Multimodal analysis device for sample material

Abstract

The invention relates to a device for multimodal analysis of sample material, e.g. from a tissue, which acquires molecule image data from the sample material in a spatially resolved fashion, e.g. usin

Description

Device for multimodal analysis of sample material Field of the invention

[0001] The invention relates to a device for the multimodal analysis of sample material, e.g. from a tissue, which acquires spatially resolved molecular image information from the sample material, e.g. using a time-of-flight mass analyzer, records spatially resolved light microscopic image information from the sample material and combines both with improved accuracy to form spatially resolved co-registered overall image information. Background of the invention

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

[0003] In MALDI mass spectrometry imaging (MSI; MALDI = matrix-assisted laser desorption ionization), co-registration of ion images with high-resolution optical microscope images is an important source of information. Previously used methods can be time-consuming and error-prone.

[0004] In addition to the precise correlation of the xy plane coordinates of each pixel for co-registration, MALDI-MSI requires information on the sample topography for every pixel, if possible. This can then be used to optimize material removal and thus data quality. This is especially true for high- and ultra-resolution MALDI-MSI. Here, the so-called transmission geometry, tMALDI, can be used. The shallow depth of field of the microscope objectives used, e.g., 1.6 micrometers when using a 50X Mitutoyo Plan Apo NUV with an infinity-corrected objective, requires precise topography determination with submicrometer accuracy.

[0005] At the current state of the art, these two tasks of spatially precise superposition and topography determination are treated separately and are independently subject to inaccuracies that limit any informative evaluation.

[0006] Various commercially available slide scanners are used to capture optical images, e.g., the Olympus VS200, Hamamatsu, etc. The term "slide" refers to the slide itself. These slide scanners are separate assemblies and primarily work with individual images rasterized across the sample surface, which are then stitched together to form a complete image. This stitching is based on previously processed image data, taking into account the search for matching image features, rather than on raw data.

[0007] Co-registration of externally acquired optical images with ion images using teaching marks added during sample preparation is also known. Various mass spectrometer manufacturers offer software programs for this type of multimodal optical / mass spectrometric imaging. Examples include Bruker's fleximaging and Waters / Micromass' High Definition Imaging.

[0008] The co-registration of optical images in MALDI-MSI serves two purposes. First, the measurement range is defined before the measurement using co-registered optical scanning. Second, in multimodal approaches, microscopic images acquired before or after the MALDI measurement are used to combine optical and mass spectrometric information. Both tasks can only be solved with relatively imprecise and time-consuming methods using the known methods. For the initial co-registration of the optical image (scan) and ablation position in the spectrometer, the user or an automated routine must first correlate optical features of an externally acquired image with the real-time image from the mass spectrometer's internal camera. This leads to an initial error in the positioning of the markers in both images.Particularly with pixel sizes of less than 10 micrometers, the optical imaging quality within the mass spectrometer often precludes precise assignment. After marking, the optical image is stretched, rotated, and subjected to skew correction without a fixed aspect ratio. These image corrections also always introduce artifacts that often lead to inaccurate co-registrations. Such systematic uncertainties in this initial co-registration have the direct consequence that the selected measurement area must always be selected with a certain safety margin. This prevents the measurement of directly adjacent areas, so that parts of the sample surface are ignored, and increases measurement times.

[0009] In the multimodal interpretation of the acquired data, the most accurate co-registration possible is essential. Only precisely correlated images allow a direct comparison of morphological and molecular information. This is especially true for high- and ultra-resolution In MALDI-MSI (pixel size < 10 micrometers), the systematic error is often several times the pixel size. This requires subsequent correction of the co-registration between the microscopic image on the one hand and the ion distribution images on the other. Since both modalities generate their contrast in very different ways, systematic errors typically remain on the order of the pixel size.

