Method for analyzing and measuring sample material on a sample holder

Abstract

The present invention relates to a method for analyzing and measuring sample materials deposited on the surface of a sample holder, comprising: (a) defining a plurality of regions on the surface, some of which are in contact with the sample material, (b1) using a desorption beam to sample a sample segment on one region to generate desorbed molecules, which are ionized and transferred to an analyzer, (b2) scanning the region along a non-linear trajectory selected from a plurality of predefined non-linear trajectories on the region by changing the orientation of the beam relative to the surface, while keeping the holder in one position, (c) using a spatial adjustment device of the sample holder to transition from the scanned region to the next region to be scanned, and (d) repeating steps (b1), (b2) and (c) until a predetermined termination condition is met. The present invention also relates to a system for analyzing ions, which has an ion generating device and a control unit.

Description

Technical Field

[0001] The present invention relates to a method for analytical measurement of extended or flat sample material deposited on the surface of a sample holder, wherein the sample material is converted into the gas phase by desorption means for spatial resolution and charged species or analyte ions are generated from the material and then transferred to an analyzer for further analysis. The method is particularly suitable for mass spectrometric imaging of (thin) tissue sections, with or without prior mobility separation. Background Art

[0002] In this background technology, reference is made to specific aspects to explain the prior art. However, this should not be construed as a limitation of the following disclosure of the invention. Useful further developments and improvements from what is known in the prior art may also be applied above and beyond the relatively narrow scope of this background technology, and will be apparent to those skilled in the art after reading the disclosure of the invention following this background technology.

[0003] Imaging mass spectrometry essentially involves scanning a flat sample, e.g., a sliced ​​(thin) tissue section, by a desorption beam that releases spatially resolved analyte molecules from the flat sample and transfers the charged molecular species or analyte ions generated from the molecules to a mass analyzer, e.g., a time-of-flight analyzer (TOF). Examples are matrix-assisted laser desorption / ionization (MALDI) or ionization by primary ion bombardment (secondary ion mass spectrometry, SIMS) coupled to a time-of-flight analyzer, MALDI-TOF and SIMS-TOF, respectively. Spatially resolved measurement data can be used to compile and display distribution maps of different analyte molecules on a flat sample.

[0004] Imaging mass spectrometry setups originally operated using a fixed beam guide and an xy translation stage that could be moved in two dimensions, and on or in which a sample holder with a flat sample was located. In order to scan a flat sample with spatial resolution in such a setup, the translation stage is moved at regular intervals so that after each movement of the stage a different region of the flat sample is in the focus of the desorption beam. As a moving mechanism, the translation stage is relatively slow and therefore the movement of the stage is very time-consuming, since the mass of the flat sample, the sample holder and the stage itself when taken together is considerable and must be moved repeatedly (in a stop-and-go cycle) at regular intervals over relatively short distances of tens to a hundred micrometers. After each movement, oscillations and vibrations occur in the heavy stage arrangement, which must be quelled before scanning with the desorption beam, otherwise the positioning accuracy of the desorption process is affected. Furthermore, the positioning accuracy of commercially available translation stages is in the single-digit micrometer range, about 2.5 to 3 micrometers, which limits the spatial resolution that can be achieved. Although piezoelectric translation stages with better positioning accuracy are known, they are very expensive to purchase and are therefore not suitable for industrial production.

[0005] An example of this mode of operation can be found in patent publication US 2008 / 0017793 A1. It discloses a method for dynamic mass spectrometry pixel imaging, the method comprising directing a laser beam onto a sample to be scanned, so that the laser beam releases an analyte from the sample. The laser beam and the sample are then moved relative to each other so that the laser beam moves substantially continuously along a predetermined path on the sample so as to release the analyte from the sample along the predetermined path. The released analyte is then mass analyzed.

[0006] The fixed positioning of the sample holder, together with the flat sample deposited thereon, and the scanning of the sample surface by changing the orientation of the desorption beam, is kinematically equivalent to a purely mobile platform and the use of a fixedly oriented desorption beam. An example is given in patent publication US 2004 / 0183009 A1, which shows a MALDI mass spectrometer comprising a laser beam guide device, by which the movement of the laser focus on the MALDI sample being analyzed can be controlled. The laser beam guide device can include a mirror device, which includes a pair of independently controlled mirrors. The first mirror can move the laser focus along the x-axis of the MALDI sample, while the second mirror can move the laser focus along the y-axis of the MALDI sample. Its purpose is especially to detect sample areas (sweet spots) with very high yields. There are practical limits to the area on the sample holder that can be scanned purely by beam displacement, because the degree to which ion generation and the transfer of ions to the connected analyzer are adversely affected increases with the decrease and flattening of the incident angle of the beam relative to the surface of the sample holder. In particular, when using a typical time-of-flight analyser with axial implantation of ions, a maximum displacement of about 100 microns from the straight-line time-of-flight axis can be produced without any further compensation of the generated ion beam.

