Methods for desorbing and ionizing sample material

Abstract

Method for desorbing and ionizing sample material placed on a sample carrier (15, 115, 215) comprising: - repeated local impact of sample material on the sample carrier (15, 115, 215) using a first energetic radiation (12, 112) and inducing local desorption of sample material into the gas phase above the sample carrier (15, 115, 215), thereby changing a relative position of the first energetic radiation (12, 112) to the sample carrier (15, 115, 215) and targeting a multitude of impact points on the sample material on the sample carrier (15, 115, 215); - Pulsed impingement of the locally desorbed sample material using a second energy radiation (12*, 112*, 212*) directed into the desorbed sample material, causing ionization and / or increasing the degree of ionization of the locally desorbed sample material, wherein a propagation direction of the second energy radiation (12*, 112*, 212*) lies in a plane that is substantially perpendicular to a surface normal of the sample carrier (15, 115, 215) and is arranged above the sample carrier (15, 115, 215), tracking the focus and / or beam waist position of the second energy radiation (12*, 112*, 212*) such that it is substantially opposite an actual point of impact on the sample material on the sample carrier (15, 115, 215), where sufficient distance is maintained between the second energetic radiation (12*, 112*, 212*) and a sample carrier surface to prevent unintentional grazing of the sample carrier (15, 115,215) or of the sample material placed thereon, and to prevent the formation of substrate; and , - Transferring ionized sample material, which has been obtained from locally desorbed sample material exposed to the second energetic radiation (12*, 112*, 212*), into an ion processing facility.

Description

Field of invention

[0001] The invention relates to methods and devices for desorbing and ionizing sample material deposited on a sample carrier. The principles of the disclosure can be used, for example, in imaging ion spectrometry, in particular in imaging ion spectrometry with ion formation using matrix-assisted laser desorption and ionization (MALDI). Background of the invention

[0002] The prior art is explained below with reference to a specific aspect. However, this should not be understood as a limitation. Useful developments and modifications of the invention may also be applicable beyond the relatively narrow scope of this introduction and will be readily apparent to experienced practitioners in this field after reading the subsequent disclosure of the invention.

[0003] The MALDI method has long been used for ion spectrometric analyses. In ultraviolet vacuum MALDI, soluble analyte molecules are embedded in a light-absorbing, crystallizing matrix substance and then irradiated with coherent ultraviolet light pulses. The UV light is absorbed by the crystallizing matrix substance, which then desorbs into a cloud of material, along with the embedded analyte molecules. Due to the properties of the desorption process and the material cloud, charge carriers are also generated and transferred to the analyte molecules, resulting in charged analyte molecules or analyte ions. These analyte ions can then be guided and analyzed using electromagnetic fields, for example, in mobility and / or mass analysis, which sorts and identifies charged molecules or ions according to their cross-section or mass-to-charge ratio.

[0004] Advantages of this established MALDI method include the very gentle ionization of analyte molecules, virtually eliminating fragmentation, and the largely uniform charge state of the resulting analyte ions, typically z = 1. However, particularly in complex samples, it has been shown that different classes of molecules respond differently to the MALDI method, especially depending on the matrix substance used. For example, certain biomolecules are sufficiently ionized to detectable levels, while others are quantitatively underrepresented in the ion currents obtained from a mixture. This varying susceptibility is evident, for instance, in the imaging mass spectrometry analysis of tissue sections and can limit the significance of the resulting measurement data.For example, it has been observed that lipids may be overrepresented in spectra of tissue sections compared to proteins and peptides.

[0005] Some time ago, a post-desorption ionization method was proposed to increase the conversion rate of, for example, low-concentration molecules. The principle is to direct an additional coherent ultraviolet light pulse laterally into the material cloud of the MALDI desorption. This approach is called MALDI-2. The interaction of the light pulses with the particles in the material cloud expands the charge carrier supply, which improves the ionization yield, particularly for low-concentration analyte molecules. However, even higher-concentration biomolecules can benefit from a post-ionization modality. For example, phosphatidylethanolamines (PEs) are hardly detected in MALDI measurements compared to phosphatidylcholines (PCs), even though both are comparably abundant in tissue. MALDI-2 strongly ionizes PEs, making them reliably detectable in the recorded spectra.

[0006] An important publication on the MALDI-2 method is the study by Jens Soltwisch et al. (Science, April 10, 2015 • Vol. 348, Issue 6231, 211-215), in which this name was coined. A wavelength-tunable post-ionization laser is used to induce secondary MALDI-like ionization processes in the gas phase. An increase in the ion yield of up to two orders of magnitude is reported for numerous lipid classes, fat-soluble vitamins, and saccharides imaged in animal and plant tissue with a 5-micrometer-wide laser spot. The pressure of the cooling gas in the ion source, the laser wavelength, the pulse energy, and the delay between the two laser pulses are identified as crucial factors influencing the initiation of the secondary ionization processes.