[0010] Furthermore, accurate co-registration with available methods requires time- and computationally intensive procedures that rely on multiple microscopic images before and after MALDI-MSI, as explained in two articles by Nathan Heath Patterson et al. They describe workflows for the registration and analysis of MALDI MSI-to-microscopy data using non-destructive MSI-compatible wide-field autofluorescence (AF) microscopy in combination with computer-assisted image registration (Anal. Chem. 2018, 90, 12395-12403) and an advanced histology-guided platform that uses MSI-compatible AF microscopy prior to histological staining or MSI measurement (Anal. Chem. 2018, 90, 12404-12413).

[0011] The work of Michael J. Taylor et al. (Metabolites 2021, 11, 200) focuses on laser ablation electrospray ionization mass spectrometry (LAESI-MS) for single-cell metabolomics. They report on the integration of a microscope into the optical path of a LAESI source to perform visually supervised in situ single-cell analysis to advance the understanding of intercellular differences within large cell populations under ambient conditions. The microscope captures optical images across the sample area in individual "tiles," which are then "stitched" together into a complete image using image processing software.

[0012] In the method presented by Michael J. Taylor et al., illumination and observation coaxial with the ablation laser beam path are described in a LAESI experiment. An autofocus routine based on image sharpness is also used. This observation of vox in reflected light is not suitable for MALDI samples due to the largely opaque matrix coating. Furthermore, observation along the sample normal would be impossible or very difficult to implement, as this space is required for ion extraction in MALDI. Observation at an angle greater than 0° to the sample normal leads to image distortions that would have to be corrected. Due to the required shifting of the individual images relative to one another, sufficiently precise co-registration is not possible with this method.

[0013] In addition to co-registration, pixel-sharp topography determination can be an important prerequisite for ultra-high-resolution MALDI-MSI, e.g., when examining highly profiled tissue such as the retina. Current methods for tissue samples or other mass spectrometric samples perform this topography determination independently. The accuracy of the topographic information available in the spectrometer is therefore partly based on error-prone co-registration. In addition, commonly used methods are not precise enough for the shallow depths of field in the sub-micrometer range used in tMALDI and are limited to relatively large areas and therefore not pixel-sharp. For example, in the timsTOF fleX from Bruker, a striped pattern is projected onto the sample surface using an auxiliary laser. Using this pattern, the distance between the focal plane of the laser and the sample surface can be determined to approximately5 micrometers. This accuracy is not high enough for the shallow tMALDI depths of field, such as the 1.6 micrometers calculated above for the 50X objective lens. Furthermore, this method averages over an area of ​​approximately 1 millimeter x 1 millimeter. Structures smaller than this are thus averaged out and excluded from the analysis.

[0014] In the study by Mario Kompauer et al. (Nat. Methods 2017, 14, 1156-1158), it is proposed that height profile determination using a point light source requires a time-consuming autofocus routine for each pixel to resolve small structures. The pixel size is currently limited to approximately 20 micrometers. Related to this is patent publication EP 3 306 639 A1 (corresponding to US 10,964,519 B2), which aims to provide a device that enables the simultaneous analysis of the two-dimensional, spatial chemical composition and topography of a sample.

[0015] In the following, some prior art documents that may be relevant to the subject matter of the present disclosure are also briefly reviewed:

[0016] The work of F. Hillenkamp et al. (Appl. Phys. 8, 341-348 (1975)) presents a high-sensitivity / current microprobe mass analyzer (LAMMA) designed specifically for the analysis of biological sample material. Building on this pioneering work, the article by H. Vogt et al. (Fresenius Z. Anal. Chem. 308, 195-200 (1981)) describes the principles and technical features of the Laser Microprobe Mass Analyzer (LAMMA) 500, which was equipped with a light microscope for visual observation of the sample in reflection through a coated electron microscopic grid as a slide.