[0007] In addition, for example, the combination of stage movement and beam orientation change is known from patent publication DE 10 2011 112 649 A1 (corresponding to GB 2 495 805 A and US 2013 / 0056628 A1), which relates to a mass spectrometer for sample ionization by matrix-assisted laser desorption (MALDI). Here, the sample is located on a movable support plate, where it is irradiated by a pulsed laser, the impact point of which on the continuously moving support plate is changed by a rotatable mirror system.

[0008] Antonín presented at the 60th ASMS Conference held in Vancouver, Canada, May 20-24, 2012 An example of an operating mode combining intermittent stage movement and changing beam orientation can be found in the poster publication "High throughput MS imaging using a fast scanning mirror" by Antonín et al. This publication concerns a prototype of a linear time-of-flight mass spectrometer with high throughput and a single fast precision scanning mirror, where a fast and precise mirror adjustment of the desorption laser within a few millimeters replaces the sample stage movement in one spatial direction. By joining the individual images together, larger areas can be visualized. This publication is followed by two expert articles from the same research group with similar, further developed content: Antonín et al. et al., Analytical Chemistry, 2014, 86, 2, 982–986 and et al., Journal of the American Society for Mass Spectrometry, (2019) 30:289-298.

[0009] A description of the different modes of operation of the SIMS-TOF mass spectrometer with pure stage movement and fixed beam orientation and with a combination of intermittent stage movement and changing beam orientation can be found in the paper by Tingting Fu (3D and High Sensitivity Micrometric Mass Spectrometry Imaging, Analytical Chemistry, Paris-Saclay University, 2017, English, NNT: 2017SACLS218, Tel. 01699065v2). The scientist strives to better understand ion production under cluster ion bombardment, investigate the MS / MS capabilities of a parallel imaging MS / MS spectrometer (PHI nanoTOF II), and use SIMS-TOF to map important wood metabolites with high spatial resolution.

[0010] Patent publication DE 10 2018 112 538 B3 (corresponding to GB 2 574 709 A and US 2019 / 0362958A1) relates to a time-of-flight mass spectrometer with a pulsed ionized sample, for example by matrix-assisted laser desorption (MALDI), wherein the sample is located on a sample holder and is irradiated and ionized one by one in a grid by a position-controlled desorption beam. An ion optical pulling lens device is located in front of the sample holder, at least one lens aperture in the device is subdivided into a plurality of segments, and a voltage source is capable of providing different voltages to these segments or some of them, depending on the impact position of the desorption beam on the support plate. The effective ion optical focusing center of the lens can then be virtually moved away from the axis, and the ion beam generated by deviating from the real lens axis can be focused into a beam substantially parallel to the real lens axis, and there is no temporal phase shift for ions of the same mass. The beam can be returned to the axis by an x / y deflection unit, for example for operating a time-of-flight mass spectrometer with a reflectron.

[0011] In view of the above explanation, there is a need to accelerate the measurement of sample materials deposited on a sample holder. In addition, there is a need to reduce unnecessary wear and tear of components involved in the relative movement of the desorption beam and the sample holder. By reading the following disclosure, those skilled in the art will immediately understand the further goals that can be achieved by the present invention. Summary of the invention

[0012] The present invention relates to a method for analyzing and measuring sample materials deposited on the surface of a sample holder, comprising the following steps: (a) defining a plurality of regions on the surface of the sample holder, some of which are in contact with the sample material, (b1) using a desorption beam to sample a slice of the sample on one region to produce desorbed molecules, which are ionized and transferred to an analyzer, (b2) scanning the region along a non-straight trajectory selected from a plurality of predefined non-straight trajectories on the region by changing the orientation setting of the desorption beam relative to the surface of the sample holder, while keeping the sample holder in one position, (c) using a spatial adjustment device of the sample holder to transition from the scanned region to the next region to be scanned, and (d) repeating steps (b1), (b2) and (c) until a predetermined termination condition is met.