[0007] The following is a brief review of some prior art publications that may be relevant to the present disclosure:

[0008] The monograph by Klaus Dreisewerd (Chem. Rev. 2003, 103, 395-425) deals, among other things, with post-ionization experiments for the characterization of the MALDI method, especially in section V. Plume Dynamics.

[0009] Patent publication WO 2010 / 085720 A1 discloses a method and a device for the efficient measurement of an ionized MALDI desorption cloud when post-ionization (POSTI) techniques are combined with a fine-vacuum MALDI ion mobility orthogonal time-of-flight mass spectrometer (MALDI-IM-oTOF-MS). Related to this is the work by Amina S. Woods et al. (J Proteome Res. 2013 April 5; 12(4): 1668-1677).

[0010] The work of M. Niehaus et al. (Nature Methods Vol. 16, 925-931 (2019)) deals with MALDI-2 mass spectrometry in transmission mode for imaging cells and tissues with subcellular resolution.

[0011] MALDI measurements of very large sample material are becoming increasingly important; consider tissue sections in imaging mass spectrometry with areas on the order of a few square centimeters, or fields of very densely packed individual preparations in high-throughput analysis, e.g., 1536 individual preparations on a MALDI sample carrier. Measurements of such loaded sample carriers can take a very long time; in the case of large tissue sections, several hours or even days. To shorten the spectral data acquisition time, a dynamic MALDI desorption laser beam operation has been proposed for the measurement procedure. This combines many rapid reorientations of the desorption laser beam to scan a predetermined finite area of ​​the sample material with a few, rather time-consuming adjustments of the MALDI sample carrier to access different areas.In this way, the entire surface of a sample carrier can be scanned more quickly than by simply adjusting the massive and therefore rather sluggish sliding table that supports the sample carrier. An example is indicated in patent publication DE 10 2018 112 538 B3 (corresponding to US 2019 / 0362958 A1 and GB 2 574 709 A), in particular with reference to... Fig. .

[0012] Scanning the sample carrier area solely using the laser beam is subject to technical limitations. Firstly, the desorption laser beam must not strike the sample material at too oblique an angle. Secondly, the ablated and ionized sample material must be transferred to other components of a connected analytical system via typically fixed interfaces. This limits the beam deflection to a distance of a few hundred micrometers between the most widely separated points of impact on a predetermined, finite area. A standard MALDI sample carrier, however, has the dimensions of a microtiter plate (127.76 mm × 85.48 mm × 14.35 mm), meaning that the available area—even with incomplete sample coverage—cannot be fully scanned by the desorption laser beam alone without spatially repositioning the sample carrier.Rather, it is usually possible to define a large number of predetermined, finite areas on the sample carrier.

[0013] Patent publication US 2022 / 0223398 A1 relates to high-resolution imaging of samples using imaging mass spectrometry and imaging of biological samples by imaging mass cytometry, in which labeling atoms are detected by IMS, and applies LA-ICP-MS (a form of IMS in which the sample is ablated by a laser, the ablated material is then ionized in an inductively coupled plasma before the ions are detected by mass spectrometry) for the analysis of various substances, such as the mineral analysis of geological samples and the analysis of archaeological samples.

[0014] In light of the foregoing, there is a need to improve methods and devices for desorbing and ionizing sample material, particularly with regard to sensitivity to weakly ionizing molecular substrates. Further problems to be solved by the invention will readily become apparent to the person skilled in the art upon reading the following disclosure. Summary of the invention

[0015] According to a first aspect, the present disclosure relates to a method for desorbing and ionizing sample material deposited on a sample carrier, comprising: - repeatedly locally impacting sample material on the sample carrier using a first energetic radiation and causing local desorption of sample material into the gas phase above the sample carrier, thereby changing a relative position of the first energetic radiation to the sample carrier and targeting a plurality of impact points on the sample material on the sample carrier;- Pulsed impingement of the locally desorbed sample material using a second energetic radiation directed into the desorbed sample material, and inducing ionization and / or increasing the degree of ionization of the locally desorbed sample material, wherein a propagation direction of the second energetic radiation lies in a plane that is substantially perpendicular to a surface normal of the sample carrier and is arranged above the sample carrier, tracking a focus and / or beam waist position of the second energetic radiation such that it is substantially opposite an actual point of impact on the sample material on the sample carrier, wherein sufficient distance is maintained between the second energetic radiation and a sample carrier surface to avoid unintentional grazing of the sample carrier or the sample material placed on it and to prevent background formation;and - Transferring ionized sample material, which has resulted from the locally desorbed sample material and the second energetic radiation, into an ion processing device.

