阅读说明：本技术 飞行时间质谱仪中具有虚拟轴追踪的解吸束控制 (Desorption beam control system with imaginary axis tracking in time of-flight mass spectrometer ) 是由 塞巴斯蒂安·博姆 于 2019-05-24 设计创作，主要内容包括：本发明涉及具有样品脉冲电离的飞行时间质谱仪,例如通过基质辅助激光解吸(MALDI),其中样品位于样品支架上并在网格中通过位置控制解吸束一个接一个地照射和电离。离子光学拉制透镜装置位于样品支架的前面,该装置中,透镜光阑中的至少一个被细分为多个区段,并且电压源能够取决于解吸束在支撑板上的冲击位置为这些区段,或者其中一些区段供应不同的电压。然后可以将透镜的有效离子光学聚焦中心虚拟地偏离轴,并将从实际透镜轴产生的离子束聚焦成基本平行于实际透镜轴的光束,并且对于相同质量的离子没有时间相移。该光束可以通过x/y偏转单元返回到轴上,例如用于操作具有反射器的飞行时间质谱仪。(The present invention relates to the time of-flight mass spectrometers ionized with sample pulse, such as by substance assistant laser desorpted (MALDI), wherein sample is located on sample holder and desorbs beam by position control within a grid and irradiates and ionize one by one.Ion-optical draws lens devices and is located at before sample holder, in the device, at least one of lens stop is subdivided into multiple sections, and it is these sections that voltage source, which can depend on the impact position of desorption beam on the supporting plate, or some of sections supply different voltage.Then can be by the effective ion optical focus center of lens virtually off-axis, and by the ion beam focusing generated from actual lens axis at the light beam for being basically parallel to actual lens axis, and there is no time-phase displacement for the ion of phase homogenous quantities.The light beam can be returned on axis by x/y deflection unit, such as operating the time of-flight mass spectrometer with reflector.)

1. the method for operating time of-flight mass spectrometer, the described method comprises the following steps:

Pulse ionization is carried out to the sample placed on sample holder in an ion source using desorption beam, wherein desorption beam is in portion It deflects in timesharing from the axis of ion source with inswept sample surfaces, also,

Ion is accelerated to enter flight path by the diaphragm as ion optical lens, wherein at least one of diaphragm is thin It is divided into multiple sections, and provides asymmetrical voltage for section, the deflection with desorption beam is mutually coordinated, so that in desorption beam spot partially By the lens of center deviation axis with ion beam is mutually accelerated into, which works the ion generated off axis in diaphragm, it is described from Beamlet is parallel to axis extension.

2. according to the method described in claim 1, the diaphragm is wherein divided into multiple half portions, tetrad or octant, and For wherein it is all or it is a part of asymmetrical voltage is provided, with desorption beam deflection mutually coordinate.

3. according to the method described in claim 1, laser beam or primary ion beam (SIMS) are wherein used as desorption beam.

4. according to the method described in claim 3, wherein ion source is to pass through the electricity of substance assistant laser desorpted (MALDI) progress From operating.

5. the x-y is inclined according to the method described in claim 1, wherein being brought back to ion beam on axis using x-y deflection unit Turn unit with the voltage supply that can be adjusted in ion source downstream, the deflection with desorption beam is coordinated.

6. according to the method described in claim 1, wherein the potential of sample holder, another accelerate diaphragm potential and/or its In be extended with flight path tof tube deflection of the potential by the supply adjustment of the voltage that can appropriately adjust, with desorption beam Coordinate.

7. according to the method described in claim 1, it is more than 50 microns that wherein desorption beam hot spot, which deviates the axis of ion source,.

8. according to the method described in claim 5, wherein computing unit control desorption beam deflection and in the segment of diaphragm, Potential is set on sample holder and/or on x-y deflection unit.

9. according to the method described in claim 8, wherein the program in the computing unit is as the position letter for desorbing beam hot spot Number calibrates the voltage that can be adjusted automatically.