[0013] According to the invention, a moving mechanism for the sample holder and a device for changing the orientation of the desorption beam advantageously operate alternately according to a predefined pattern to scan sample material distributed on an area of ​​the sample holder as quickly as possible and with minimal wear and tear. The desorption beam assembly particularly comprises an electro-optical adjustment device for changing the spatial orientation of the light beam relative to the surface of the sample holder in two spatial directions along a plane parallel to the surface of the sample holder. The device operates rapidly and has a relatively small adjustment amplitude in the range of three-digit nanometers to single-digit micrometers. The sample holder assembly particularly comprises a motion mechanism for spatially moving the sample holder relative to the desorption beam. In contrast, due to the relatively large mass to be moved, the mechanism operates slowly and requires a rest time after the movement occurs, for example, about 0.8 seconds, so that any oscillations or vibrations that may shake the sample holder and impair the desorption process can subside.

[0014] The desorption beam is moved non-linearly over an area in a plane which is substantially parallel to the surface of the sample holder on which the sample material has been deposited, while the sample material is sampled with the desorption beam. At the latest when the movement radius of the electro-optical adjustment device for scanning a certain area in two spatial directions along a plane parallel to the sample holder surface reaches its limit, the movement mechanism can be started to scan further areas, such as adjacent areas. The number of time-consuming stepwise movements of the sample holder assembly can thus be advantageously reduced. It must also be taken into account that when using a time-of-flight analyzer with axial ion implantation without any compensation of the generated ion beam, as described in patent publication DE 10 2018 112 538 A1, for example, any displacement of the desorption beam from the optimal center of the ion optics should not exceed 100 microns to avoid errors in the time-of-flight measurement.

[0015] In a preferred embodiment, in step (b2), the area can be scanned between a first orientation and a last orientation of the desorption beam relative to the sample holder surface, wherein after each transition in step (c), the scanning of the next area starts from the last orientation of the desorption beam. In other words, the desorption beam can scan the area in step (b2) starting from the first impact position on the area until the last impact position, wherein, after each transition in step (c), the scanning of the next area starts from the first impact position on the area corresponding to the last impact position on the previously scanned area. According to this embodiment, the need to reset the orientation of the desorption beam to a uniform orientation relative to the sample holder surface after or during each transition from one area to the next area can be avoided by simply retaining the last set beam orientation (last impact position, if applicable) so that the scanning of the range of each sequential anchoring area does not start with the same orientation setting of the beam relative to the sample holder surface (same impact position, if applicable). Using this advantageous operating mode, wear on electro-optical components used to adjust the beam orientation beyond the movement required to scan the sample material can be reduced. Furthermore, during the transition from one area to the next (including, if necessary, a period to allow interfering oscillations and vibrations to subside), scanning and sampling can be continued immediately after the adjustment of the movement mechanism has been completed, without having to wait for the desorption beam to be reoriented, which provides additional time advantages. If desired, this particularly preferred embodiment can even be combined with a steady or continuous movement of the sample holder, as is known from the patent publication DE 10 2011 112 649 A1 mentioned in the background art, which should therefore also be regarded as part of the present disclosure.

[0016] In various embodiments, the desorption beam can be an ion beam or a light beam. The ion beam can be a primary ion beam for bombarding the sample material, which will result in the formation of secondary ions (SIMS). The ion beam can have a continuous or pulsed ion current. The light beam can be a coherent light beam, such as from a solid-state laser (especially Nd:YAG), a gas laser (nitrogen, N2) or a light-emitting laser diode. For this photo-optical desorption, the sample material can be specially prepared to exhibit the best possible light absorption characteristics, for example, using a matrix material for MALDI ionization. The light beam can have a continuous or pulsed photon stream. If necessary, the ionization process can be assisted by a post-ionization procedure after desorption, such as MALDI-2 (Jens Soltwisch et al., Science, April 10, 2015, Vol. 348, No. 6231, 211-215) or secondary neutral mass spectrometry (SNMS), for example using a laser pulse that is purposefully directed laterally into the desorption cloud after desorption before the ions are transferred to the analyzer. The light beam, in particular the laser beam, can have a modified beam cross section, for example, with several individual intensity peaks, as described in patent publications DE 10 2004 044 196 A1 (corresponding to GB 2 421 352 A and US 2006 / 0071160 A1) and DE 10 2005 006 125 A1 (corresponding to GB 2 423 187 A and US 2006 / 0186332 A1) taking laser beams as an example).

