Apparatus for performing a desorption scan of an analyte material on a sample carrier

Abstract

The present invention relates to an apparatus and method for performing a desorption scan on an analyte material placed on a sample carrier, which may include the following operating modes: (a) setting the position of the carrier to be close to the impact area at which the beam is aligned for local desorption of the analyte material; (b) determining the actual position of the carrier after setting the position; (c) comparing the determined actual position with the target position of the carrier to determine any deviation; (d) if a deviation is detected, adjusting the beam orientation so that the beam is aligned to the impact area that would have been produced on the carrier in the absence of a deviation; (e) applying the beam to the impact area to locally desorb the analyte material and convey it to an analyzer; and (f) checking whether a predetermined end condition is satisfied, and if not, repeating steps (a)-(e) for subsequent non-conforming impact areas.

Description

Technical Field

[0001] The present invention relates to an apparatus and a method for desorption scanning of an analyte material placed on a (flat, plate-like) sample carrier. The apparatus and method are particularly useful for ion spectrometry of desorbing analyte materials, for example, mass spectrometry, migration spectrometry, and migration-mass spectrometry. Background Art

[0002] This introduction discusses the state of the art with reference to a specific aspect. However, this is not intended to limit the following disclosure of the present invention. Beneficial developments and modifications of what is known from the state of the art may also apply outside the relatively narrow scope of this introduction and will become apparent to those skilled in the art after reading the disclosure of the present invention following this introduction.

[0003] Mass spectrometry imaging (MSI; sometimes also referred to as IMS = imaging mass spectrometry) essentially involves scanning a flat analyte material, for example, a (thin) tissue section of a microtome section, using a desorption beam that releases spatially resolved analyte molecules from the sheet-like analyte material and transfers the charged molecular species or analyte ions generated by the molecules to a mass analyzer, for example, a time-of-flight (TOF) analyzer. Examples include ionization by matrix-assisted laser desorption (MALDI) or ionization by primary ion bombardment (secondary ion mass spectrometry, SIMS), which are coupled to a time-of-flight analyzer, MALDI-TOF, and SIMS-TOF, respectively. The spatially resolved measurement data can be used to compile and display a distribution map of different analyte molecules on the two-dimensional analyte material.

[0004] For example, patent publications US 2008 / 0017793 A1 and WO 02 / 084577 A1 disclose imaging mass spectrometry devices that initially operate with a fixed beam guide and an X-Y translation stage that can move in two dimensions and on which or in which a sample carrier with a two-dimensional analyte material is located, but for a single preparation. In order to scan the two-dimensional analyte material in a spatially resolved manner in such a device, the translation stage is adjusted at regular intervals so that after each movement of the stage, different regions of the flat analyte material are in the focus of the desorption beam.

[0005] As a kind of motion mechanism, the translation stage is relatively sluggish, so moving the platform is quite time-consuming because, when considered comprehensively, the flat analyte material, the sample carrier and the platform itself stack up to a considerable mass, which has to regularly repeat relatively short distances of a few micrometers (in the stop-motion cycle) for movement. In addition, the best positioning accuracy of commercially available stepper motor-driven translation stages suitable for use under negative pressure is in the single-digit micrometer range, about 2.5 to 3 micrometers, which limits the achievable spatial resolution. Piezoelectric platforms with better positioning accuracy to meet the requirements of high spatial resolution and that can also withstand negative pressure are known, but the purchase price is very expensive, so they are not suitable for industrial production. In addition, due to some major structural differences, the high-precision platforms developed for microscopes are difficult to be applied to desorption ion sources that almost always operate under negative pressure.

[0006] Stationarily positioning the sample carrier together with the flat analyte material placed thereon and scanning the surface of the analyte material by changing the orientation of the desorption beam is, from a kinematic perspective, equivalent to purely moving the platform and using a desorption beam with a stationary orientation. An example is provided in patent publication US2004 / 0183009A1. However, there are practical limitations to the area on the sample carrier that can be scanned purely by beam movement because as the beam incident angle decreases and flattens relative to the surface of the sample carrier, ion generation and ion transport through a suitable interface into the connected analyzer are increasingly adversely affected. In particular, using a typical time-of-flight analyzer with axial ion injection, the offset from the straight time-of-flight axis through the flight tube is limited to about ±100 micrometers without additional compensation for the generated ion beam, while the area of a tissue section is about on the order of several square centimeters. A pixel square with a side length of 100 micrometers can be placed 100 times in a tissue square with a side length of 1 centimeter, which means that pure beam adjustment is not sufficient to scan the entire surface of a tissue sample of this size.

[0007] In addition, the combination of platform movement and changing the beam orientation is known, for example from patent publication DE 10 2011112 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 samples are located on a movable carrier plate where they are irradiated by a pulsed laser, and the impact point of the pulsed laser on the continuously moving carrier plate is changed by a rotatable mirror system.

[0008] The following disclosure provides additional explanations of the technical background of the present disclosure:

[0009] The publication by Konstantin Aizikov et al. (Review of Scientific Instruments 82, 054102 (2011)) concerns a vacuum-compatible sample positioning device for MALDI Fourier transform ion cyclotron resonance mass spectrometry.

[0010] The study by Andre Zavalin et al. (J Mass Spectrom. November 2012; 47(11)) reported the direct imaging of single cells and tissues at subcellular spatial resolution using transmission geometry MALDI-MS.

[0011] The article by the authors Mario Kompauer et al. (Nature Methods 2017, DOI: 10.1038 / NMETH.4433) “Autofocusing MALDI mass spectrometry imaging of tissue sections and 3D chemical topography of nonflat surfaces” presented an autofocusing atmospheric pressure MALDI-MSI system for studying 3D sample surfaces with topographic aspect ratios of up to 50, which was designed to maintain MALDI laser focus, flux, and ablation spot size invariant during sample height variations by adjusting the sample stage position according to the sample height profile at each measurement point. This approach should allow a lateral resolution of ≤10 µm for three-dimensional samples.

[0012] The article by Brian J. Malys et al. (J. Am. Soc. Mass Spectrom. (2018) 29, 422 - 434) concerns the diagnosis and correction of mass accuracy and signal intensity errors caused by initial ion position variations in MALDI-TOF MS.

[0013] The study by Michelle Piotrowski et al. (J. Am. Soc. Mass Spectrom. (2019) 30, 489 - 500) proposed a method for determining the location of ion generation in MALDI-TOF MS by analyzing the laser image of the sample surface.

