bei Atmosphärendruck
US7737395B2 (en) * 2006-09-20 2010-06-15 Agilent Technologies, Inc. Apparatuses, methods and compositions for ionization of samples and mass calibrants
CZ304648B6 (cs) * 2007-12-03 2014-08-20 Masarykova Univerzita Způsob přípravy vzorků pro atomové spektrometrické techniky
US8704169B2 (en) 2011-10-11 2014-04-22 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Direct impact ionization (DII) mass spectrometry
WO2016142681A1 (fr) 2015-03-06 2016-09-15 Micromass Uk Limited Analyse spectrométrique de microbes
JP6783240B2 (ja) 2015-03-06 2020-11-11 マイクロマス ユーケー リミテッド 生体内内視鏡的組織同定機器
EP3265822B1 (fr) 2015-03-06 2021-04-28 Micromass UK Limited Analyse tissulaire par spectrométrie de masse ou par spectrométrie de mobilité ionique
CN112964625B (zh) 2015-03-06 2024-06-07 英国质谱公司 细胞群体分析
CN107533032A (zh) 2015-03-06 2018-01-02 英国质谱公司 用于从块状组织直接映射的原位电离质谱测定成像平台
CN107530064B (zh) 2015-03-06 2021-07-30 英国质谱公司 气态样品的改进电离
CN110706996B (zh) 2015-03-06 2023-08-11 英国质谱公司 用于改进电离的碰撞表面
EP3570315B1 (fr) 2015-03-06 2024-01-31 Micromass UK Limited Analyse par spectrométrie de masse par ionisation par évaporation rapide et spectrométrie de masse par ionisation par electronébulisation par désorption d'échantillons de biopsie
CA2981085A1 (fr) 2015-03-06 2016-09-15 Micromass Uk Limited Analyse spectrometrique
GB201517195D0 (en) 2015-09-29 2015-11-11 Micromass Ltd Capacitively coupled reims technique and optically transparent counter electrode
US11454611B2 (en) 2016-04-14 2022-09-27 Micromass Uk Limited Spectrometric analysis of plants
EP3909067A1 (fr) * 2019-01-11 2021-11-17 Helmholtz-Zentrum Potsdam - Deutsches GeoForschungsZentrum GFZ Stiftung des Öffentlichen Rechts des Lands Brandenburg Source d'ions comprenant un échantillon structuré pour ionisation améliorée
CN111912895A (zh) * 2019-05-09 2020-11-10 岛津分析技术研发（上海）有限公司 真空下的质谱成像装置及方法
WO2022133593A1 (fr) * 2020-12-21 2022-06-30 Atomic Energy Of Canada Limited / Énergie Atomique Du Canada Limitée Appareil de source d'ions laser compact et procédé correspondant

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5625184A (en) * 1995-05-19 1997-04-29 Perseptive Biosystems, Inc. Time-of-flight mass spectrometry analysis of biomolecules
US5663561A (en) * 1995-03-28 1997-09-02 Bruker-Franzen Analytik Gmbh Method for the ionization of heavy molecules at atmospheric pressure
GB2348049A (en) * 1999-03-17 2000-09-20 Bruker Daltonik Gmbh An ion source for mass spectrometry

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3125335A1 (de) * 1981-06-27 1983-01-13 Alfred Prof. Dr. 4400 Münster Benninghoven Verfahren zur analyse von gasen und fluessigkeiten
US5965884A (en) 1998-06-04 1999-10-12 The Regents Of The University Of California Atmospheric pressure matrix assisted laser desorption
US6744041B2 (en) 2000-06-09 2004-06-01 Edward W Sheehan Apparatus and method for focusing ions and charged particles at atmospheric pressure
US6683301B2 (en) 2001-01-29 2004-01-27 Analytica Of Branford, Inc. Charged particle trapping in near-surface potential wells
EP1402561A4 (fr) 2001-05-25 2007-06-06 Analytica Of Branford Inc Source d'ions maldi atmospherique et sous depression
US6707036B2 (en) 2002-03-21 2004-03-16 Thermo Finnigan Llc Ionization apparatus and method for mass spectrometer system
US6707039B1 (en) 2002-09-19 2004-03-16 Agilent Technologies, Inc. AP-MALDI target illumination device and method for using an AP-MALDI target illumination device

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5663561A (en) * 1995-03-28 1997-09-02 Bruker-Franzen Analytik Gmbh Method for the ionization of heavy molecules at atmospheric pressure
US5625184A (en) * 1995-05-19 1997-04-29 Perseptive Biosystems, Inc. Time-of-flight mass spectrometry analysis of biomolecules
GB2348049A (en) * 1999-03-17 2000-09-20 Bruker Daltonik Gmbh An ion source for mass spectrometry

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of WO2004112074A2 *

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EP1639622A4 (fr) 2007-11-21
CA2527886C (fr) 2014-01-14
EP1639622B1 (fr) 2016-11-16
WO2004112074A2 (fr) 2004-12-23
WO2004112074A3 (fr) 2005-10-06
CA2527886A1 (fr) 2004-12-23

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