10. time of-flight mass spectrometer, the time of-flight mass spectrometer have for using desorption beam to being placed on sample holder Sample carry out pulse ionization ion source, wherein the ion source have as ion optical lens diaphragm, to accelerate Ion enter flight path and to position control so that desorption beam deviate ion source axis,

Wherein,

At least one of described diaphragm is subdivided into multiple sections, and supplying at least some sections of diaphragm should be able to be independent The voltage of adjusting so that asymmetrical voltage in respective section generate in off-axis desorption beam spot generated ion from Effective lens centre of axis, and ion is parallel to described by the lens centre with ion beam, the ion beam is mutually accelerated into Axis extends.

Technical field

The present invention relates to the time of-flight mass spectrometer ionized with sample pulse, the sample is located on bracket, wherein Multiple individual samples or multiple sites on spatially extended sample are irradiated and are ionized one by one within a grid, example Such as by pulse laser, there is the laser of the location-controlled for substance assistant laser desorpted (MALDI) to focus；Or pass through The primary ion beam of location-controlled for Secondary Ion Mass Spectrometry (SIMS).

Background technique

The prior art is explained below with reference to particular aspects, especially MALDI flight time mass spectrum.However, this should not be by It is interpreted as limiting.Known useful further development and modification can also be in the relatively narrow models of this introduction from the prior art It uses except enclosing, and will be apparent for those skilled in the art after reading following disclosure.

10 2,011 112 649 B4 of patent specification DE (" Laserspotsteuerung in MALDI- Massenspektrometern"；A.Holle et al.；Corresponding to 8,872,103 B2 of GB 2 495 805 B and US) it explains How in MALDI mass spectrograph, the positioning of laser facula is controlled between spectra collection twice, is allowed in the form of a grid The sample that scanning space extends, such as tissue sample, to generate the mass spectrum image of sample.It is located in 100 microseconds and carries out, therefore Allow per second 10⁴A mass spectrographic acquisition rate.Mass spectrum image corresponds to color image, wherein each point (each pixel) of image Comprising full mass spectrum rather than chromatography.

10 2,011 112 649 B4 of patent specification DE and its all the elements will be included herein by reference.Until drawing The prior art for entering laser facula control is also so to be described in detail in the specification.

Laser facula control has been that imaging mass spectrum brings promotion.Its linear consistent movement with sample holder is together It carries out, to scan up to 1 square centimeter or bigger of tissue surface.But there are hundreds if not thousands of samples on sample holder The high-throughput mass spectrography of product also benefits from laser facula control.

Unfortunately, usually by stepping motor generate sample holder movement never substantially uniformity and usually Also it will receive the interference of oscillatory process.Therefore, mass spectrographic acquisition is carried out using static and stable sample holder to be advantageous.But It is for static sample holder, laser facula control can only scan the maximum 100 microns squares for multiplying 100 microns, because such as Fruit ion beam is by the way that with the drawing lens of the distance deviation lens axis more than 50 microns, which corresponds at the position Flight axis flight path, then the ion of phase homogenous quantities no longer by drawing lens with mutually accelerate.Due to phase shift, Xiang Tongzhi The no longer same Xiang Feihang of the ion of amount, thus they slightly different times reach detector, the result is that mass resolution by Limitation.

Ion in ion source is accelerated to different speed due to its different quality.Lighter ion ratio is heavier Ion reaches ion detector earlier.It in ion detector, measures ion stream and is digitized, carry out two per nanosecond and arrive Eight measurements.The flight time of ion is determined according to measurement result, and the quality of ion is determined according to the flight time.Such as this field Known to technical staff, velocity focusing reflector can use to improve resolution ratio.The ion acceleration especially postponed (is in German Delay is extracted) ion that can effectively in addition focus phase homogenous quantities again, although it is initially as caused by the plasma cloud extended The wide in range distribution of primary power.It corresponds to the prior art, by about 30 to 1000 individual time of flight spectrum of a sample It is added together to the mass spectrum to form summation time of flight spectrum and therefrom obtain sample.Good time of-flight mass spectrometer is used at present The mass resolution of R=m/ Δ m > 50 000 is realized in the wide mass range of 1000Da < m/z < 4000Da.Present quality Precision reaches the numerical value of the magnitude of hundred a ten thousandth of quality (1ppm).