[0014] The disclosure by Jeffrey M. Spraggins et al. (Anal Chem. November 19, 2019; 91(22) 14552 - 14560) explained an investigation of high-performance molecular imaging using MALDI trapped ion mobility time-of-flight (TimsTOF) mass spectrometry.

[0015] The paper by Kyunghan Kim et al. (JLMN - Journal of Laser MicroNanoengineering Vol.7, No.2, 2012) proposed a method for instant synchronization of a galvanometer scanner and a linear stage for rapid and large - area material fabrication, such as for flexible printed circuit boards. However, there was no intention to further use or investigate the laser - ablated materials. The position and velocity information of the linear stage were transmitted to the control board of the galvanometer scanner via encoder signals, and the control board calculated the step size of the laser beam movement by subtracting the original CAD data (CAD = computer - aided design) from the movement of the linear stage. Summary of the Invention

[0016] In view of the above discussion, there is a need for such devices and methods that, although the movement accuracy of some of the components used is designed to meet certain requirements for the spatial resolution of scanning, can accurately locally desorb analyte materials over an extended surface area and also meet high requirements. For those skilled in the art, further objectives achievable by the present invention become immediately clear upon reading the following disclosure.

[0017] According to a first aspect, the present invention relates to a device for desorption scanning of analyte materials placed on a sample carrier, comprising: - a beam device arranged and configured to direct a beam for locally desorbing analyte materials onto an impact area on the sample carrier; - an interface arranged and configured to receive the locally desorbed analyte materials and convey them to an analyzer or an analytical device; - a translation device arranged and configured to change the position of the sample carrier relative to the interface (along at least one dimension) in order to move to different, predetermined non - coincident impact areas; - a position monitoring device arranged at or near the translation device and configured to collect data for determining the actual position of the sample carrier; and - a control system arranged and configured to communicate with the beam device, the translation device, and the position monitoring device, and to control and coordinate their operation. The control system coordinates the operation of the beam device and the translation device such that each impact area is arranged so that the locally desorbed analyte materials can be received by the interface. The control system uses the data from the position monitoring device to detect any deviation of the actual position from the desired or target position of the sample carrier, and if such a deviation is detected, derives an adjustment of the beam orientation therefrom in order to direct the beam onto the impact area that would have been produced without the deviation.

[0018] The present inventors have recognized that the imprecision of the movement of the translation device, which results from the translation device being designed to meet lower spatial resolution requirements, can be compensated for by timely and rapid adjustment of the orientation of the desorption beam. This makes it possible to reliably achieve a spatial resolution as low as 5 - 10 micrometers when scanning the analyte material under investigation, even using simple and robust translation facilities. This improves the performance of analytical devices such as mass spectrometers and / or ion mobility - mass spectrometers, especially in imaging mass spectrometry and high - throughput analysis of single preparations that are very closely spaced on a sample carrier, such as in the characterization of candidate drugs. The principles of the present disclosure apply to both the step - wise movement (in stop - motion cycles) and the quasi - continuous movement of the translation device (e.g., performing many consecutive individual steps at a high frequency, which is considered to be almost continuous).

[0019] In various embodiments, the position monitoring device can be configured as a linear rotary encoder or an incremental encoder, a capacitive sensor system, or an optical sensor system for light. For example, an incremental encoder itself can be based on optical technology and provide a resolution in the range of a few hundred nanometers, especially through interpolation. However, any type of robust position sensing with sufficient resolution is suitable for determining data on any positioning error and for further processing to adjust the orientation of the desorption beam.

[0020] In an optical sensing implementation, the position monitoring device can utilize high - precision interferometry of coherent light. For example, reflective feature mechanisms can be provided for the translation device itself, especially the piezoelectric stage, or for the sample carrier, especially on its narrow sides, onto which the coherent light is directed. The light reflected from these reflective feature mechanisms can be received by a photoreceiver and the interference pattern with the emitted light can be examined, especially the consecutive maxima can be calculated. Using the data thus collected, the position of the sample carrier with nanoscale precision can be directly obtained if the sample carrier itself is monitored, or indirectly if the reflective feature mechanisms are located on the translation device. Suitable devices can determine the position along one spatial direction x, two spatial directions xy, or even three spatial directions xyz, for example using some appropriately aligned optical paths and reflective feature mechanisms. In another variant, the position monitoring device can also operate using high - resolution camera images of the area in which the translation device moves the sample carrier, with a dedicated image evaluation program instantaneously comparing the actual position and the target position of the sample carrier. The camera collects data by observing the position of the translation device and / or the sample carrier, preferably in incident light; however, it can also operate in transmitted light if one or more components are substantially transparent. Multiple camera images taken from different angles can also be used and then combined. In this way, multi - dimensional positioning errors can be included and compensated for when adjusting the beam orientation.

[0021] In various embodiments, the beam device may include a laser beam generator or an ion beam generator. The ion beam may be a primary ion beam for bombarding the analyte material, which results in the formation of secondary ions (SIMS). The ion beam may have a continuous or pulsed ion current. The laser beam, as a coherent light beam, may be emitted from, for example, a solid-state laser (especially Nd:YAG), a gas laser (nitrogen, N2), a dye laser, or a light-emitting laser diode. For such photon-optical desorption, the analyte material may be specially prepared to exhibit optimal light absorption characteristics, for example, by using a matrix material for MALDI ionization. The laser beam may have a continuous or pulsed photon flux. If necessary, the ionization during the desorption process may be supported by a post-ionization mode, such as the so-called MALDI-2 (Jens Soltwisch et al., Science, April 10, 2015 - Volume 348, Issue 6231, 211 - 215) or secondary neutral mass spectrometry (SNMS). For example, shortly after desorption, before the desorbed analyte material is transferred to the analyzer through the interface, a laser pulse laterally irradiated onto the desorption cloud may be used to achieve the post-ionization mode. The desorption laser beam may have a modified beam cross-section, for example, having several independent 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).