[0017] Each region can have multiple range elements or pixels, and the desorption beam can sample a sample segment on a range element along a desorption path selected from a plurality of predefined desorption paths. The selected desorption path is preferably non-linear to take advantage of relatively fast and easy adjustment of the orientation of the desorption beam in two spatial directions along a plane parallel to the sample holder surface to scan the range elements. If the desorption beam is operated in a discontinuous or pulsed mode, each range element or each region can have multiple (separated or spaced apart) sampling points. The sampling points can be irradiated with one or more pulses to produce desorbates that may contain charged species or analyte ions. A range element or pixel is typically the unit of minimum spatial resolution in a graphical representation of data measured over a large range. Data from this range element or region can be merged even if it contains several separate sampling points. In post-processing, the spatial resolution can always be retrospectively reduced in a graphical representation or a molecular content distribution map of the sample material by combining measurement data acquired spatially separately, for example, from different sampling points.

[0018] The desorption path selected over a range element may comprise a plurality of changes in direction over the range element. Changes in direction may in particular comprise (almost) right angles or other acute or obtuse angles. The change in direction may be abrupt, i.e. at an angle, but may also be almost continuous, for example, along a circular trajectory, a cycloid or any other type of curved trajectory.

[0019] The sample material may be a tissue section. The molecular content of a thin tissue section is preferably measured with spatial resolution in order to create and visualize a distribution map of molecules of interest, for example biomolecules such as peptides, lipids (phospholipids and glycolipids), oligosaccharides, sterols, glycans, secondary metabolites or fat-soluble vitamins, as well as other possible non-tissue molecules such as active drugs (drug substances) and / or their degradation products. The expert will consider that the sample material may also have a tissue microarray (TMA) or a separately prepared array, for example sample material with matrix preparation by the dry droplet method for subsequent MALDI ionization. These areas may then include hydrophobic or hydrophilic areas on a metal or other conductive sample support, which are separated from each other and therefore isolated by hydrophilic or hydrophobic areas on the sample support, such as the AnchorChip from Bruker. TM The form of the board.

[0020] In various embodiments, the area and / or range elements can have a polygonal contour. Thus, an array of multiple area and / or range elements can be generated that almost completely covers the basic area of ​​the sample holder and the sample material deposited thereon. Examples are rectangles, such as squares, and polygons with more angles, such as hexagons. The contour of the area and / or range elements can be uniform over the entire sample holder. However, in principle, it is also possible to define area and / or range elements of different shapes in the same measurement if this is advantageous for the application. The contours of the area and range elements can also be designed differently, for example, a uniform rectangle or square for the area and a uniform hexagon for the range elements in the individual areas. The freedom of range is almost unlimited for the expert.

[0021] In different embodiments, the analyzer may be a mobility analyzer, a mass analyzer or a combined mobility-mass analyzer.In general, the term ion spectrometer and measurement methods may be used, which may include mobility separation, mass separation or a combination of both.

[0022] Ion mobility analyzers separate charged molecules or molecular ions according to the ratio of their collision cross section to the charge, sometimes expressed as Ω / z or σ / z. The basis is the interaction between the ion species and the electric field, which couples to the charge of the ions, and the simultaneous action of a buffer gas, which affects the average cross-sectional area of ​​the ions. In particular, drift tube mobility separators with an electrostatic field gradient are known, which drive the ions through a substantially stationary gas. Here, the drift velocity of the ion species is given by the propulsive force of the electric field and the deceleration force of the collision with the gas particles. Also common are trapped ion mobility separators (TIMS), which have a continuous laminar gas flow that drives the ions forward, which is offset by a gradually changing electric field gradient and a correspondingly variable deceleration force. Traveling wave mobility separators are also worth mentioning.

[0023] On the other hand, a mass analyser separates charged molecules or molecular ions according to their mass-to-charge ratio, which is usually designated as m / z. In addition to the above-mentioned time-of-flight analysers, which can be equipped with linear and reflectron setups, other types of mass dispersive separators can be used, such as quadrupole mass filters (single quadrupole), triple quadrupole analysers ("triple quadrupole"), ion cyclotron resonance (ICR) cells, Kingdon type analysers, e.g. (Thermo Fisher Scientific) etc. Of course, separators of the above type can be coupled so that ionic species can be separated multidimensionally, ie according to more than one physicochemical property, such as m / z and Ω / z or σ / z.