[0017] The work by Bernhard Spengler et al. (J Am Soc Mass Spectrom 2002, 13, 735-748) concerns a scanning microprobe matrix-assisted laser desorption ionization (SMALDI) mass spectrometer for resolved LDI and MALDI surface analysis in the submicrometer range. For overview observation, the sample can be imaged with a standard light microscope at a magnification of approximately 400x. An area of ​​approximately 500 micrometers x 400 micrometers is displayed on a video monitor. The sample is not illuminated and observed from the side, but rather through the objective lens due to geometric constraints. Because the objective lens was not corrected for chromatic aberration, only monochromatic light, namely a He-Ne laser with an output power of 15 mW, could be used to image the sample.

[0018] Patent publication US 2003 / 0222212 A1 discloses a method and system for generating a correlated optical image of an ion desorption region of a sample substrate sampled using light with foci down to the micrometer or submicrometer range for matrix-assisted laser desorption / ionization (Figure 3).

[0019] Patent publication US 2006 / 0289734 A1 presents a method for generating a sharp image of a region on a sample plate for a matrix-based ion source, comprising: positioning the region in a field of view of an imaging device; generating a first image having an in-focus region and an out-of-focus region using the imaging device; generating a second image having an in-focus region and an out-of-focus region using the imaging device; and generating a final sharp image using the in-focus regions of the first and second images.

[0020] US Patent No. 7,180,058 B1 protected an ion source for a mass spectrometer, comprising: a radiation source for generating a beam of radiation; beam focusing optics configured to focus the beam of radiation onto a sample disposed on a front surface of a sample carrier. The beam focusing optics have a focal length of less than 25 millimeters and are positioned adjacent to a rear surface of the sample carrier. The sample carrier is transparent at the wavelength of the beam of radiation to transmit the beam of radiation. Also included is an ion optical device positioned adjacent to the front surface of the sample carrier and configured to transport ions generated by irradiation of the sample. Also included is a viewing optic for acquiring an image of the sample, which is arranged adjacent to the rear surface of the sample carrier.

[0021] The patent publication DE 10 2007 006 933 A1 (corresponding to US 2008 / 0191131 A1 and GB 2 446 699 A) describes a method by which the distance between the sample surface and the first accelerating electrode on the flight path can be adjusted in a MALDI axial time-of-flight analyzer using knowledge of the position of the sample surface, which is determined by evaluating the images of a digital camera, relative to the digital camera.

[0022] Patent publication US 2009 / 0146053 A1 describes a mass spectrometer for performing mass analysis while simultaneously microscopically observing a two-dimensional region of a sample. The observation position for selecting a target region while simultaneously observing an image of the sample captured with a CCD camera is separated from the analysis position for performing the mass analysis, in which laser light is emitted onto the sample. The sample is placed on a stage, which is said to be precisely movable between the observation position and the analysis position by a stage drive mechanism.

[0023] Patent publication US 2011 / 0315874 A1 discloses a mass spectrometer that is said to be capable of efficiently performing imaging mass spectrometry over a spatial area that extends beyond the field of view of a microscopic observation unit.

[0024] Patent publication US 2011 / 0266438 A1 discloses a mass spectrometer capable of obtaining a microscopic observation image with high spatial resolution in real time during mass analysis without interfering with the analysis. An opening is formed in a stage, onto which a transparent sample plate is placed. A microscopic observation unit, comprising an optical observation system and a CCD camera, is provided below the stage to observe the back of the sample through the stage opening and the transparent sample plate. The observed image is displayed on the screen of a display unit.

[0025] In the study by Andre Zavalin et al. (J Mass Spectrom. 2012 November; 47 (11), 1473-1481), the authors conclude that with a transmission geometry configuration, a laser beam in the ion source of a MALDI mass spectrometer can be focused to dimensions of less than 1 micrometer, thus enabling the direct imaging of heterogeneous tissue sections and individual cells with subcellular resolution. The subcellular MS images were validated by co-registration of individual images acquired using microscopic methods, such as optical phase contrast, DIC (differential interference contrast), and brightfield images. Individual optical microscope images were acquired both before and after also recorded after the MS imaging experiment. Figure 1 of the present disclosure shows an adaptation of Figure 2 of this document. Reference numerals denote: 1 - chamber; 2 - UV laser; 3 - aperture; 4 - beam attenuator; 5 - white light source; 6 - CCD camera; 7 - xy translation stage; 8 - rear observation window; 9 - microscope objective; 10 - time-of-flight analyzer flight tube.