[0022] In various embodiments, the beam device may include an adjustable beam orientation element that is controlled to adjust the beam orientation. Preferably, the beam orientation element includes a galvanometer mirror for the laser beam or an opposing pair of DC electrodes for the ion beam. For the laser beam, in particular, a combination of a reflective element such as a mirror, a refractive element such as a prism, and / or an electro-optic or acousto-optic deflection element such as an electro-optic crystal may be used, which is capable of changing or offsetting the position where the desorption beam strikes the sample carrier surface or the analyte material thereon in two spatial directions in a plane parallel to the sample carrier surface, for example, by tilting or otherwise changing the spatial orientation. For the ion beam, an opposing deflection electrode system supplied with a potential may be used, which surrounds the ion beam path and can generate a variable potential gradient in the space between the two opposing electrodes of the electrode pair. An example is two opposing electrode pairs that are 90° to each other. If the potentials applied to the two opposing electrodes of an electrode pair are the same, then the ion beam passing through this pair of electrodes will not experience any lateral deflection. If the potentials are different, the primary ions will be deflected according to the potential difference during their flight.

[0023] In various embodiments, the impact area can be (i) scanned substantially completely by the desorption beam, or (ii) subdivided into a plurality of sub-areas that are scanned by the beam in a predefined order. The "macroscopic movement" generated by the translation device, such as a typewriter-like or serpentine movement through the sample carrier towards the linearly arranged impact areas that can be carried out quickly one after another step by step and, if necessary, also quasi-continuously, can be superimposed with the "microscopic movement" through the individual impact areas, for example by purely changing the orientation of the beam to scan the sub-areas of the impact area. The latter design in particular allows for a relatively fast and easy adjustment of the orientation of the desorption beam in two spatial directions in a plane parallel to the surface of the sample carrier. An impact area or a sub-area thereof is typically the smallest spatial resolution unit in the graphical representation of the data acquired in a larger area of the analyte material with the analytical device. However, the data from the subdivided impact areas do not necessarily have to be considered separately but can also be combined. In post-processing, the spatial resolution in the graphical representation or distribution map of the molecular content of the sampled analyte material can always be retrospectively reduced by combining the measurement data obtained spatially separately, such as measurement data from different sampling sites.

[0024] In various embodiments, the interface can have an axis that is substantially parallel to the surface normal of the sample carrier and conveys the locally desorbed analyte material along this axis to the analytical device. It is advantageous for the interface to be arranged such that its projection includes the impact area and the desorption site on the sample carrier, since the desorbed analyte material always moves away from the surface of the sample carrier and thus already has a velocity component in the direction of the interface without any further action. However, it is also possible to additionally or alternatively actively drive the charged desorbed analyte material towards the interface, for example by using a suitable static or pulsed switched potential gradient between the sample carrier and the interface.

[0025] In different embodiments, the beam device can be arranged and configured such that the desorption beam impinges on the sample carrier at an angle of incidence with respect to the surface normal. This embodiment is preferred when the desorbed analyte material is received from the interface along the surface normal of the impact area and the desorption position and conveyed to the analytical device. An example is a time-of-flight analyzer with axial acceleration in the entry flight path, such as a mass spectrometer from Bruker.

[0026] In various embodiments, the translation device may be arranged and configured to change the position of the sample carrier in the xy plane, which is substantially perpendicular to (i) the axis of the interface and / or (ii) the surface normal of the sample carrier. In a particular embodiment, the axis of the interface and any surface normal of the sample carrier, regardless of its origin, are preferably at least parallel, even identical; the latter especially with respect to the surface normal emanating from the adjusted impact area. Preferably, the beam device is also arranged and configured to track the position of the beam focus on the analyte material in response to a derived adjustment of the beam orientation in a direction substantially perpendicular to the xy plane. The beam device may in particular include an adjustable imaging lens for tracking the beam focus. A preferred translation device is an xy translation stage or a similar actuator, which is designed to adjust or move the sample carrier, together with the analyte material placed thereon or therein, in two spatial directions parallel to the plane of the sample carrier surface. The larger the movement radius of the adjustment device for changing the desorption beam orientation, the lower the requirements regarding the minimum step size of the translation stage can be, since the movement of the desorption beam can cover the impact area between two activations of the translation stage. The conventional step size of the translation stage can be in the range of two-digit to three-digit micrometers.

[0027] In various embodiments, the movement mechanism for the sample carrier, especially the one implemented in or on the translation stage, may also include the possibility of adjustment in a third spatial direction (z-axis), for example, to adapt the optimal focus of the desorption beam on analyte materials of different thicknesses or heights, as described in the applicant's patent publication DE 10 2007 006 933 A1 (corresponding to US 2008 / 0191131 A1 and GB 2 446 699 A). When irradiated at an angle, this z-adjustment also results in an adjustment in the spatial directions xy perpendicular to it (i.e., with respect to the sample carrier surface), which can be compensated for by the adjustment of the beam orientation described herein.

[0028] In various embodiments, the impact area may be selected to allow the surface normal of the sample carrier emanating from the impact area to pass through the interface, to ensure that locally desorbed analyte material can be conveyed to the analysis device.

[0029] In various embodiments, the analyzer or analysis device may be a mobility analyzer, a mass analyzer, or a mobility-mass combination analyzer. In general, the terms ion spectrometry analyzer and measurement method may be used, which may include mobility separation, mass separation, or a combination of both.

[0030] The mobility analyzer separates charged molecules or molecular ions based on their ratio of collision cross-section to charge, sometimes expressed as Ω / z or σ / z. It is based on the interaction between the ion species and the electric field coupled to the charge of the ion, as well as the simultaneous influence of the buffer gas interacting with the average cross-sectional area of the ion. In particular, drift tube mobility separators with a static electric field gradient are known, which drive ions through a substantially stationary gas. Here, the drift velocity of the ion species is determined by the driving force of the electric field and the decelerating force of collisions with gas particles. Also common are trapped ion mobility separators (TIMS), which use a continuous laminar gas flow to drive the ions forward, and the gas flow is counteracted by a gradually varying electric field gradient with a corresponding variable decelerating force. Traveling wave mobility separators are also worth mentioning.

[0031] On the other hand, mass analyzers separate charged molecules or molecular ions based on their mass-to-charge ratio (commonly expressed as m / z). In addition to the previously mentioned time-of-flight analyzers, for which linear and reflector devices can be selected, or devices for axially or orthogonally accelerating ions into the flight path, other types of mass dispersion separators can also be used, such as quadrupole mass filters (single quadrupole), three-sector quadrupole analyzers ("triple quadrupole"), ion cyclotron resonance cells (ICR), Kingdon-type analyzers, such as (Thermo Fisher Scientific), and others. Of course, separators of the types mentioned previously can be coupled so that ion species can be separated multidimensionally, i.e., according to more than one physicochemical property, such as m / z and Ω / z or σ / z.