[0016] The height of the plane above the sample carrier can be in the range of 300–1000 micrometers, particularly 500 micrometers. Preferably, the position of the plane above the sample material and sample carrier is substantially constant, e.g., with only small deviations in the orientation of the first energy radiation of a few degrees. In certain embodiments, it may be possible to briefly and temporarily change the height of the plane above the sample material and sample carrier, e.g., to increase or decrease it. During desorption and ionization under reduced pressure, the environment in which the sample carrier with the sample material is stored can be maintained at a pressure in the range of 0.5–10 hectopascals, e.g., by suitably connected pumps. The time interval or delay between the triggering of the first energy radiation and the second energy radiation is preferably in the range of 0.5–1000 microseconds.The propagation directions of the first energy radiation and the second energy radiation can be substantially perpendicular to each other, in particular exhibiting an angle between them within a range of, for example, 45–135 degrees. The first energy radiation can be incident light, i.e., incident from a side of the sample carrier on which the sample material is placed, or transmitted light, i.e., incident from a side of the sample carrier facing away from the side on which the sample material is placed.

[0017] In various embodiments, the direction of incidence of the first energy beam can be changed relative to a surface normal of the sample carrier, and a multitude of point of impact can be targeted. This method accelerates the scanning of a sample carrier covered with sample material, since changes in beam orientation, e.g., using reflective optical elements such as galvanometric micromirrors, can be performed much faster and with less effort than moving the very heavy transfer table on which the sample carrier and / or sample material are placed and / or prepared. Movements of the transfer table are preferably performed when the range of motion of the first energy beam over a predetermined, finite area on the sample carrier has been exhausted.

[0018] In various embodiments, the sample material can be prepared with a light-absorbing matrix substance. Depending on the requirements, a MALDI method in reflected light (reflected light) or transmitted light (transmitted light) can be used for desorption. The MALDI method requires a specific sample preparation with a light-absorbing matrix substance, e.g., sinapic acid, 2,5-dihydroxybenzoic acid, α-cyano-4-hydroxycinnamic acid, or 2,5-dihydroxyacetophenone, all of which strongly absorb in the ultraviolet spectral range. Suitable for the first energy radiation is, for example, laser light from a nitrogen laser at a wavelength of approximately 337 nanometers or from a frequency-tripled solid-state Nd:YAG laser at approximately 355 nanometers. The second energy radiation can comprise laser light pulses with a wavelength of, for example, 266 nanometers.In general, all wavelengths below the two-photon limit for ionizing the matrix material used are suitable for the second energy radiation, typically wavelengths less than or equal to 290 nanometers for matrix materials with ionization energies around eight electron volts. The energy of the first energy radiation is preferably in the range of 0.1–50 microjoules, with the lower limit being particularly applicable for small laser foci on the sample material, such as those adjustable in transmission MALDI or transmitted-light MALDI. The energy of the second energy radiation can, for example, be in the range of 100–600 microjoules; 300–500 microjoules is particularly preferred.

[0019] In various embodiments, the first energetic radiation can be directed using a transmitted light optics system, which is arranged and designed such that the first energetic radiation, incident from the rear, impacts the sample material after passing through the sample carrier. The design as a transmitted light optics system allows the front-side desorption and ion formation region to be kept free of beam-guiding elements that could interfere with the ion extraction. The ion extraction from the ion formation region can be linear, parallel to a surface normal of the sample carrier, or it can involve changes in direction, e.g., deflections of 90°, which can be achieved by suitably arranged deflection electrodes.Furthermore, a transmitted light optics system allows for a tighter focusing of the initial energy beam for highly localized ablation of the sample material, thus achieving significantly higher spatial-lateral resolutions than with reflected light optics systems such as the reflective MALDI. With a laser beam, ablation areas, and therefore image elements or pixel areas, with diameters in the single-digit micrometer range and – with particularly careful fine-tuning – even in the sub-micrometer range can be realized, e.g., 0.5–5 micrometers in diameter.

[0020] In various configurations, the sample material can consist of a multitude of point preparations or a single, planar tissue section. In particular, a microtomized tissue section can be used as the sample material. Examples include brain tissue and retinal tissue, e.g., from rodents. The sample material can be sectioned, in particular, from a frozen tissue sample or formalin-fixed, paraffin-embedded (FFPE) tissue, which may require further preparation steps prior to analysis, e.g., deparaffinization and de-crosslinking, also known as antigen retrieval. The thickness of a tissue section to be analyzed can be 2–20 micrometers, and for transmitted-light MALDI applications, in particular 2–15 micrometers. For reflected-light or reflected-light MALDI, the sections can also be thicker, e.g., 2–40 micrometers.The analysis of tissue sections is gaining increasing importance, particularly in clinical applications for identifying pathological tissue conditions and differentiating them from non-pathological conditions, or for assessing cellular responses to pharmaceuticals. A large number of point preparations can, for example, comprise a dense field of 1536 or more individual preparations on the sample carrier, produced using a dried-droplet method. One application area is, for example, drug candidate identification for pharmacological investigations.