[0024] A plurality of deflection elements can be used to change the orientation of the desorption beam. For light-optical beams, a system of reflective elements can be used, such as mirrors, refractive elements, such as prisms, and / or electro-optical or acousto-optical deflection elements, such as electro-optical crystals, which can change or move the position of the desorption beam in two spatial directions along a plane parallel to the sample holder surface that impacts the sample holder surface or the sample material thereon, for example, by tilting or otherwise changing the spatial orientation. For ion beams, a system of deflection electrodes supplied with an electric potential can be used, which surrounds the ion beam path and can produce a variable electric potential gradient in the space between two electrodes in a pair of electrodes. An example is two electrode pairs, 90° to each other. If the electric potential applied to the two opposing electrodes of a pair of electrodes is the same, the primary ion beam passing through the pair of electrodes will not experience any lateral deflection. If the electric potentials are different, the primary ions deflect during flight according to the action of the electric potential difference.

[0025] The selected trajectory can contain multiple changes of direction over the area and / or range elements. This allows to scan flat or two-dimensional areas of sample material by quickly changing the orientation of the desorption beam and with virtually no wear and tear, since the number of step-by-step movements of the rather heavy sample holder assembly can be reduced accordingly. The changes in direction can in particular include (almost) right angles or other acute or obtuse angles. The change in direction can be abrupt, i.e. at an angle, but also almost continuous, for example along a circular trajectory, a cycloidal trajectory or any other type of curved trajectory. The trajectory of the area to be scanned next can correspond to the trajectory of the area already scanned, but in the opposite direction. In general, the trajectory can vary from area to area, if this is advantageous for the application.

[0026] In various embodiments, the sample holder can be moved in space by a translation stage. Preferred is an xy translation stage, which is designed to translate or move the sample holder, including sample material placed on or in the translation stage, in two spatial directions parallel to the plane of the sample holder surface. The larger the movement radius of the adjustment device for changing the orientation of the desorption beam, the lower the requirement for the minimum step size of the translation stage, because the movement of the desorption beam can cover the surface area between two activations of the translation stage. The step size of the translation stage is typically in the range of two to three digit micrometers. In various embodiments, in particular, the moving mechanism for the sample holder arranged in or on the translation stage can also include a device for moving along a third spatial direction (z-axis), for example, to adjust the optimal focus of the desorption beam to adapt to sample materials of different thicknesses or heights.

[0027] In various embodiments, information obtained from the area and / or range elements sampled using the analyzer can be combined to form a spatial distribution map of ions or underlying molecular substrates across the sample material. For example, this allows the distribution of sample molecules themselves and molecules that are foreign to the sample material to be displayed on the sample material in a non-optical microscopy manner with label-free measurements, compared to staining methods in optical microscopy.

[0028] The termination condition may be met when all regions and / or range elements have been scanned and sampled. In extreme cases, sampling may proceed to such an extent that the sample material is more or less completely removed due to the influence of the desorption beam on all regions and / or range elements, so that the desorption beam will no longer be able to produce desorbed molecules. Another example of a termination condition may be that a number of regions of particular interest have been scanned and sampled, which number is smaller than the total number of defined regions, so that parts of the sample material remain unmeasured, e.g. regions of different cell types in a (thin) tissue section. A termination condition may also be defined as a failure during the execution of the method, e.g. because a mirror has become stuck.

[0029] The present invention also relates to a system for analyzing ions, comprising an ion generating device and a control unit, which is designed and configured to perform the method described above on the ion generating device. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The present invention can be better understood by referring to the following description. The elements in the figures are not necessarily drawn to scale, but are mainly intended to illustrate the principles of the present disclosure (mainly schematic). In the figures, the same reference numerals represent corresponding elements in different views.

[0031] Figure 1 The design of a MALDI time-of-flight mass spectrometer (adapted from the prior art) suitable for carrying out methods according to the principles of the present disclosure is schematically shown.

[0032] Figure 2 A schematic cross-sectional view of a scanning sequence with movement of the desorption beam and the sample holder relative to each other is shown in side view. Figure 1 The results were performed in a MALDI time-of-flight mass spectrometer.

[0033] Figure 3A Example patterns of non-straight trajectory of the desorption beam across several regions are shown in plan view.

[0034] Figure 3B Also shown in plan view are example patterns of non-linear desorption paths of desorption beams across multiple range elements in one region, and example patterns of non-linear trajectories across multiple regions containing multiple range elements.

[0035] Figure 3C Further example patterns of non-linear desorption paths of desorption beams through several range elements in one region, as well as example patterns of non-linear trajectories through multiple regions containing multiple range elements, are also shown in plan view. DETAILED DESCRIPTION

[0036] While the invention has been shown and described with reference to various embodiments, those skilled in the art will recognize that various changes in form and details may be made without departing from the scope of the technical teachings as defined in the following claims.