For many years, the laser technology for MALDI time of-flight mass spectrometer has greatly improved.Not only by laser facula point It is cleaved into several intensity peaks and is widely used under " smartbeam " this title, but laser irradiation frequency is also from purple Initial 20 times per second of outside line nitrogen laser increase to 10000 times per second for using ultraviolet solid-state laser today, it means that Only 100 microseconds can be used for obtaining time of flight spectrum, and change the position of laser facula.By nanosecond per in detector five times Ion stream is measured, single time of flight spectrum is made of 500000 measurements.As already mentioned, pass through measurement again and again The individual time of flight spectrum being added together from one sample acquisition 30 to 1000, to form summation time of flight spectrum.So Afterwards thus to obtain the mass spectrum of sample.

The special applications of the technology with high laser irradiation rate are " the imaging mass spectrography " of thin tissue section, use Hundreds of thousands of a mass spectrums are up in obtaining from thin tissue section.As original color image includes whole chromatogram one in each pixel Sample, mass spectrum image include complete mass spectrum in each pixel.Nowadays, using the pixel separation from 50 microns to 20 micron, and And following target is 10 microns even 5 microns of separation.From one square centimeter of thin tissue section, with 50 microns of resolution Rate obtains 40000 mass spectrums, and under 10 microns of resolution ratio, there are 1,000,000 mass spectrums.In addition, the mass spectrum of a pixel Usually obtained by being added together to each time of flight spectrum of 30 to 1000 laser irradiations to form summation time of flight spectrum , the mass spectrum of pixel is then obtained from the spectrum.The quantity for each time of flight spectrum being added together in each case is bigger, Detection limit and signal-to-noise ratio become better.However, being not always that the single of arbitrarily large quantity can be obtained and be superimposed from same point to fly Row time spectrum, because sample usually exhausts quickly.

In addition, purpose of today also resides in the uniform utilization for realizing the Free Region of sample sites, and therefore using available Analyte molecule obtain individual time of flight spectrum.For today be used for using substance assistant laser desorpted (MALDI) into The preparation of the thin tissue section of row ionization applies one layer of small host material crystal on slice, solvable in slice Property peptide and protein are transported to the crystal of top layer.Using the preparation of these thin layers, if speckle patterns do not move, laser Analyte molecule under hot spot exhausts after three to five laser irradiations.Herein, the laser point guidance of location-controlled facilitates Different, still not used point is burnt every time.However, up to the present, in order to realize to the highly uniform of sample surface Burn, need additional mobile example bracket.But due to oscillation, as a consequence it is hardly possible to realize the highly uniform fortune of sample holder It is dynamic.

In view of the foregoing, it needs to promote on relatively large region, such as partly to the matter on one square millimeter of area The latticed acquisition of spectrum, and sample holder remains static, to analyze the sample with high spatial density, such as tissue sample For mass spectrum to be imaged.This allows to longer time interval mobile example bracket and allows to make sample for some time The vibrational stabilization of bracket is without big loss in efficiency.

Summary of the invention

In view of the introduction, this disclosure relates to a kind of method for operating time of-flight mass spectrometer comprising following step It is rapid :-using desorption beam, such as laser beam (especially for MALDI) or primary ion beam (especially for SIMS), to from The sample deposited on sample holder in component carries out pulse ionization, wherein axis of the desorption beam in part-time from ion source is inclined Turn with inswept sample surfaces, also ,-accelerate ion to enter flight path by the diaphragm as ion optical lens, wherein light At least one of door screen is subdivided into multiple sections (for example, half portion, tetrad or octant), and provides asymmetry for section Voltage (especially for all sections, or regions some at least within provide individual voltage), the deflection with desorption beam is mutually assisted Adjust so that the ion that off-axis generates in desorption beam spot by the lens of center deviation axis with being mutually accelerated into ion beam, the lens It works in diaphragm, the ion beam is parallel to axis extension.