[0021] In various embodiments, the sample support can be a glass plate, a metal plate, or a ceramic plate. Preferably, the surface of the sample support carrying the sample material is electrically conductive to establish an electrical reference potential and to enable and / or simplify the handling of the desorbed and ionized sample material. This design is particularly advantageous for axial extraction of the ionized sample material from the ion formation region, i.e., extraction performed substantially parallel to a surface normal of the sample support. Suitable examples include polished steel plates or those with lyophilic anchoring surfaces in a lyophilic environment, such as Bruker's AnchorChips™. For the application of first-energy radiation in transmitted light, indium tin oxide-coated glass slides (ITO) can be used.

[0022] In various embodiments, the first energy radiation and / or the second energy radiation can be supplied by a pulsed laser. In particular, the sample material can be pulsed with the first energy radiation. The pulse rate can be in the range of a few hertz, e.g., 1–20 pulses per second, up to 10 3 or 10 4The frequency of the second energy radiation can be adjusted to match the frequency of the first energy radiation, and each individual desorption cloud can be irradiated with a suitable time delay of a few microseconds, which, starting from the exposure to the first energy radiation, promotes the formation of a desorption cloud. The time delay can be, for example, 0.5–100 microseconds, depending on the height of the propagation plane above the sample carrier and the pressure level, and preferably 5–20 microseconds, particularly under fine vacuum pressures of a few hectopascals and a propagation plane height above the sample carrier of approximately 500 micrometers.

[0023] In various embodiments, the relative position of the sample carrier to the first energy radiation and / or the propagation direction of the second energy radiation can be changed or adjusted using one or more mirrors and / or one or more lenses. For the first energy radiation, for example, an optical setup with a Keplerian telescope can be used, as described in patent publication DE 10 2011 112 649 A1 (corresponding to GB 2 495 805 A and US 2013 / 0056628 A1). For the second energy radiation, the use of galvanometric micromirror pairs is preferred, wherein each micromirror can be rotated about an axis of rotation to change the direction of reflection of a reflected beam.The use of at least one pair of rotatably flexible mirrors allows the beam orientation to be changed so that a nearly parallel offset compared to a preset standard beam orientation is possible, while simultaneously maintaining the same altitude of the propagation direction above the sample material and sample carrier. In this way, the orientation of the second energy radiation can be quickly and reliably adjusted to the varying points of impact on the sample material on the sample carrier, ensuring that a spreading desorption cloud is always optimally irradiated. By providing an additional pair of rotatably flexible galvanometric micromirrors, whose axes of rotation are oriented perpendicular to those of the other micromirrors, the altitude of the propagation direction of the second energy radiation can also be changed briefly and temporarily, e.g., raised or lowered.

[0024] In various embodiments, the ion processing device can be configured as an analyzer, in particular as a mobility analyzer, mass analyzer, or coupled mobility-mass analyzer. Ion-carrying intermediate stages, such as high-frequency voltage ion conductors like rod multipoles or RF funnel arrangements, can be arranged upstream of the actual analyzer or multiple analyzers connected in series, and also in various sections between such serially connected analyzers. Likewise, different analyzers and intermediate stages can be operated at different vacuum levels.

[0025] An ion mobility analyzer separates charged molecules or molecular ions according to their collision cross-section-to-charge ratio, sometimes denoted as σ / z or Ω / z. This is based on the interaction of the ion species with an electric field that couples to the charge of the ions, combined with the action of a buffer gas that acts on the mean cross-sectional area of ​​the ion. Drift tube mobility separators with a static electric field gradient are particularly well-known; these drive ions through a substantially stationary gas, where the drift velocity of an ion species results from the driving force of the electric field and the braking force of the collisions with the gas particles.Equally common are trapping ion mobility separators (TIMS) with a continuous laminar gas flow that propels the ions, counteracted by a stepwise changing electric field gradient with a correspondingly varying braking force. Travelling wave mobility separators can also be mentioned.

[0026] A mass analyzer, in turn, separates charged molecules or molecular ions according to their mass-to-charge ratio, usually denoted as m / z. Time-of-flight analyzers can be used, for which both linear and reflector setups, and / or those with orthogonal acceleration into the flight path, can be employed. Other types of mass-dispersing separators can also be used, e.g., quadrupole mass filters (single quads), triple quadrupole analyzers ("triple quads"), ion cyclotron resonance (ICR) cells, Kingdon-type analyzers such as the Orbitrap® (Thermo Fisher Scientific), and others. It is understood that analyzers and separators of the aforementioned types can be coupled to separate ion species multidimensionally, i.e., according to more than one physicochemical property such as m / z and σ / z or Ω / z.