[0037] Figure 1Schematic diagram of a MALDI time-of-flight mass spectrometer (adapted from DE 102011 112 649 A1) with a time-of-flight analyzer 1 and a laser system 2, which uses a mirror system 7, 8 to control the direction of the laser beam relative to the surface of a sample holder 13 carrying the sample material. The laser beam, usually in the form of pulses, is generated in a beam generating unit 3, which contains a laser crystal 4 and, if necessary, a device 5 for laser frequency doubling. The laser beam has an intensity peak in a pattern generator 6 and is deflected in two spatial directions by two galvanometers 7 and 8 in a mirror system so that the position where the laser beam impinges on the surface of the sample holder can be moved. The deflected laser beam is then expanded in a Keplerian telescope 9 and moved in parallel according to the angular deflection. The emitted laser beam is then guided into the objective lens 11 by a mirror 10 with a reduced deflection angle so as to be completely centered again. According to the angular deflection, the beam passes through the center of the objective lens 11, but at a slightly different angle, thereby changing the position where it impinges on the sample holder 13. It should be noted here that the beam guidance within the Kepler telescope 9 is more complex and, for simplicity reasons, the illustration does not reproduce it realistically, although the illustration does correctly reproduce the external influence of the telescope on the laser beam.

[0038] The desorbed charged molecules and ions produced in the desorption cloud of the impinging laser beam are accelerated by the voltages on the diaphragms 14 and 15 to form an ion beam 18 which passes through two deflection capacitors 16, 17 to correct its trajectory and is focused by a reflector 19 (MALDI TOF arrangement) onto a detector 20. A control unit 22 suitable for implementing the principles of the present disclosure and which may be appropriately programmed communicates with the time-of-flight analyser 1 and the laser system 2 and coordinates their operation relative to each other.

[0039] Figure 2 A schematic cross-sectional view of a scanning sequence with movement of the desorption beam 23 and the sample holder 13 relative to each other is shown in side view, as can be seen in FIG. Figure 1 Reference numeral 21 corresponds to the ion optical axis along which the charged species or analyte ions are accelerated onto the flight path by the extraction voltage applied to the first accelerating membrane 14, see Figure 1 ion beam 18.

[0040] On the sample holder 13, several areas a, b, c, d, etc. are defined, which subdivide the sample material (not shown) deposited thereon, e.g. a (thin) tissue section prepared with a MALDI matrix, into several segments. In order 24 from top to bottom, the desorption beam 23 is initially directed towards the left edge of area c. The sample material located here can be desorbed and ionized by the colliding desorption beam 23 and then extracted along the ion optical axis 21 by applying an electrical extraction potential to the first accelerating electrode 14. Afterwards, the orientation of the desorption beam 23 is changed so that an adjacent site on area c becomes a target. While this happens, the sample holder 13 remains stationary. After two further sampling steps of area c using the desorption beam 23 whose orientation has been slightly changed while the position of the sample holder 13 remains stationary, the right edge of area c is reached.

[0041] It should be pointed out here that the side view should not give the impression that during sampling, area c is swept by the desorption beam in only one spatial direction in a plane parallel to the sample holder surface (in Figure 2 According to the present disclosure, the relatively small mass and thus rapid change of direction of the desorption beam 23 on the surface of the sample holder 13 in both spatial directions is utilized to minimize the number of times the sample holder has to be moved for a more or less complete scan of the expanded or flattened sample material (arrow 33). Figure 2 In the representation of , the second direction is perpendicular to the drawing plane, that is, entering or leaving the drawing plane. This will be referred to below Figure 3A -C explains in more detail.

[0042] When area c has been scanned and thus sampled at several positions by adjusting the direction of the desorption beam 23 in two spatial directions parallel to the sample holder surface, the sample holder 13 can be moved to another position (arrow 33) by its moving mechanism (not shown) so that the position where the desorption beam 23 impinges is located on the edge of area b, in this case, adjacent to the previously scanned area c. After the sample holder has been moved, the scanning and sampling pattern of area b can be very similar to the pattern used to scan and sample the previous area c. However, it is advantageous not to change the direction of the desorption beam 23 relative to the sample holder surface during the sample holder movement 33, but to keep it as it is. In this preferred embodiment, the subsequent scanning and sampling of area b starts from the edge area on the right. The desorption beam 23 is then oriented in the opposite direction (in the Figure 2 The sequence 24 in FIG. 2 changes several times from right to left, including a movement perpendicular to the drawing plane, which is only implicit due to the side view, until the desorption beam 23 is oriented towards the left edge of the region b and the sample material located there has, for example, already been sampled for the last time.