Therefore, above-mentioned purpose is particularly solved by the way that drawing lens devices are placed on before sample holder, wherein At least one lens stop is subdivided into multiple sections, such as half portion, tetrad or octant, and voltage source can be these Section or sections some at least within provide different voltage.It then can be by effective focusing center of lens virtually off-axis Line；According to the deflection of desorption beam, the ion beam for deviateing the generation of actual lens axis, which can focus to become, is parallel to actual lens axis Light beam, and the ion of phase homogenous quantities does not have time-phase displacement.

When focusing center's strong deflection, the slightly ob-round shape of equipotential lines around center.This causes in two phases The case where mutually there are different focusing forces on vertical direction, and the ion beam for creating substantially uniformity is a challenge.If example Such as, lens stop is divided into multiple octants with eight separately controllable voltage sources, then can produce in particular round Focusing center.In the simplest embodiment, furthermore by diaphragm be subdivided into three sections (each section cover about 120 °) or Bigger odd number section it is also conceivable that, although this asymmetrical design is not preferred because it is caused in order to Mobile lens center and the calculating of deflection voltage carried out is very complicated.Diaphragm can be subdivided into multiple sections, for example, eight Fission, what four sections in only one of them subset, such as eight were supplied with the function of the deflection as desorption beam can be single The voltage solely adjusted.

In various embodiments, x-y deflection unit can be used to bring back to ion beam on axis, the x-y deflection unit With the adjustable voltage supply in ion source downstream, the deflection with desorption beam is coordinated.When this is particularly suitable for reflector flight Between mass spectrograph, the incidence angle that especially wherein incidence point and ion beam enter reflector can influence reflex behavior.

In various embodiments, the current potential that sample holder can be adjusted by variable voltage source, the deflection phase with desorption beam Coordinate.Due to virtual lens do not have identical off-axis focal length, and due to potential well have different depth, cannot for from Son provides identical accelerating curve, it is thus possible to also need to change voltage (and/or another acceleration voltage on sample holder And/or the other parts of the tof tube where flight path), to generate the identical dependence with mass of ion to the flight time The time of flight spectrum of property.

May and it is envisioned that make desorb beam hot spot deviate ion source axis be more than 50 microns, be especially up to 250,300 or even 500 microns (and focusing center that diaphragm is virtually tracked by each voltage of appropriate adjustment).Work as acceleration When the diameter of bore for spending diaphragm is 3 to 5 millimeters, effective focusing center can also move about half millimeter.

In various embodiments, computing unit can control the deflection of desorption beam and in the segment of diaphragm, sample branch Potential is set on frame and/or on x-y deflection unit (if it is necessary, and in the other parts of tof tube).Most preferably, Program in computing unit calibrates adjustable voltage as the position function of desorption beam hot spot automatically.The flight time matter of these types Spectrometer has computing unit, which desorbs beam by process control.These programs can also be turned by suitable digital-to-analogue Parallel operation (DACs) control diaphragm section on voltage, the correction voltage on sample holder, x-y deflection unit (if present) and/ Or the voltage on tof tube other component.

Present invention is equally related to a kind of time of-flight mass spectrometer, have for using desorption beam to being put on sample holder The sample set carries out the ion source of pulse ionization, and wherein the ion source has the diaphragm as ion optical lens, to accelerate Ion enters flight path and to position control so that desorption beam deviates the axis of ion source.It is characterized in that, in diaphragm At least one at least some section for being subdivided into multiple sections and diaphragm can be separately adjustable voltage supply so that accordingly Asymmetrical voltage on section generates off-axis effective lens centre for generated ion in off-axis desorption beam spot.It should By ion with ion beam is mutually accelerated into, the axis which is parallel to ion source extends for lens centre.It should be appreciated that tying above The embodiment that conjunction this method illustrates also can be applied to the time of-flight mass spectrometer as device.

Detailed description of the invention

The present invention may be better understood by reference to illustrated below.Element in diagram is not drawn necessarily to scale, and It is mainly used for showing the principle of the present invention (mainly schematical).