[0027] In various embodiments, the focus and / or beam waist position of the second energy radiation can be adjusted (i) perpendicular to and / or (ii) along the propagation direction of the second energy radiation. Preferably, the beam waist or focus position along the propagation direction of the second energy radiation is achieved using a lens system in the beam path, which includes at least one movable optical lens with which the focal length setting of the overall optical system for the second energy radiation can be adjusted. For tracking perpendicular to the propagation direction of the second energy radiation, pairs of galvanometric micromirrors are preferably used, each of which can be rotated about its own axis of rotation and thus change the direction of reflection of the second energy radiation.

[0028] Changing the height of the beam waist or focus above the sample material and support can be advantageous when a very large deflection of the first energy radiation from a standard point of impact is set. Due to the divergence of the second energy radiation, there is a risk that, with very large deflections of the point of impact, areas on the sample material and support may collide with peripheral regions of the second energy radiation, thereby generating interfering background in the spectral data. By setting a temporarily and briefly higher height above the sample material and support for such extreme deflections, a compromise can be found between reducing this risk of ionic or chemical background in the spectral data and maintaining a favorable beam path for the second energy radiation.It may also be advisable to temporarily adjust the height above the sample material and sample carrier, for example, to direct the interaction of the second-energy radiation and the desorbed sample material to a region of the desorption cloud where the particle density is high enough to significantly promote charge carrier formation and transfer to uncharged molecules in the cloud, thereby increasing the yield of ionized desorbed sample material. Such an embodiment is also attributable to the invention.

[0029] According to a further aspect, the present disclosure relates to a device for desorbing and ionizing sample material placed on a sample carrier, comprising: - a desorption device for generating and guiding the first energy radiation; - an ionization device for generating and guiding the second energy radiation; - a first adjustment device for setting and changing the relative position of the first energy radiation to the sample carrier; - a second adjustment device for setting and tracking the focus and / or beam waist position of the second energy radiation; and - a control system that communicates with the desorption device, ionization device, first adjustment device and second adjustment device and is designed and programmed to coordinate and execute a procedure as described above. Brief description of the illustrations

[0030] For a better understanding of the invention, reference is made to the following illustrations. The elements in the illustrations are not necessarily shown to scale, but are primarily intended to illustrate the principles of the invention (mostly schematically). In the illustrations, corresponding elements in the different views are identified by the same reference numerals. Fig. schematically shows the setup of an ion spectrometer in whose source area laser-assisted post-ionization is used (adapted from DE 10 2016 124 889 A1, corresponding to GB 2 558 741 A and US 2018 / 0174815 A1). Fig. This schematically illustrates an operating mode of an ion source in which an initial energetic radiation is moved across a field of sample material placed on a sample carrier to different impact and ablation points. Fig. schematically shows the ion source from Fig. with activated post-ionization modality and the separation of the impact / deposition point and focus and / or beam waist of the second energetic radiation. Fig. schematically illustrates the adjustment of the propagation direction of the second energetic radiation when the points of impact and absorption of the first energetic radiation are offset in a direction perpendicular to the propagation direction of the second energetic radiation. Detailed description

[0031] While the invention has been presented and explained with reference to a number of embodiments, those skilled in the field will recognize that various changes in form and detail can be made to it without deviating from the scope of the technical teaching defined in the attached claims.

[0032] Fig. Figure 10 shows an ion spectrometer that uses a post-ionization modality and in which principles of the present disclosure can be implemented, and serves for contextualization.

[0033] The representation in Fig. Figure 1 shows a simplified diagram. The typical normal operation with intermediate storage and possible collision fragmentation of ions in the ion storage unit (19) is as follows: In an ion source with a laser system (11), the first laser light pulse beam (12), which enters the source through a window (not shown), ionizes the sample material (16) on the sample carrier (15). This ionized sample material is then forced into a typical radio frequency (RF) ion funnel (17) by a potential at the electrode (14). The point of impact of the first laser light pulse beam (12) on the sample material can be varied within certain limits by changing the direction of incidence of the first laser light pulse beam (12), as explained previously.Ion generation can be supported by a second laser pulse beam (12*) which is focused laterally into the desorption cloud of the removed sample material spreading above the sample carrier (15) in a timely manner, before a beam collector (30) captures it away from the sample carrier (15). The focus and / or beam waist position of the second laser pulse beam (12*) is adjusted so that it is substantially opposite the current point of impact on the sample material in order to ensure optimal interaction between the energy in the second laser pulse beam and the particles in the desorbed material cloud.