[0043] The desorption beam 23 remains in this last orientation setting until the sample holder 13 has been moved to a subsequent position (arrow 33) in which the region a is located within the movement radius of the desorption beam 23. Here again, the position at which the desorption beam 23 strikes for subsequent scanning and sampling of the region a is initially located at the left edge. Unnecessary resetting of the orientation of the desorption beam 23 during the sample holder movement 33 can thus be avoided. The scanning and sampling pattern of the region a can be similar to the pattern used for the region c, but can also be different.

[0044] Figure 3A By way of example, a plan view of a non-linear trajectory 30 spanning a plurality of predefined regions a, b, c, ... is schematically shown, these regions having different (separated from each other) sampling points 31 on the regions a, b, c, ... The solid arrows 32 within the regions a, b, c, ... indicate the direction of movement of the scan when the orientation of the desorption beam (not shown) relative to the surface of the sample holder is changed. The individual points 31 along the solid arrows 32 represent sampling points in the regions a, b, c, ... of the desorption beam operating in a pulsed mode. Information about the abundance or number of charged species and analyte ions generated by the desorption beam impinging on these sampling points 31 can be combined when registered by the detector of the mobility and / or mass spectrometer for a graphical representation of the molecular content, and therefore the regions a, b, c, ... are used as units of minimum spatial resolution in this representation.

[0045] from Figure 3A As can be seen in the figure, the scanning and sampling of the first area a starts from the upper left corner in the figure, and then follows a zigzag or zigzag route direction while performing several 90° changes until it reaches the position in the lower right corner. When the scanning and sampling of the first area a are completed, the desorption beam maintains its orientation relative to the surface of the sample holder, and the moving mechanism of the sample holder is gradually adjusted (dashed arrow 33 outside the area) so that another area b enters the moving radius of the desorption beam. The subsequent scanning and sampling of the new area b starts from a position in the area corresponding to the position where the scanning and sampling of the previous area a ends, which is located in the lower right corner in this example. For clarity, the sampling points 31 in the second area b have been omitted except for the first and last ones. In this example, the desorption beam follows non-linear trajectories on areas a, b, c,..., which extend in opposite directions in successive areas. However, this is not always the case. If a person skilled in the art considers it to be beneficial for a specific application, a personalized, non-linear trajectory or other suitable patterns for each area a, b, c,... can also be selected for the scanning and sampling of the desorption beam.

[0046] The time savings achieved by the procedure described here compared to a setup with fixed beam orientation and an xy translation stage as the only movement mechanism can be illustrated using a numerical example. Figure 3AThe region a in contains 15 sampling points, so 14 stage movements are required to completely scan the region a. Therefore, after the sample holder is moved step by step by the corresponding parts of the moving mechanism, the waiting time for the oscillation and vibration mentioned in the background technology totals 14x(~)0.8 seconds=11.2 seconds. On the other hand, changing the direction of the desorption laser beam is much faster, i.e. 14x(~)0.002 seconds=0.028 seconds, which is a significant speedup.

[0047] The reduction in the load on the electro-optical device for changing the orientation of the desorption beam can also be illustrated by means of a numerical example. In the example shown, in each zone, from the first impact point to the final impact point on this zone, the electro-optical adjustment device must change the movement direction of the scanning desorption beam eight times. If the orientation of the desorption beam is to be consistently reset relative to the sample holder surface or a consistent impact position in this zone during or after each transition from one zone to the next, as suggested by the prior art, this would require one additional movement or adjustment, i.e. nine instead of eight movement changes, an increase of 12.5%. In particular, when the scanning area is 1 cm 2 And the area is 10μm x 10μm = 100μm 2 When (thin) tissue sections are cut, according to Figure 3A The pattern in must scan 100 million areas, which will result in 12,500,000 essentially unnecessary movements of the optoelectronic adjustment device of the desorption beam. By adjusting the scanning pattern according to the preferred embodiment of the present disclosure, the service life of the adjustment device limited by wear can be correspondingly extended.

[0048] Figure 3B Another example pattern for scanning and sampling an area containing sample material according to the principles of the present disclosure is shown. Figure 3A In contrast to the example in Figure 3B Not only is the subdivision into defined regions a, b, c, ... shown, but also a plurality of range elements 34 in each region. The range element 34 can be used as a unit (pixel) of minimum spatial resolution for a graphical representation of the molecular content measured from the sample material. The regions of the range elements 34 are scanned and sampled according to a predetermined non-linear desorption path 35 (solid arrow), thereby generating predetermined, non-linear scanning and sampling trajectories over a plurality of range elements 34 in a region. The transition from one region to the next is carried out using a movement mechanism of the sample holder, while many range elements in a region are scanned simply by changing the orientation of the desorption beam in two spatial directions parallel to the surface of the sample holder.