[0026] The study by Marcel Niehaus et al. (Nat. Methods 2019, 16, 925-931) uses MALDI-2 mass spectrometry in transmission mode to image cells and tissues with subcellular resolution. Microscopic observation of the sample in transmitted light is possible via the same beam path of the MALDI laser, using a single LED as the light source, a beam splitter, and a CCD (charge-coupled device) camera.

[0027] In light of the foregoing, there is a need to improve the co-registration of spatially resolved molecular image information and spatially resolved microscopic image information, particularly with regard to its accuracy and the complexity of designing the optical assemblies. Further problems to be solved by the invention will become readily apparent to those skilled in the art upon reading the following disclosure. Summary of the invention

[0028] According to a first aspect, the present disclosure relates to a device for the multimodal analysis of sample material, comprising: - a desorption optics system arranged and configured to expose the sample material arranged on one side of a sample carrier to a first radiation and to cause desorption of the sample material into the gas phase, wherein the desorbed sample material is ionized; - an analyzer arranged away from the sample carrier and configured to receive the desorbed and ionized sample material and process it into spatially resolved molecular image information; - a transmission-incident light optics system arranged and configured to record spatially resolved light microscopic image information of the sample carrier and sample material using a second radiation reflected through the transparent sample carrier;wherein the second radiation is emitted by a light source which is arranged on a side of the sample carrier facing away from the sample material and is designed such that the second radiation, when incident on the sample carrier, does not pass through an optical component which is passed through by the first radiation when incident on the sample carrier, and - a computing unit which is arranged and designed such that it communicates with the desorption optical system, the analyzer and the transmission incident light optical system and which calculates the spatially resolved, To combine molecular image information and spatially resolved light microscopic image information to produce spatially resolved co-registered overall image information.

[0029] Microtomized tissue sections can be used as sample material. Examples include brain and retinal tissue. The sample material can be cut from frozen tissue or formalin-fixed, paraffin-embedded (FFPE) tissue, which may require further processing 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, particularly 2–15 micrometers for tMALDI applications. For incident or reflected light MALDI, the sections can also be thicker, e.g., 2–40 micrometers.The multimodal analysis of tissue sections is becoming increasingly important, particularly in clinical applications for the detection of pathological conditions of a tissue and their differentiation from non-pathological conditions, or the cellular response to the administration of pharmaceutical substances.

[0030] The desorption optics system can comprise a laser desorption ion source (LDI), which can be designed, in particular, as a MALDI source. Depending on the requirements, a MALDI process in reflected light (in reflection) or transmitted light (in transmission) can be used for ionization. The MALDI process requires 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 absorb strongly in the ultraviolet spectral range, e.g., laser light from a nitrogen laser at a wavelength of approximately 337 nanometers or a frequency-tripled solid-state Nd:YAG laser at approximately 355 nanometers.

[0031] The sample material can be exposed to the first radiation in a pulsed manner. The pulse rate of a pulse sequence can be in the range of a few Hertz, e.g. 1-20 pulses per second, up to 10 3 or 10 4 Hertz.

[0032] The analyzer can be a mobility analyzer, a mass analyzer, or a combined mobility-mass analyzer. Generally speaking, ion spectrometric analyzers and measurement methods can be used, which can include mobility separation, mass separation, or a combination of both.

[0033] An ion mobility analyzer separates charged molecules or molecular ions according to their collision cross-section-to-charge ratio, sometimes referred to as Q / z or o / z. This is based on the interaction of the ion species with an electric field that couples to the charge of the ions, with the simultaneous exposure of a buffer gas that acts on the average cross-sectional area of ​​the ion. Drift tube mobility separators with Static electric field gradients drive ions through an essentially static gas, with the drift velocity of an ion species resulting from the driving force of the electric field and the decelerating force of the collisions with the gas particles. Also common are trapping ion mobility separators (TIMS), which use a steady laminar gas flow that propels the ions, counteracted by a gradually changing electric field gradient with a correspondingly variable decelerating force. Traveling wave mobility separators may also be mentioned.