[0032] In different embodiments, the interface can include electrodes to which a potential can be applied to direct local desorption of the charged analyte material. These can be diaphragm electrodes, for example, plates of a conductive material with pores. These designs are particularly suitable as extraction electrodes for accelerating the desorbed analyte material into the flight path. Additionally or alternatively, electrodes to which a high-frequency voltage is applied can also be used, as is the case, for example, in high-frequency voltage ion funnels, devices designed to condense spatially widely dispersed ions into a relatively thin beam. The potential can be applied permanently to the connectable electrodes, thereby creating a permanent potential gradient, and / or it can be switched in a pulsed manner, for example, timed to coincide with desorption events in the impact area on the sample carrier.

[0033] The analyte material can be a tissue section. It is preferred to measure the molecular content of (thin) tissue sections with spatial resolution of their molecular content in order to create and visualize a distribution map of molecules of interest, such as biological macromolecules, such as peptides, lipids (phospholipids and glycolipids), oligosaccharides, sterols, saccharides, secondary metabolites or fat-soluble vitamins, but also including other possible non-tissue molecules, such as active pharmaceutical substances (drug substances) and / or their degradation products. Those skilled in the art will appreciate that the analyte material can also be an array of tissue microarrays (TMAs) or individual preparations, such as sample material prepared according to the dried droplet method for subsequent MALDI ionization. Then, the impact areas can include lyophobic or hydrophilic areas on a metallic or other conductive sample carrier, which are isolated from each other by lyophobic or hydrophilic areas on the sample carrier, such as the AnchorChip from Bruker TM in the form of a plate.

[0034] In various embodiments, the impact areas can have a polygonal contour. Thus, an array of impact areas can be created that covers the underlying sample carrier surface and the analyte material placed thereon with little to no gaps. Examples are rectangles, such as squares, and also polygons with more corners, such as hexagons. The contour of the impact areas can be uniform across the entire sample carrier. However, in principle, different shaped impact areas can also be defined during the same scan, provided that this is useful for the application. The contour of the impact areas can also be designed in different ways. The scope is almost limitless for those skilled in the art.

[0035] In different embodiments, the information obtained from the sampled impact areas by an analyzer can be combined to form a spatial distribution map of ions or the underlying molecular matrix on the analyte material. This enables label-free measurement in a non-optical microscopy manner to illustrate the distribution of endogenous and exogenous molecules on the analyte material, which is different, for example, from staining methods in optical microscopy.

[0036] According to another aspect, the present invention relates to a method for desorption scanning of an analyte material placed on a sample carrier, comprising the steps of: (a) positioning the sample carrier (along at least one dimension) to approach an impact area at which a beam is aligned for local desorption of the analyte material; (b) determining the actual position of the sample carrier after the positioning; (c) comparing the determined actual position with a desired or target position of the sample carrier to determine any deviation; (d) if a deviation is detected, adjusting the beam orientation so that the beam is directed to the impact area that would have occurred on the sample carrier in the absence of the deviation; (e) applying the beam to the impact area to locally desorb the analyte material and deliver it to an analyzer or an analyzing device; and (f) checking whether a predetermined end condition is met, and if not, repeating steps (a)-(e) for subsequent non-conforming impact areas.

[0037] In the context of the present disclosure, determining the actual position of the sample carrier after the positioning in step (b) may include collecting data at a high clock frequency (e.g., in the range of 10 kHz or higher) to determine the actual position of the sample carrier during the decay time of the movement of a high-quality translation device assembly when the actuator of the translation device performing the positioning has stopped acting after completing a step movement. Then, the high clock frequency of collecting data from the position monitoring device allows timely actuation of the beam device for alignment correction while the subsequent oscillations of the translation device are still decaying. The fast response of the beam device on the order of kilohertz can compensate for small-scale offsets of the translation device near the equilibrium position caused by the oscillations, so that the beam can be quickly applied to the stepped impact area. This method can save several milliseconds of decay waiting time for each stepped position setting; this is particularly advantageous when scanning large-area tissue sections, as this may require millions of stepped position settings.

[0038] The final condition may be met when the desorption beam has been applied to all planned impact areas on the sample carrier or the analyte material. In an extreme case, the beam may be applied to a point where the analyte material has been substantially completely removed by the action of the desorption beam in all planned impact areas, so that the desorption beam cannot produce additional desorbed analyte material. Another example of an end condition is when some impact areas of particular interest, which may be smaller than the total area of the extended analyte material, have been scanned, leaving some portions unaffected by the beam, e.g., areas of different cell types in a (thin) tissue section. The termination condition may also essentially be defined as a failure occurring during the execution of the method, e.g., because a mirror is stuck in the beam path.

[0039] Since the method according to the present invention is suitable as an operating mode of the device according to the present invention, the embodiments described above with reference to the device are also applicable to the method in a corresponding manner. Description of the Drawings

[0040] The present invention can be better understood by reference to the following drawings. The elements in the drawings are not necessarily to scale, but are mainly for the purpose of illustrating the principles of the present invention (mainly schematic). In the drawings, the same reference numerals denote corresponding elements in different views.

[0041] Figure 1 For the purpose of classifying the content of the present invention, there is shown a schematic diagram of an LDI axial reflector TOF arrangement having and capable of implementing the principles of the present invention.

[0042] Figure 2A There is schematically shown the challenge of the present invention regarding the use of a translation device whose performance characteristics do not meet the requirements of the scanning spatial resolution.

[0043] Figure 2B There is shown a microscopic image of the surface of a sample carrier scanned without beam alignment correction.

[0044] Figure 2C There is schematically shown an operating mode in accordance with the principles of the present invention.

[0045] Figure 2D There is shown similar to Figure 2B a microscopic image of the surface of a sample carrier scanned, with beam alignment correction.

[0046] Figure 3 For further classifying the content of the present invention, there is shown an LDI-TIMS-Q-FRAG reflector-OTOF arrangement having and capable of implementing the principles of the present invention. Detailed Description

[0047] Although the present invention has been shown and described with reference to several embodiments, those skilled in the art will recognize that various modifications in form and detail can be made without departing from the scope of the technical teachings as defined in the appended claims.