[0034] The ions then enter the RF quadrupole rod system (18), which can be operated both as a simple ion guide system or ion conductor and as a mass filter for selecting a type of precursor ion to be fragmented. The unselected or selected ions are then fed into the high-frequency quadrupole ion storage unit (19) and can be fragmented by energetic collisions, depending on their acceleration. The ion storage unit (19) is gas-tight and is supplied with a collision gas such as nitrogen or argon via the gas supply (20) to focus the ions by collisions and concentrate them along the axis.

[0035] At predetermined times, ions are extracted from the ion reservoir (19) through an extraction lens (21), shaped into a fine primary ion beam (22), and directed to the ion pulser (23). The ion pulser (23) pulses a segment of the primary ion beam (22) orthogonally into the drift section, which is at a high electrical potential, thereby generating the new ion beam (24). The ion beam (24) is reflected velocity-focused in the reflector (25) and measured in the detector (26). The mass spectrometer is evacuated by connected pumps (27), (28), and (29).

[0036] In Fig. Figure 1 shows how ionized sample material, which moves away from the sample carrier (15) on which it is initially stored, generally along a surface normal of the sample carrier (15), is deflected by means of a deflecting electrode (14) and—assisted by the gas flow from the ion formation area in the fine vacuum into a subsequent compartment maintained at lower pressure—is directed into an attached ion guide, here implemented as an HF funnel (17). Those skilled in the field will understand that this design is not to be considered limiting. Likewise, an extraction direction of the ionized desorbed sample material is conceivable that is substantially parallel to a surface normal of the sample carrier (15). With regard to Fig. This could mean that the sample carrier (15) is positioned substantially opposite the wide end of the RF funnel (17) (e.g., by rotating the sample carrier clockwise by 90°). The deflecting electrode (14) could then be removed or retained by repurposing the central opening as the extraction electrode. The beam paths of the first and second laser pulse beams (12, 12*), possibly by removing and / or adding suitable deflecting mirrors, as well as the position of the beam catcher (30) for the second laser pulse beam (12*), would have to be adjusted accordingly in such a modified design.

[0037] Fig. This figure schematically and exemplarily illustrates a removal and desorption process in an ion source. A sample carrier (115), which in this example carries a multitude of individual samples of sample material, is arranged on a sliding table (132). The sliding table (132) can be designed to adjust the position of the sample carrier (115) along up to three spatial axes xyz, two of which span an xy-plane perpendicular to the plane of representation, and the third z-axis can run from bottom to top in the plane of representation. However, the number of actual actuations of the sliding table (132) is quite low, since it is inherently very massive, so that a movement takes a considerable amount of time from execution until any vibrations generated during the movement have subsided.It is more advantageous to move the transfer table (132) only when sample material located outside a predetermined, finite area on the sample carrier (115) that can be covered with beam guidance means is to be exposed to a desorption beam (112). This operating mode can be described as a so-called hybrid or combined "stage scan" and "laser scan" approach.

[0038] In this example, a first laser system (111) is arranged obliquely above the transfer table (132) and is designed to direct a laser beam (112) in various orientations (solid, dotted, and dashed contours) to predetermined locations within a predetermined, finite area on the sample carrier (115) without moving the transfer table (132). This can be, for example, a configuration as a reflected-light MALDI or reflected-light MALDI. The latter designation arises from the fact that the desorbed and ionized sample material leaves the sample carrier (115) in the broadest sense opposite to the direction of incidence of the first laser beam (112). The finite area can, for example, have a diameter or an edge length of 100–1000 micrometers.A guide element arranged above the sample carrier (115), such as the indicated RF ion funnel (117), is able to collect the desorbed and electrically charged sample material and feed it to a connected analyzer (not shown), optionally via intermediate stages and / or using axial extraction or extraction with changes in direction. The RF ion funnel (117) can be continuously or pulsedly connected to extracting electrical potentials, synchronized with the desorption pulses of the initial energy radiation (112).

[0039] Furthermore, a post-ionization modality in the form of a second laser system (111*) is shown, which is arranged and designed such that a second laser beam (112*) can be focused laterally into a desorption cloud of sample material, e.g. according to the so-called MALDI-2 method, see Fig. The contour and outer dimensions of the second laser beam (112*) are not necessarily shown to scale here, particularly with regard to its divergence. In practice, the user is well advised to ensure that the second, laterally incident laser beam (112*) maintains a sufficient distance from the sample carrier surface to avoid unintentional grazing of the sample carrier (115) or the sample material placed on it and to prevent the formation of background in the spectral data.