[0034] A mass analyzer, in turn, separates charged molecules or molecular ions according to their mass-to-charge ratio, usually referred to as m / z. Time-of-flight analyzers can be used, which can be designed with either linear or reflector configurations and / or with axial or orthogonal acceleration into the flight path. Other types of mass-dispersing separators can also be employed, 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. 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 Δ / z or Δ / z.

[0035] Linking spatially resolved molecular image information and spatially resolved light microscopic image information can mean superimposing the two pieces of image information in a spatially accurate manner and displaying them pictorially or graphically, e.g., on a computer screen. In this way, a user can be enabled to visually recognize, in a spatially resolved manner, matching and / or differing feature characteristics or structures in the image information of the various modalities. Linking within the meaning of the present disclosure can, however, also include further processing of the image information, for example, creating an overview map of the sample material on which a key figure is plotted in a gray or color scale, which results from the calculation of the spatially resolved intensities of one or more ion species of interest m / z and the spatially resolved intensities of one or more wavelengths λ from the electromagnetic spectrum.An ion species of interest can be, for example, a biomolecule or biopolymer, such as a protein, peptide, lipid, polynucleotide, or polysaccharide. The spatially resolved, co-registered overall image information preferably covers the entire sample material, e.g., the entire area of ​​an analyzed tissue section. The image information grids of the various modalities are usually dimensioned differently, with the optical modality allowing a finer grid, i.e., image elements with smaller dimensions, than the mass analysis modality. Depending on the design of the light micro- For scopic observation optics, the optical resolution is typically in the submicrometer range, e.g., 500-700 nanometers, whereas the pixel size (or lateral resolution) for mass analysis ablation is typically in the low micrometer range, e.g., 1-10 micrometers. In particular embodiments, linking may involve adapting the image information data of the spatially higher-resolution modality to the grid of the lower-resolution modality.

[0036] In various embodiments, the desorption optics system can comprise a transmitted-light optics system arranged and designed such that the first radiation impinges on the sample material after passing through the sample carrier. The transmitted-light optics system design allows the ion formation region to be kept free of beam-guiding elements that could interfere with ion extraction. Furthermore, a transmitted-light optics system enables a stronger focusing of the first radiation for the very localized ablation of the sample material, allowing significantly higher spatial resolutions to be achieved than with reflected-light optics systems such as reflection MALDI. Using a laser beam, ablation areas and thus image element or pixel areas with diameters in the single-digit micrometer range and—with particularly careful fine-tuning—even in the submicrometer range can be realized.The terms “picture element” and “pixel” have the same meaning in the present disclosure and are used synonymously.

[0037] In various embodiments, an observation axis of the transmitted-light optical system and an optical axis of the first radiation upon incidence onto the sample carrier can be aligned using a dichroic and / or dielectric mirror. This enables dual use of some optical components, such as lenses and / or mirrors, in guiding the first radiation upon incidence onto the sample carrier and the second radiation upon reflection from the sample material by the sample carrier.

[0038] In various embodiments, the device can be configured for the use of a conductively coated glass plate as a sample carrier. Indium tin oxide-coated glass plates (indium tin oxide, ITO) are particularly suitable, as their conductivity allows the generation of an electrical reference potential on their surface supporting the sample material. This potential is helpful in the further processing of the generated ionized sample material based on electrical potentials and fields.