[0048] Figure 1Schematically shown is a MALDI time-of-flight mass spectrometer (modified from DE 10 2011 112 649 A1 cited in the introduction) for classifying the present disclosure. It includes a time-of-flight analyzer 1 and a beam device 2, which has mirror systems 7, 8 for controlling the orientation of the laser beam relative to the surface of the sample carrier 13 carrying the analyte material. A generally pulsed laser beam is generated in the beam generation unit 3, which includes a laser crystal 4 and, if necessary, a device 5 for doubling the laser frequency. Then intensity peaks are provided in the pattern generator 6 (for example, according to the principle set forth in DE 10 2004 044 196 A1; corresponding to US 2006 / 0071160 A1 and GB 2 421 352 A), and are deflected in two spatial directions in the mirror system by two galvo mirrors 7 and 8 such that the impact position of the laser beam on the sample carrier surface can be moved.

[0049] Then, the deflected laser beam is expanded in the Kepler telescope 9 and parallel-shifted according to the angular deflection. The outgoing laser beam is guided into the objective lens 11 through the mirror 10 at a reduced deflection angle and is now centered again completely. According to the angular deflection, the beam passes through the center of the objective lens 11 but at a slightly different angle, thus moving its impact position on the sample carrier 13. It should be noted here that the beam guidance within the Kepler telescope 9 is more complex, and for simplicity, the figure does not actually reproduce it, although the figure correctly depicts the external influence of the telescope on the laser beam.

[0050] Desorbed charged molecules and ions generated in the desorption cloud of the incident laser beam are accelerated at the interface towards the mass analyzer by a potential to form an ion beam 18, which has diaphragms 14 and 15 pulse-connected to a potential. The ion beam passes through two deflection capacitors 16, 17 for path correction and is focused onto a detector 20 in a reflector 19 (LDI axial reflector TOF device). The sample carrier 13 is coupled to a translation device 22, for example placed on a translation stage, which can be adjusted, for example, in one, two or even three spatial directions xyz so as to approach the impact area of the analyte material on the sample carrier targeted by the laser beam. A position monitoring device 24, such as a linear rotary encoder or an incremental encoder, a capacitance sensor system or an optical sensor system, is attached to or in the vicinity of the translation device 22, which is capable of independently determining data on the actual position with respect to the translation device 22 and / or the sample carrier with high precision, for example with a nominal resolution of 0.1 µm. Preferably, a position monitoring device is used whose nominal position determination accuracy exceeds the smallest possible step size of the translation device by more than a factor of ten. If the smallest step size of the translation device is 5 µm, this results in a nominal accuracy of the position monitoring device ≤ 0.5 µm; correspondingly, a smallest step size of 3 µm means a nominal accuracy ≤ 0.3 µm, etc.

[0051] A control system 26 adapted to implement the principles of the present invention and programmed accordingly communicates with both the position monitoring device 24 and the adjustable mirrors 7, 8 of the beam device 2 and the translation device 22 and coordinates the operation of the latter two, in particular their operation relative to each other. The impact area on the sample carrier is arranged such that the analyte material desorbed and ionized by the beam can pass through the interface in the form of two diaphragm electrodes 14, 15. In the illustrated arrangement, the surface normal of the sample carrier 13 starting from the set impact area passes through the pores of the diaphragm electrodes 14, 15 and is substantially parallel to the ion beam 18. In this example, the first diaphragm electrode 14 or more precisely its pores define a geometric acceptance frame for receiving and transporting the locally desorbed analyte material, since material desorbed on the sample carrier 13 outside the contour of the projection of the pores onto the sample carrier surface can no longer be reliably evaluated using time-of-flight to mass imaging calibration, and furthermore, compared to such material desorbed within the projection contour of the pores on the sample carrier 13, it suffers a significant loss of resolution.

[0052] The problems solved by the present disclosure and their solutions will be explained below with reference to Figure 2A - Figure 2D The sample carrier is covered with an analyte to be desorbed, for example, a two-dimensional tissue section of a microtome section or an array of two-dimensional extensions of individual sample preparations at positions marked for this purpose, the latter being, for example, the AnchorChip from Bruker TMOn one of the type plates. The desorption beam is used to scan different impact areas of the analyte material in order to locally release molecules and ions, which are transported via the interface to the analyzer for analysis. The sample carrier is connected to a translation device that can change its position relative to the interface along at least one spatial direction, preferably along two spatial directions in a plane, and further preferably along three spatial directions.

[0053] The position of the sample carrier can be defined, for example, as its geometric center or any other fixed point on or within it. The position and size of the sample carrier on the translation device are usually preset or known through instrument conditions, such as mounting recesses or positioning feature devices such as clamps, so that the position change of the translation device can be quickly and easily converted into a position change of the sample carrier. Similarly, the position and size of the analyte material on the sample carrier are known (e.g., in the case of an AnchorChip TM plate) or can be predetermined (e.g., in the case of a tissue section whose exact cut shape and profile are difficult to predict). If needed, for example, if there is uncertainty in this regard, or if confirmation is required, they can be optically monitored or scanned, for example, using a camera and suitable reference marks usually located in the edge region of the sample carrier.

[0054] In the standard orientation, the desorption beam impinges on a specific area of the sample carrier surface, which is essentially determined by the position of the translation device. Beam deflection from this standard orientation is possible and specified within specific spatial limits; the latter especially when a series of alternating translation device movements and beam orientation changes are used for the purpose of accelerating the execution to perform the scanning of the analyte material, see, for example, the German application 102021114934.7 recently filed by the applicant (corresponding to US 17 / 739,251 and UK 2208059.2). If the beam is to be applied to an area of the sample carrier surface that is beyond the movement radius of the beam device, the translation device will move the sample carrier in the appropriate direction to restore the proximity of the desorption beam to the analyte material. During the measurement cycle, many such movements of the translation device may typically occur quasi - continuously at a high step frequency, which in principle also increases the cumulative risk of occurrence of positioning errors, even if, as often claimed by translation stage manufacturers, the probability of positioning errors in a single movement is low.

[0055] Especially when scanning tissue sections, but also in the case of very densely arranged individual preparations, adjacent impact regions of the beam should be close together, but still spatially separated from each other. In particular, in the imaging of flat analyte materials, spatial proximity is particularly important in order to extract as much analyte information as possible from the analyte material. At the current state of the art, pixels or individual preparations with a size of 5 - 10 micrometers and a comparable pixel or preparation size are considered challenging. At the same time, it is equally important to be able to clearly assign the analyte information to a narrowly defined surface area of the analyte material as the origin point, especially in regions where there are significant changes in the analyte composition of the analyte material, such as from one tissue type to another, or from one individual preparation to another.