Fig. 1 is the schematic diagram of MALDI time of-flight mass spectrometer according to prior art, with ToF analysis instrument 1 With the laser system 2 for controlling position of the laser facula of light pulse on sample holder 13 by mirror system 7,8.It is produced in light beam Laser pulse is generated in raw unit 3, which includes laser crystal 4 and if necessary also include for frequency multiplication Device 5；The laser pulse is divided into spot pattern in pattern generator 6；And by two galvanometer mirrors 7 and 8 in mirror surface It is upward deflected in system in two spaces side.Then, the laser beam of deflection extends in Kepler's mirror tube 9, and inclined according to angle It is parallel for walking around.Then, the laser beam of outgoing is directed into object lens 11 by mirror surface 10 with reduced angular deflection, so as to complete It is complete placed in the middle.Depending on angular deflection, light beam passes through object lens 11 at center, but with slightly different angle, thus mobile light Position of the spot pattern on sample holder 13.The ion generated in the plasma cloud of laser spot pattern is by diaphragm 14 and 15 On voltage accelerate, to form ion beam 18, which is corrected its track by two deflection capacitors 16 and 17 and will It is focused in reflector 19 on detector 20.It should be noted there that the light beam guidance in Kepler's mirror tube 9 is more multiple It is miscellaneous, and for simplicity, diagram is unable to actual reproduction, although seeing from the outside, diagram has correctly reappeared mirror tube pair really The influence of laser beam.

Fig. 2 and Fig. 3 depicts the equipotential lines in ion optical lens, is made of in the example shown multiple quadrants.Such as All four quadrants of fruit all provide identical voltage U1=U2=U3=U4, then equipotential lines is circle, and effective focusing center is located at Intermediate (Fig. 2).If asymmetricly applying voltage, such as U1=U2 ≠ U3=U4, i.e., have in this example and be organized in pairs, to the greatest extent Pipe depends on concrete condition it is also contemplated that perfectly asymmetric voltage (U1 ≠ U2 ≠ U3 ≠ U4), potential minima can also occur Offset, therefore, effective focusing center of lens is displaced outwardly a small distance (Fig. 3) from centre.The depth of focus power and potential well Degree also changes herein, but they can by sample holder (or if feasible on flight path Other apertured electrodes or tof tube itself) it is compensated using slightly different ion accelerating voltage.

Fig. 4 depicts the enlarged drawing of the ion source of the device according to Fig. 1, but the drawing lens 14 here in Fig. 1 are thin It is divided into two lens stops 14a and 14b, and the section of two equipotential surfaces 22 has carried out superposition to show the function of lens.It will Voltage is applied to lens stop, so that the formation of equipotential surface 22 is penetrated across the potential of diaphragm 14a, to form ion lens.Solution It inhales beam (not shown) and generates ion on the axis 21 of device here；The ion beam slightly dissipated is by lens forming collimated light beam.Phase The ion 24 of homogenous quantities forms the forward position perpendicular to beam axis.

In Fig. 5, the ion deflection axis 21 of beam (not shown) generation device is desorbed.However, lens 14a, 14b are generated again Collimated light beam, the collimated light beam are tilted relative to axis, and are redirect on axis by deflection unit 16,17.In such case Under, the ion 25 of phase homogenous quantities does not re-form the forward position perpendicular to ion beam axis.This means that they cannot reach ion simultaneously Detector；Resolution ratio reduces.

In Fig. 6 for illustrative purposes, lens stop 14c is portrayed as quadrant diaphragm, as shown in Figure 3.Voltage is applied Onto lens, so that equipotential surface 23 forms the effective focusing center (the focusing potential well penetrated) of off-beams axis and is formed slightly The ion of micro- diverging, these ions form collimated light beam except the axis 21 of the device again.The light beam is now parallel to axis 21 and prolongs It stretches and can be by doubling deflection unit 16a, 17a, 16b, 17b returns to axis 21, such as in order to most preferably enter reflection Device.By the focusing center of mobile lens, fly the ion 26 of phase homogenous quantities in the forward position perpendicular to the axis of ion beam again Row.Therefore, the ion of phase homogenous quantities reaches detector simultaneously；Although desorption beam is deflected across sample surfaces, keep differentiating Rate.

Fig. 7 shows the pattern of the laser facula with nine independent intensity peaks for MALDI ionization.This pattern is special It is not advantageous, because it combines highly sensitive and low sample consumption.The diameter at each peak is about 5 microns；Between between each peak Every also reaching 5 microns.