[0049] and Figure 3A, each desorption path 35 in this example comprises a plurality of directional changes, according to which a rectangular oscillating-type "micromotion" across the individual range elements 34 is superimposed on a "macromotion" of a meandering or serpentine scanning of the areas a, b, c, ... . The transition from the last range element in a row to the adjacent range element in the next row involves a brief movement of the desorption beam in the opposite direction. However, due to the advantageous adjustment properties of the desorption beam, this does not present any problems and requires hardly any time. For the sake of clarity, Figure 3B The sampling points at which the pulsed desorption beam can strike are omitted, but it can be assumed that the sampled material can be sampled at least once along each solid arrow. The advantageous principle of performing each spatial adjustment of the sample holder while maintaining the orientation of the desorption beam is also implemented in this example. In the grid of similar areas a, b, c, ... and similar range elements 34 in these areas shown here, this means that the scanning and sampling of one area starts with such an orientation of the desorption beam relative to the sample holder surface and therefore starts with such an impact position on the sample holder, i.e., the scanning and sampling of the previous area ended at this orientation and impact position.

[0050] Figure 3C Another example embodiment of a scanning and sampling pattern of a range on a sample holder is shown, which is divided into a plurality of regions and a plurality of range elements contained therein. Figure 3B The description can be easily transferred to Figure 3C In the embodiment of the present invention, no further details are given here.

[0051] The invention has been described above with reference to different specific example embodiments. However, it will be appreciated that various aspects or details of the described embodiments may be modified without departing from the scope of the invention. For example, these examples only mention pulsed operation of a desorption beam. However, stable or continuous operation is also compatible with the principles of the present disclosure, in particular in the case of SIMS ionization using a continuous primary ion beam. Furthermore, the features and measures disclosed in conjunction with the different embodiments may be combined as desired, if this is feasible for a person skilled in the art. Furthermore, the above description serves only as an illustration of the invention and not as a limitation of the scope of protection, which is limited only by the appended claims, taking into account any equivalent forms that may exist.

Claims

1. A method for analyzing and measuring a sample material deposited on a surface of a sample holder, the method comprising the following steps: (a) defining a plurality of regions on the surface of the sample holder, some of which are in contact with the sample material, (b1) sampling a sample segment over an area using a desorption beam to produce desorbed molecules, which are ionized and transferred to an analyzer, (b2) scanning the area along a non-linear trajectory selected from a plurality of predefined non-linear trajectories over the area by changing the orientation of the desorption beam relative to the surface of the sample holder, while maintaining the sample holder in one position, (c) using a spatial adjustment device of the sample holder to transition from one scanned area to the next area to be scanned, and (d) repeating steps (b1), (b2) and (c) until a predetermined termination condition is satisfied, The area in step (b2) can be scanned between a first orientation and a final orientation of the desorption beam relative to the sample holder surface, wherein after each transition in step (c), the scanning of the next area starts from the final orientation of the desorption beam.

2. The method according to claim 1, wherein the desorption beam can be an ion beam or a light beam. 3 . The method according to claim 1 , wherein each region may have a plurality of range elements, and the desorption beam may sample a sample segment on one range element along a desorption path selected from a plurality of predefined desorption paths. The method of claim 1 , wherein the sample material comprises a tissue section. The method according to claim 1 , wherein the area and / or range elements have a polygonal outline. The method of claim 1 , wherein the analyzer is a mobility analyzer, a mass analyzer, or a combined mobility-mass analyzer. The method of claim 1 , wherein a plurality of deflection elements are used to change the orientation of the desorption beam.

8. The method of claim 1, wherein the selected trajectory comprises a plurality of direction changes over an area and / or range element.

9. The method according to claim 1, wherein the sample holder is moved in space by a translation stage.

10. A method according to claim 1, wherein information obtained from the area and / or range elements of sampling using the analyzer can be combined to form a spatial distribution map of ions or underlying molecular substrates across the sample material.

11. The method of claim 1, wherein a termination condition is met when all region and / or range elements have been scanned and sampled.

12. A system for analyzing ions, the system comprising an ion generating device and a control unit designed and configured to perform the method according to claim 1 on the ion generating device.

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