[0040] The alignment and focusing of the second laser beam (112*) is usually fixed, meaning it cannot be changed or adjusted without complex human intervention. This implies that there is only one optimal position for a desorption cloud to be hit as effectively as possible by the second beam (112*). The goal is always ionization, or at least increasing the degree of ionization, should the desorption process itself involve ionization, as in MALDI preparation. Therefore, the ablation or desorption site would always have to be moved into the fixed focus or beam waist position of the second laser. However, this can only be achieved by moving the transfer stage (132), which, as mentioned above, is cumbersome and time-consuming. If the alignment of the first laser beam (112) to the sample carrier (115), or rather to a surface normal (134) of the sample carrier (115), see Fig. If the first beam (112) is deflected, the point of impact and desorption, as well as the focus or beam waist position of the second beam (112*), may no longer align optimally, and this can lead to a loss of effectiveness, for example, due to only marginal overlap between the second beam (112*) and the desorption cloud, or because beam divergence prevents a critical beam fluence for interaction with the desorbed sample material from being achieved. In an extreme case, with a very strong deflection of the first beam (112) from a standard point of impact, the second beam (112*) could even completely miss the desorption cloud, which, based on experience, does not propagate exactly along a surface normal (134) of the sample support (115) in reflected-light or reflected MALDI with oblique beam incidence, but is slightly distorted against the direction of incidence of the first beam (112). The beneficial, post-ionizing effect of the second beam (112*) would then of course be absent.

[0041] Fig. The figures schematically and exemplarily illustrate the problem when the first beam (112) is deflected onto impact points that are aligned along the propagation direction of the second beam (112*). With the sliding table (132) stationary, the first beam (112) is directed at different impact points at three different angles of incidence relative to the sample carrier (115), see figure. Fig. The focal position of the second beam (112*) is substantially opposite only the central section of the sample material and is therefore optimal; with deflection (Δ) to the right and to the left in the Fig. The second beam (112*) does not intersect the desorption cloud with its beam waist or its area of ​​least circumference, which in most cases coincides with the focus position. This can lead to parts of the desorbed sample material not interacting with the second beam (112*) or to the fluence at the interaction site remaining below a critical threshold, so that the beneficial charge carrier increase effect may only occur incompletely. Adjusting the focus or beam waist position along the propagation direction of the second beam (112*) is necessary to remedy this problem, for example, by using movable optical lenses (140) arranged in the beam path. The lens (140) in Fig. This is to be understood as a schematic placeholder. A more complex lens system with multiple optical elements can be arranged in its place to change the beam waist and / or focus position along the direction of propagation.

[0042] The situation is more serious if a change in the orientation of the first energetic beam (112) deflects the point of impact on the sample carrier (115) along a direction substantially perpendicular to the propagation direction of the second beam (112*). In such a situation, the problem of the complete spatial separation of the second beam (112*) and the desorption cloud arises much more quickly than a loss of focus due to deflection along the propagation direction of the second beam (112*). This case is in Fig. Illustrated schematically and with examples.

[0043] Fig. The schematic top view shows the second laser system (211*), which produces the second energy beam (212*), on the left. At the opposite end of the figure, on the right, is a beam catcher (230) designed to absorb excess photonic energy and remove it from the arrangement, thus preventing any interfering stray light that could, for example, affect other components or assemblies. Also shown is the sample carrier (215) with sketched-out impact points indicating ablation and desorption sites of sample material, as well as an optical guidance system comprising mirror pairs (238, 238') and imaging lenses (240). The sample material on the sample carrier (215) is depicted here as a tissue section. The beam path of the second beam (212*) in different settings is illustrated with solid, dotted, and dashed-dotted lines.The mirrors (238, 238') are designed such that they can each be rotated about their own axis of rotation, the axis of rotation of mirrors (238) being different from that of mirrors (238'), and these axes of rotation are preferably perpendicular to each other. For example, the axes of rotation of mirrors (238) can be perpendicular to the plane of the drawing, whereas the axes of rotation of mirrors (238') lie in the plane of the drawing. The rotationally flexible design of the mirrors (238) ensures that the direction of propagation of the second energetic radiation (212*) can be adjusted in a fixed plane parallel to the surface of the sample carrier (215) without changing the height above the sample carrier (215). The rotationally flexible design of mirrors (238') also allows the height of the beam plane above the sample carrier (215) to be adjusted.The surface area of ​​the mirrors (238, 238') is dimensioned such that different angular deflections of the second beam (212*) can be translated into a spatial offset (Δ) of the beam axis along two spatial directions perpendicular to the general propagation direction of the second energetic radiation (212*). Furthermore, the lens system (240) can comprise a plurality of imaging lenses (240), at least one of which can be moved in the propagation direction of the second beam (212*). In this way, the focus or beam waist position of the second beam (212*) above the sample carrier (215) can be adjusted in the propagation direction of the second beam (212*) along the three spatial directions, as previously described with respect to the... Fig. explained.

[0044] A guidance system (242) communicates with the adjustment mechanism (not shown) of the sample carrier (215), the second laser system (211*), the adjustment mechanism of the first beam (not shown), the mirror pairs (238, 238') and the lens system (240) and coordinates their operation so that the point of impact and ablation on the sample carrier (215) and the beam waist or focus position of the second beam (212*) are always substantially opposite each other. The communication is indicated by the dash-dotted lines (244).