Fig. 8 illustrates how to be ionized using MALDI with 32 laser irradiations (square of lower right), uses the figure of Fig. 7 Case is accurately sampled having a size of 60 × 60 microns of square pixels.In general, before sample exhausts, it can be to thin section matrix Coating carries out about four to five times samplings, therefore the summation light of about 120 to 150 individual spectrals can be obtained from the pixel Spectrum.

Specific embodiment

Although by reference to multiple embodiment description and interpretation present invention, it will be recognized to those skilled in the art that In the case where not departing from the range of technical teaching defined in appended claims, it can be carried out in form and details Various changes.

The inspiration that the present invention is controlled by fast laser hot spot, as shown in Figure 1.Fig. 1 is according to patent specification DE 10 The schematic diagram of the MALDI time of-flight mass spectrometer of 2011 112 649 B4, with ToF analysis instrument 1 and laser system 2, the light pulse on the sample holder 13 in the control mass spectrograph of revolving mirror 7,8 can be turned to by means of two in laser system Laser spot position.Generate laser pulse in beam generation unit 3, beam generation unit include laser crystal 4 and if It needs to further include the device 5 for frequency multiplication, which is separated into dot pattern in pattern generator 6, and passes through two Galvanometer mirror 7 and 8 is upward deflected in two spaces side.Then, the laser beam of deflection extends in Kepler's mirror tube 9, and according to Angular deflection moves in parallel.Then, the laser beam of outgoing is directed into object lens 11 by mirror surface 10 with reduced angular deflection, So as to completely placed in the middle.Depending on angular deflection, light beam passes through object lens 11 in centre, but with slightly different angle, to move Dynamic position of the spot pattern on sample holder 13.The ion generated in the plasma cloud of laser spot pattern is by diaphragm 14 Accelerate with the voltage on 15, to form ion beam 18, the ion beam is by two deflection capacitors 16 and 17 to correct its track And it is focused on detector 20 in reflector 19.It should be noted there that the light beam guidance in Kepler's mirror tube 9 is more Complexity, diagram is unable to actual reproduction, although seeing from the outside, diagram has correctly reappeared influence of the mirror tube to laser beam really.

It is further noted that the linear operation of Time-of flight analyzer 1 is can be in the case where not using reflector 19 The imagination.In this case, detector is positioned directly facing bracket 13, is reflected without any ion beam.In this set In, deflection capacitor can be unnecessary.

According to embodiment, hot spot controls the laser light that can produce distance center positive/negative 300,400 or even 500 microns The deflection of spot, without the significant distortion of spot area.However, up to the present, it has not been possible to not produced to mass resolution Wide deflection is realized in the case where raw negative effect, because drawing lens 14 make ion beam be offset to the ions of phase homogenous quantities from center The degree being no longer present in the forward position perpendicular to ion beam direction.This means that being no longer possible to be maintained at the ion of center generation The high-quality resolution rate of beam.The desorption of any recognizable mass resolution deterioration can be used without with high-quality resolution rate The deflection of beam is about positive/negative 50 microns.

If sample support plate need to remain static during operation, only possible scanning is distinguished by the prior art 100 microns × 100 microns of measurement point.In order to obtain only one square millimeter of mass spectrum image, needed under the stable time appropriate Want 100 sample support plates mobile.This is even it cannot be guaranteed that each measurement point accurately abuts, because of the movement of sample support plate Precision is limited in about one to four microns.One square centimeter of tissue area needs sample holder 10000 times movements.

As described above, the purpose of the present invention is promote the scanning of surface region relatively large on stationary sample bracket to analyze For mass spectrographic tissue sample to be imaged, but the high throughput point for there are thousands of small independent samples is also used on sample support plate Analysis.Surface area can be such as 1000 microns × 1000 microns, i.e., about 1 square millimeter.Then, deflection of the beam from central axis is desorbed It will be positive/negative 500 microns.This allows to only with longer time interval mobile example support plate and allow sample support plate Vibrational stabilization for a period of time, without losing the plenty of time.Then, the tissue for one square centimeter, it is only necessary to 100 fortune It is dynamic, rather than 10000 movements according to prior art.The time of vibrational stabilization is easily half second or so；One square li The acquisition time of rice tissue regions will extend only 50 seconds, therefore less than one minute.