[0056] Using commercially available translation devices, these high requirements can only be met to a limited extent. In particular, if the requirements for the spatial resolution of the scan are so high that impact regions with dimensions of a few micrometers need to be scanned and then also displayed as different image elements in the graphical evaluation, the limited precision of the components used to date becomes significant. Figure 2A The sample carrier 13' is schematically shown, which bears the analyte material (not shown) and is to be scanned by the desorption beam 30 according to a predefined grid of impact regions. The planned impact regions 32 are represented by dashed squares, see the following description. The corresponding grid floor plan includes both the positions of the impact regions (e.g., central positions or predefined positions at the edges or corners) and the spacing and arrangement of the regions relative to each other, and this grid floor plan can be specified by the user or created automatically or semi - automatically by a computer program. For simplicity, this example assumes a grid with a linear array of planned impact regions 32, although the principle can of course be applied to two - dimensional scanning. It is also assumed that the desorption beam 30 impacts the impact regions in a fixed orientation, i.e., there is no micro - movement of the orientation of the beam 30 in the impact region in order to sweep a specific region within the movement radius of the beam 30, as described in parts of the prior art as a combination of "beam scanning mode" and "platform scanning mode". However, the principles of the present disclosure can still be applied in a corresponding manner to these kinds of embodiments.

[0057] In addition to the planned impact regions 32, Figure 2A the actual impact regions 34 are also shown in the form of differently shaded squares with solid outlines. In the top step, the actual impact region (vertically hatched) and the planned impact region coincide. This means that in further data and signal processing, the information of the region elements from the analyte material is correctly spatially assigned. This also means that the information is obtained from analyte material that has not been sampled previously; in other words, the analyte material has not been altered by a previous desorption at this position.

[0058] The intermediate step shows the problem of the translation device (not shown) when the movement to the subsequent impact area is insufficient and, for example, insufficient due to a positioning error. Such a positioning error may be due to the inhomogeneity of the magnetic field, which may be caused by the design of the vacuum motor. This is shown by the compression arrow 36A. The actual impact area (diagonal hatching) no longer coincides with the planned impact area 32; instead, the two areas only overlap by about half, and this is aggravated by the fact that the edge area of the actual impact area contacts the edge area of the previously properly irradiated impact area (vertical hatching). If scanning is performed in this orientation, the desorption result will be a mixture of unsampled and already sampled material, which leads to an adverse spatial confusion of the underlying analyte information. Depending on the degree of desorption, for example, in the case where the analyte material at the previous bombardment position is completely laser ablated, part of the beam may also hit the bare sample carrier surface, which particularly distorts the quantitative information.

[0059] In the bottom step, another problem of the translation device is shown when the movement from the previous impact area to the subsequent adjacent impact area does not follow the specifications but lasts too long, for example, due to a positioning error. This is represented by the stretching arrow 36B. In this example, the actual impact area (cross hatching) also no longer coincides with the planned impact area 32; again, the two areas only overlap by about half, where the actual area protrudes into the area of the sample carrier 13' and the analyte material that has not yet been sampled by the desorption beam 30. In both of the above cases, the planned and actual desorption sites no longer match, which results in an adverse mapping error in the measured data to be evaluated.

[0060] To illustrate these problems in practice, Figure 2B A microscopic image of ablation pits caused by laser bombardment on a sample carrier widely and uniformly prepared with the MALDI matrix substance α-cyano-4-hydroxycinnamic acid (HCCA) is shown. Each ablation pit is the result of multiple bombardments of the same site, in this case 30 times, which is often used in MALDI data acquisition to increase the signal-to-noise ratio. For the purpose of display, the image is color-inverted, so the ablation pits that are planned to be spaced 10 microns horizontally and vertically appear as white dots. The movement sequence for sampling different impact areas only includes the movement sequence of the translation stage on which the prepared sample carrier is located. On the other hand, the alignment of the laser beam remains stationary, which is inconsistent with the principle of the present disclosure. As a result, the components used in this example show a large number of irregularities in what is actually planned as a regular row and column grid, especially at the beginning of the scan lines. There, the actual impact areas partially merge with each other. In the further course of the row, visible gaps greater than the planned uniform spacing of 10 microns exist between the individual impact areas. The column fidelity, which is planned to be an ideal grid, can only be described as insufficient in actual implementation and for specific requirements.

[0061] Based on Figure 2A and Figure 2B the problems occurring in Figure 2C a method according to the principles of the present disclosure is shown. It is important for the implementation of this method that the beam device never operates with a stationary alignment of the desorption beam 30, but is controlled in such a way that the detected misalignment of the translation device is compensated for by recalculating the position coordinates of the impact area on the surface of the sample carrier and thus recalculating the beam alignment. Information about the misalignment of the translation device is provided by a position monitoring device (not shown), for example, a high-precision linear incremental encoder, a high-precision capacitance or optical sensor system, which transmits its measurement data to a control system, which can be, for example, a distributed system consisting of many control devices assigned to individual components, such as laser control, translation stage control, etc. Alternatively, the control system can include a central control device, which can be designed as a microprocessor or a similar computing unit. If necessary, these computational processes can also be executed in the operating system of a computer, which coordinates the analyzer, especially the ion source, connected via an interface according to a predefined scan run and, if necessary, using distributed control devices.

[0062] After each movement of the translation device, the position monitoring device collects data about its actual position, from which the position of the sample carrier 13' and the analyte material placed thereon can be directly derived, or collects data about the actual position of the sample carrier itself and transmits the data to the control system. The control system compares the actual position with the nominal, desired or target position generated by a previously defined grid plan. If no deviation is detected, or only a deviation within a certain tolerance is detected, a control command can be sent to trigger the desorption beam 30 at the proximity position. These tolerances can be measured, for example, according to whether a distinguishable distance is maintained between two adjacent impact areas of the analyte material and thus the origin positions.