[0045] The principles of the invention make it possible, in particular, to enlarge the areas on the sample material that can be treated for material removal simply by adjusting the first energy radiation beam, without having to move the heavy and inert sliding table on which the sample carrier is mounted. In this way, larger deflections in the beam path of the first energy radiation beam can be set. This can help to accelerate the spatially resolved processing of a sample carrier covered with sample material compared to methods known from the prior art, since the total number and frequency of the sample carrier movements required for scanning can be reduced even further.

[0046] The invention is described above with reference to various specific embodiments. It is understood, however, that various aspects or details of the described embodiments can be modified without deviating from the scope of the invention. Furthermore, the features and measures disclosed in connection with different embodiments can be combined as desired, provided this appears practical to a person skilled in the art. Moreover, the above description serves only to illustrate the invention and not to limit the scope of protection, which is defined exclusively by the accompanying claims, taking into account any existing equivalents.

Claims

[1] Method for desorbing and ionizing sample material placed on a sample carrier (15, 115, 215), comprising: - repeated local impact of sample material on the sample carrier (15, 115, 215) using a first energetic radiation (12, 112) and inducing local desorption of sample material into the gas phase above the sample carrier (15, 115, 215), thereby changing a relative position of the first energetic radiation (12, 112) to the sample carrier (15, 115, 215) and targeting a multitude of impact points on the sample material on the sample carrier (15, 115, 215); - Pulsed impingement of the locally desorbed sample material using a second energy radiation (12*, 112*, 212*) directed into the desorbed sample material, causing ionization and / or increasing the degree of ionization of the locally desorbed sample material, wherein a propagation direction of the second energy radiation (12*, 112*, 212*) lies in a plane that is substantially perpendicular to a surface normal of the sample carrier (15, 115, 215) and is arranged above the sample carrier (15, 115, 215), tracking the focus and / or beam waist position of the second energy radiation (12*, 112*, 212*) such that it is substantially opposite an actual point of impact on the sample material on the sample carrier (15, 115, 215), where sufficient distance is maintained between the second energetic radiation (12*, 112*, 212*) and a sample carrier surface to prevent unintentional grazing of the sample carrier (15, 115,215) or of the sample material placed thereon, and to prevent the formation of substrate; and, - Transferring ionized sample material, which has been obtained from locally desorbed sample material exposed to the second energetic radiation (12*, 112*, 212*), into an ion processing facility. [2] Method according to claim 1, wherein an incident direction of the first energetic radiation (12, 112) is changed relative to a surface normal of the sample carrier (15, 115, 215) and a plurality of impact points are targeted. [3] Method according to claim 1 or claim 2, wherein the sample material has been prepared with a light-absorbing matrix substance. [4] Method according to any one of claims 1 to 3, wherein the sample material comprises a plurality of point preparations or a planar tissue section. [5] Method according to any one of claims 1 to 4, wherein the sample support (15, 115, 215) is designed as a glass plate, metal plate or ceramic plate. [6] Method according to any one of claims 1 to 5, wherein the first energetic radiation (12, 112) and / or the second energetic radiation (12*, 112*, 212*) is supplied by a pulse laser. [7] Method according to any one of claims 1 to 6, wherein the relative position of the sample carrier (15, 115, 215) to the first energetic radiation (12, 112) and / or the propagation direction of the second energetic radiation (12*, 112*, 212*) is changed or tracked using one or more mirrors (238, 238') and / or one or more lenses (140, 240). [8] Method according to any one of claims 1 to 7, wherein the ion processing device is configured as an analyzer, in particular as a mobility analyzer, mass analyzer or coupled mobility-mass analyzer. [9] Method according to any one of claims 1 to 8, wherein the focus and / or beam waist position of the second energetic radiation (12*, 112*, 212*) is tracked (i) perpendicular to and / or (ii) along the propagation direction of the second energetic radiation (12*, 112*, 212*). [10] Device for desorbing and ionizing sample material placed on a sample carrier (15, 115, 215) comprising: - a desorption device (111) for generating and guiding a first energetic radiation (12, 112); - an ionization device (111*, 211*) for generating and guiding a second energetic radiation (12*, 112*, 212*); - a first adjustment device for setting and changing the relative position of the first energetic radiation (12, 112) to the sample carrier (15, 115, 215); - a second adjustment device for setting and tracking the focus and / or beam waist position of the second energetic radiation (12*, 112*, 212*); and - a control system (242) that communicates with the desorption device (111), ionization device (111*, 211*), first adjustment device and second adjustment device and is designed and programmed to coordinate and execute a method according to any one of claims 1 to 9.

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