The mass spectrum the time it takes for obtaining one square centimeter of tissue regions depends on selected pixel size, desorption The pattern or profile of beam and the irradiation quantity on each sample site.For example, if selecting laser facula as shown in Figure 7 Pattern, and Pixel Dimensions multiply 60 square microns for 60, then one square centimeter of tissue regions include nearly 28000 pixels. If each pixel is sampled using 32 laser irradiations, the total acquisition time under 10000 spectra collections per second is about 90 seconds.In addition to this there are also 50 seconds stabilization times.If four overlapping scans of acquisition are on same site to exhaust sample, This causes total time to be about seven minutes.

If the axis of ion deflection ion source is generated and is focused off axis by virtual ion optical lens center, such as Fig. 6 Shown, ion cannot pass through with Fig. 4 intermediate ion by paraxial identical acceleration profile.Therefore, the ion 24 in Fig. 4 and figure The energy of ion 26 in 6 is slightly different.The length of flight path can also change with the continuous deflection of desorption beam, especially When using deflection unit.Therefore, off-axis ion has the flight time slightly different with the ion of phase homogenous quantities on axis.It is logical Cross current potential (and other apertured electrodes or tof tube itself on flight path when necessary slightly modified on sample support plate Part), can give the ion of phase homogenous quantities but different spaces origin the unified flight time.Generally speaking, when desorption beam When mobile, the voltage being not only on lens section, and there are also the current potential of sample support plate and deflection unit 16a, 17a, 16b and Deflection voltage (and when necessary in the other parts of tof tube) on 17b must track this offset, so as to will be by not It is added together to form summation spectrum with the single spectrum of difference that desorption beam deflection obtains.

When focusing center's strong deflection is far from axis, the shape of slightly oval, such as Fig. 3 are presented around the equipotential lines at center It is middle to be used as shown in example.This leads to the presence of the case where different focusing forces in two mutually orthogonal directions, and can not produce The ion beam of the raw substantially uniformity with the ion to fly in parallel.For example, being supplied if lens stop is divided into eight voltages The octant answered then can produce actually circular focusing center, this eight voltage supplies can individually control (not shown).

In view of being organized in pairs for four sections shown in Fig. 3, it is also conceivable to which diaphragm is subdivided into only to two half-unit is not ( It shows).The effective ion optical lens center of this diaphragm can only be moved along the axis perpendicular to the line of demarcation between two halves as a result, It is dynamic.However, since the deflection that the desorption beam spot on sample holder reaches +/- 50 microns not will lead to distinguishing for mass resolution Deterioration, even if not tracking effective ion optical lens center, according to an embodiment, such as still can with desorption beam Elongate area on inswept sample, short-and-medium axle is especially in +/- 50 microns of the maximum value, and long axis is in maximum deflection It is mobile, it still can be compensated by Mobility Center (+/- 500 microns about maximum), therefore such as covering maximal side is 100 microns × 1000 microns of rectangle.

All these voltages should be recalibrated at least once with the control of the variation of the movement of desorption beam, but select Fixed time interval repeats more preferable.May be used herein rapid location control come automatically, process control determine desorption Shu Guang The optimum voltage of each position of spot.Optimum voltage is by mass spectrometric maximum sensitivity and the best quality resolution ratio being achieved in To define.The special sample of the time of flight spectrum and millions of desorption beam irradiations of uniform strength can will be provided within a few hours Product are for this purpose.These samples are known, such as the liquid application for the peptide being dissolved in glycerol can be used herein.It uses These glycerol samples, fresh analyte molecule constantly pass through liquid and are diffused into the position under specific desorption beam hot spot to supplement Supply.By this method, on the one hand, all correction voltages of diaphragm section, light beam deflection, coriolis acceleration and tof tube Current potential, and on the other hand, desorb the impact position of beam, between correlation can completely automatically determine.