[0063] On the other hand, if a position deviation is detected, especially in the three-digit nanometer range or larger, the difference along the affected spatial direction is determined and input as an offset for recalculating the alignment or orientation of the desorption beam 30. Then, this realignment or alignment adjustment or orientation adjustment is performed by an adjustable beam directing element in the beam guiding path, for example, by tilting a galvanometer beam guiding mirror in the case of laser ablation (see, for example, Figure 1 the reference numerals 7, 8 in Figure 2C ), or by changing the potential difference applied to a pair of opposing deflection electrodes in the case of a desorption primary ion beam. In Figure 2CIn the top step, no adjustment of the beam orientation is required because there is no positional deviation. In the intermediate step, the beam orientation must be deflected slightly against the direction of movement of the translation device in order to align the planned and the actual impact areas. In the bottom step, the beam alignment is deflected slightly in the direction of movement in order to aim at the planned impact area. If a position correction is carried out by means of the beam device when required, which is done within a few milliseconds without any problematic time loss, then, and only after that, the beam is triggered for local desorption of the analyte material.

[0064] In particular, a mathematically simple case of recalculation or correction of the beam orientation is when the desorption beam impinges on the sample carrier surface at an angle of incidence close to zero degrees relative to the surface normal, because then the deflection of the beam from the standard orientation directly in the impact plane corresponding to the sample carrier surface can be treated equally in each direction. Mathematically, it becomes slightly more complex when the desorption beam impinges on the sample carrier at a non-zero or 180-degree angle of incidence with respect to the surface normal (depending on the viewing angle), as Figure 1 schematically shown in the axial TOF arrangement in. There, charged molecules and ions are preferably extracted from the ion generation region along the surface normal of the sample carrier. For example, by the method described in DE 10 2018 112 538 B3 (corresponding to US 2019 / 0362958 A1 and GB 2 574 709 A), a slight deviation from this ion extraction axis can be compensated in terms of mass resolution. In the standard orientation of such an axial TOF setup, the angle of incidence can be 30° - 40°.

[0065] In the case of such an oblique incidence in the standard alignment of the beam, the appropriate tilting of the mirror or other adjustment of the beam directing element not only changes the incidence position of the beam on the sample carrier, but also its shape, and in particular, no longer fully guarantees that its focus (defined as the region of the smallest spatial extent having the maximum spatial energy density simultaneously) is located in or on the analyte material at the position where it is to exert its desorption force. This is all the more true since, compared to an incidence parallel to the surface normal, there is a preferred direction of oblique incidence, which manifests itself in that, for a given same angular increment, a change in the beam alignment in the preferred direction has a greater impact on the shape and focus of the beam than a change in the reverse (or perpendicular to) preferred direction. As a result, in these kinds of embodiments, the control system must take into account the oblique incidence in the calculations to ensure that the desorption beam still correctly finds its target after adjusting the incidence angle of the desorption beam according to the detected misalignment of the translation device. Depending on the degree of translation misalignment to be corrected by the optical or ion optical device of the light, adjusting the alignment of the desorption beam may also involve adjusting its focus in the z direction perpendicular to the xy surface of the sample carrier. For example, for an electromagnetic beam, an imaging lens arranged in the beam path and whose position in the beam path can be adjusted can be used to change the focus position, or for a primary ion beam, a single lens arranged in the beam path and whose working potential can be adjusted can be used to change the focus position.

[0066] Figure 2B The results of a scan without correction of the laser beam alignment are shown, whereby the accuracy of the scan is determined by the precision of the translation stage designed for lower requirements, similar to Figure 2B , Figure 2D The results of the same experiment are shown, but with beam alignment correction as a function of the translation misalignment of the translation stage or the sample carrier, which is determined from the data of the position monitoring device. Again, this is a color-inverted illustration for illustrative purposes. It can be clearly seen that the ablation pits (white dots) are located in regularly arranged rows and columns. The spacing between the individual points is substantially uniform, approximately 10 micrometers. There is no obvious overlap of multiple impact regions. Such a setup enables ensuring a spatially precise image of the analyte material. Of course, the findings from this example can also be applied to more complex desorption setups, for example, using a desorption beam with a diameter of approximately 3 - 5 micrometers on the analyte material, a translation device moving in steps of approximately 5 micrometers, and a spacing of approximately 5 - 10 micrometers between adjacent impact regions being scanned, which is of the same order of magnitude as the movement step of the translation device.

[0067] For further classification of the present invention, Figure 3Schematic diagram of another possible arrangement of the device according to the present invention, where the analyzer connected through the interface is a migration spectrometer - mass spectrometer (changed from the article by Jeffrey M. Spraggins et al. cited in the introduction). Its setup and operation are briefly explained below:

[0068] A laser system is designed as a beam device 2* with various beam guiding elements for pulsed bombardment of the analyte material on the sample carrier 13*. The sample carrier 13* can be scanned step by step to obtain spatially resolved measurement signals from the analyte material, such as from an extended two - dimensional analyte material, like a planar tissue section or a separately prepared sample array, such as a locally applied tissue homogenate preparation.

[0069] The sample carrier 13* is coupled to a translation device 22*, for example, placed on a translation stage, which can be adjusted in one, two, or even three spatial directions xyz to approach the position targeted by the laser beam on the analyte material on the sample carrier. A position monitoring device 24* is connected to or located near the translation device 22*. This position monitoring device is, for example, a linear rotary encoder or an incremental encoder, a capacitive sensor system, or an optical sensor system, which can independently determine the actual position data of the translation device 22* and / or the sample carrier 13* with high precision, for example, with a nominal resolution of 0.1 micrometer. When the position of the translation device itself is determined, the position of the sample carrier 13* and thus the position of the analyte material can be easily obtained.

[0070] A control system 26*, suitable for implementing the principles of the present invention and programmed accordingly, communicates with the position monitoring device 24* and the adjustable beam guiding elements (not shown) in the beam device 2* and the translation device 22*, and particularly coordinates the operation of the latter two. The impact area on the sample carrier 13* is set such that the analyte material desorbed and ionized by the beam is received by an interface in the form of a high - frequency voltage ion funnel 40 and spatially compressed into a thin ion string for further transmission through the arrangement. In the shown arrangement, the surface normal of the sample carrier 13* starting from the set impact area passes through the pores in the electrodes of the funnel 40. The electrode assembly of the first funnel element, or more precisely, its pores, sets the geometric acceptance frame for receiving and transmitting the locally desorbed analyte material in this example. This acceptance range is larger than Figure 1 the interface in the example, but nevertheless, in this case, the analyte material cannot be desorbed at any arbitrary position on the sample carrier 13*. Correspondingly, compared with the material desorbed within the projection profile of the maximum funnel opening on the sample carrier 13*, the material desorbed outside the projection profile of the first funnel opening on the surface of the sample carrier 13* cannot be reliably transferred to the connection tunnel area of the migration spectrometer, or can only be transferred with losses.