Here term " pixel " is commonly used, from wherein obtaining mass spectrum.The term needs slightly detailed consideration reconciliation to release. One point of pixel not instead of sample, the region of selected size, such as 10 × 10 micrometre squares or 60 × 60 micrometre squares. It is ionized especially for MALDI, for obtaining each time of flight spectrum of sample, always uses laser in accurate identical position Point or laser dot pattern are unfavorable, because sample exhausts quickly herein.For thin layer preparation, approximately three to five times It can be exhausted after laser irradiation.It would thus be advantageous to the Free Region of scanning element, so that sample is equably burnt.Possible In the case where, it should not be set as compact arranged pattern each laser facula in continuous laser irradiation, because this can It can lead to the excessive local heating of specimen material.Therefore, if it would be possible, such scan pattern should be selected, the scanning Pattern avoids the hot-spot of specimen material, and also assures that sample is equably burnt on available pixel region.As showing Example, Fig. 8 are depicted by means of the laser spot pattern with 9 intensity peaks for this scan pattern uniformly burnt, In with accurate 60 microns of edge length sample area square in, one layer of sample is with 32 laser irradiation phases in total When equably burning.Promote this scanning by the rapid location control to laser facula or laser spot pattern, and also Other kinds of desorption beam can be applied to.

Finer square can also be scanned, but LASER SPECKLE is closely put together, is that can not keep away Exempt from.Using the pattern of nine intensity peaks, therefore the pros of 30 microns of edge lengths can be scanned in eight laser irradiations Shape.It, can be in each case by 40 individual time of flight spectrum if the yield of sample, which allows to burn five, burns layer It is added together to form the summation time of flight spectrum of the finer sample area.Intensity peak there are four only having can be used Speckle patterns scanning has the square of 18 microns of edge lengths.Finer square burns the space for increasing organization chart picture Resolution ratio, although being unfavorable for detectable limit and signal-to-noise ratio；It but in many cases, then can be by finer combination of pixels The pixel region of Cheng Geng great, unless the different mass spectrums from very delicate institutional framework unexpectedly appear in finer region In.

In extreme circumstances, such as intensity peak of 5 micron diameters and each position 5 times can be used in this method Laser irradiation to measure surface with maximum resolution, so that mass spectrum can also show even most fine structure.If do not had here There is apparent fine structure, these mass spectrographic group can then be combined by data processing again has compared with low spatial resolution Pixel, to realize better signal-to-noise ratio.This allows to obtain the weak signal with low resolution retrospectively from data With with high-resolution strong signal.

It is ability for most preferably preparing the method for sample and for the mass spectrographic optimal acquisition of various analysis tasks and processing Known to field technique personnel, and do not need to be described in detail herein.For the imaging mass spectrum on thin tissue section, for example, having Conductive surface and be applied with host material microlite layer particular sample slice preparation in document DE 10 2,006 019 Individually illustrate in 530 B4 (M.Sch ü renberg etc.) and 10 2,006 059 695 B3 of DE (M.Sch ü renberg).Document DE 10 2,010 051 810 (D.Suckau etc.) illustrates how that the part digestion for carrying out protein is used in combination to generate digestion peptide In the protein of identification thin tissue section.10 2,008 023 438 A1 of document DE (S.-O.Deininger etc.) explains height How Chong Die with mass spectrum image resolution vision image is.Document DE 10 2,010 009 853 A1 (F.Alex-androv) explanation How using Mathematical treatment largely muting protein map picture generated on tissue sections.

The present invention is described by reference to different specific example embodiments above.It should be appreciated, however, that not departing from this In the case where invention scope, the various aspects or details of described embodiment can be modified.Particularly, described here to have The arrangement of the lens stop of quadrant is not intended to the desorption beam hot spot never on the axis of lens devices and generates with same phase Ion parallel ion beam only possible arrangement.Other than MALDI, the electricity of other pulse patterns can also be used From, such as SIMS.Therefore, the present invention should not necessarily be limited by these arrangements.In addition, if being feasible to those skilled in the art , then it can according to need combination and combine feature and method disclosed in different embodiments.In addition, above description is merely to illustrate this Invention, not as limiting of its scope, protection scope be defined solely by the appended claims, and consider there may be Any equivalent program.