[0071] After generation, the charged analyte material enters the mobility spectrometer 42 through the high-frequency voltage funnel 40. The mobility spectrometer has an accumulation region 42A and a subsequent analysis region 42B. For example, the principle of this dual design is described in the applicant's patent publication US 2016 / 0231275 A1. An inert gas flows through the two regions of the mobility spectrometer 42 (from left to right in the figure). The charged analyte material is driven therein by the gas flow against an opposing electric field. In the analysis region 42B, the charged analyte material is separated at corresponding different positions along the axis, depending on their respective mobilities.

[0072] The stepwise reduction of the electric field strength in the analysis region 42B of the mobility analyzer 42 enables the sequential transport of the charged analyte material separated by mobility. After mobility analysis in the analysis region 42B, the charged analyte material collected simultaneously in the accumulation region 42A is transferred to the analysis region 42B. The charged analyte material emerging from the analysis region 42B first passes through the transfer multipole 44 and then enters the quadrupole mass filter 46. Here, the charged analyte material can be selected for further analysis, while other charged analyte materials can be filtered out. Subsequently, the charged analyte material is transferred to the collision cell 48, where the selected charged analyte material can be fragmented by being accelerated into a neutral gas.

[0073] In the collision cell 48, the charged analyte material and / or any charged fragments generated therefrom are temporarily stored before being introduced into the pulser 50 of the time-of-flight analyzer 52 in a synchronous and orthogonal injection manner. There, perpendicular to the entry direction, the analyte material or charged fragments are accelerated into the flight path of the reflector time-of-flight analyzer. At the end of the flight path, after reversing the flight direction in the reflector (indicated by the arrow), a detector (not shown) receives various time-resolved and thus mass-resolved data packets and outputs them as instantaneous time-of-flight data, which can subsequently be reprocessed into mass (m) or mass / charge ratio (m / z).

[0074] The present invention has been described above with reference to different, specific embodiments. However, it should be understood that various aspects or details of the described embodiments can be modified without departing from the scope of the present invention. In addition, if feasible for those skilled in the art, the features and measures related to different embodiments can be combined as needed. Furthermore, the above description is only a demonstration of the present invention and not a limitation of the scope of protection, which is fully defined by the appended claims, taking into account any possible equivalents.

Claims

1. A device for performing a desorption scan on an analyte material placed on a sample carrier, comprising: - A beam device, which is arranged and configured to direct a beam for locally desorbing an analyte material onto an impact area on a sample carrier; - An interface, which is arranged and configured to receive the locally desorbed analyte material and convey it to an analysis device; - A translation device, which is arranged and configured to change the position of the sample carrier relative to the interface so as to access different predetermined, non - completely overlapping impact areas; - A position monitoring device, which is arranged at or near the translation device and is configured to collect data after each movement of the translation device to determine the actual position of the sample carrier; and - A control system, which is arranged and configured to communicate with the beam device, the translation device, and the position monitoring device, and to control and coordinate their operations, wherein the control system coordinates the operations of the beam device and the translation device such that each impact area is arranged so that the locally desorbed analyte material can be received by the interface, and wherein the control system uses the data of the position monitoring device collected after each movement of the translation device to detect any deviation between the actual position and the desired position of the sample carrier, and if such a deviation is detected, the control system derives an adjustment of the beam orientation therefrom so as to direct the beam onto the impact area that would have been produced without the deviation.

2. The device according to claim 1, wherein the position monitoring device is a linear encoder or an incremental encoder, a capacitive sensor system or an optical sensor system of light.

3. The device according to claim 1, wherein the beam device comprises a laser beam generator or an ion beam generator.

4. The device according to claim 1, wherein the beam device comprises an adjustable beam orientation element, and the beam orientation element is controlled to adjust the beam orientation.

5. The device according to claim 4, wherein the beam orientation element comprises a galvanometric mirror for a laser beam, or a pair of opposing DC electrodes for an ion beam.

6. The device according to claim 1, wherein the impact area (i) is substantially completely scanned by the desorption beam, or (ii) is subdivided into a plurality of sub-regions, and these sub-regions are scanned by the beam in a predetermined order.

7. The device according to claim 1, wherein the interface has an axis substantially parallel to the surface normal of the sample carrier, and conveys the locally desorbed analyte material to the analysis device along the axis.

8. The device according to claim 1, wherein the beam device is arranged and configured such that the desorption beam impinges on the sample carrier at an incident angle with respect to the surface normal.

9. The device according to claim 7, wherein the translation device is arranged and configured to change the position of the sample carrier in the xy plane, and the xy plane is substantially perpendicular to (i) the axis of the interface and / or (ii) the surface normal of the sample carrier.

10. The device according to claim 9, wherein the beam device is further arranged and configured to track the position of the beam focus on the analyte material in response to the resulting adjustment of the beam orientation in a direction substantially perpendicular to the xy plane.

11. The device according to claim 10, wherein the beam device comprises an adjustable imaging lens for tracking the beam focus.

12. The device according to claim 1, wherein the impact area is selected to allow the surface normal of the sample carrier emitted from the impact area to pass through the interface, so as to ensure that the locally desorbed analyte material can be conveyed to the analysis device.

13. The device according to claim 1, wherein the interface comprises an electrode, and a potential can be applied to the electrode to direct local desorption of the charged analyte material.

14. A method for performing a desorption scan on an analyte material placed on a sample carrier, the method being carried out using the device according to any one of claims 1 to 13, the method comprising the following steps: (a) Set the position of the sample carrier to approach the impact area at which the beam is aligned for local desorption of the analyte material; (b) Determine the actual position of the sample carrier after setting the position; (c) Compare the determined actual position with the target position of the sample carrier to determine any deviation; (d) If a deviation is detected, adjust the beam orientation so that the beam is aligned onto the impact area that would be produced on the sample carrier without the deviation; (e) Apply the beam to the impact area to locally desorb the analyte material and convey it to the analyzer; and (f) Check whether a predetermined end condition is met, and if not, repeat steps (a) - (e) for subsequent non - completely overlapping impact areas.

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