[0039] In various embodiments, the light source can be arranged and designed such that the spatially resolved light microscopic image information is recorded substantially shadow-free. In a highly magnified image, the light source facing away from the sample carrier can The surface of the sample material, possibly covered by matrix substance, may appear highly jagged. Highly directional illumination of such a highly structured surface can mislead automated image analysis algorithms due to shadows cast on it, as the actual topography can only be clearly identified on the side of the structures facing the incident light. The shadowed areas, however, provide little support for image-based feature detection due to the small intensity differences and the resulting uniform appearance.The light source can be designed in several parts by having more than one light generator, the radiation of which is then combined for the observation of the spatially resolved light microscopic image information on the sample material, or it can also have one light generator, the light of which is then spatially spread out using one or more diffusers before being radiated onto the sample material.

[0040] In various embodiments, the light source can be ring-shaped and surround an observation axis of the transmission-incident optical system. A ring-shaped configuration around the observation axis, in particular, enables backlight incidence onto the sample carrier such that an observation point, e.g., an image element or pixel with an area of ​​0.01-1 square micrometer, on the possibly highly structured sample material can be illuminated with light from an extended solid angle range and thus at a multitude of different angles of incidence. Such a configuration is potentially suitable for avoiding shadows and the previously mentioned associated disadvantageous effects.

[0041] In various embodiments, the light source can comprise a plurality of light-emitting diodes. The light from each individual light-emitting diode can contain a mixture of different wavelengths, which together create a white color impression. However, it is also possible to design one or more light-emitting diodes so that they emit light with color characteristics deviating from white, e.g., one or more of the complementary colors red-cyan, green-magenta, and blue-yellow. Monochromatic light simplifies the optical design for beam guidance, since the optical components used do not have to process an extended wavelength range but can be optimized for a narrowband. Using light of different colors to record the spatially resolved microscopic image information can have advantages for the perception and automated evaluation of specific sample features.For example, monochromatic light can make it easier to locate contrast or sharpness maxima in optical images. It is possible to design all LEDs identically, e.g., with white light characteristics. In variants, LEDs of different colors can be placed next to each other. Together, they produce light of a first color, e.g., white—if all are activated and emit light—and, if some are activated, a second color, e.g., white. are not activated – produce light of a different color, e.g., green. A user can then selectively switch the color characteristic that best suits their planned experiment and the sample material used, without having to make complex modifications to the device.

[0042] In various embodiments, the light source can be arranged and designed such that the second radiation does not pass through an imaging and / or deflecting optical component upon incidence on the sample carrier. This simplifies the structure of the transmitted incident light optics and enables the light source to be arranged in the immediate vicinity of the sample carrier or its holder. Optical components are understood to mean, in particular, refractive (refracting) and reflective (reflection, “mirroring”) components such as lenses, mirrors, optical fibers, etc. A microscope objective as a whole is also to be regarded as an optical component. Purely light-transmitting plates, e.g. made of glass, through which light or electromagnetic waves pass under largely paraxial conditions, i.e. at angles that deviate only slightly from a surface normal of the plate, are not to be regarded as optical components within the meaning of the present disclosure.In particular, a sample carrier holder that supports the sample carrier, for example, in a transmitted-light desorption optical system in which the first beam impinges on the back of the sample carrier, is not considered an optical component within the meaning of the present disclosure. The light-transmissive sample carrier is also not considered an optical component within the meaning of the disclosure.

[0043] In various embodiments, the device can further comprise a movement mechanism for the sample carrier, which is arranged and designed to move the sample carrier along at least one spatial direction relative to a direction of incidence of the first radiation and / or the second radiation. The movement mechanism can in particular comprise an xy translation stage on which the sample carrier is placed and which spatially adjusts the sample carrier, together with the sample material placed thereon, along two spatial directions x and y, which extend substantially perpendicular to a surface normal of the sample carrier and substantially perpendicular to a microscopic observation direction. Preferably, the movement mechanism is also designed to spatially adjust the sample carrier in a third spatial direction z, which is perpendicular to the aforementioned spatial directions x and y.In particular, the movement mechanism can be designed and arranged to operate under negative pressure. A typical pressure, applicable, for example, for vacuum MALDI, is substantially greater than a high vacuum (> 10'). 3 hectopascals) and less than about 10 2 Hectopascals (< atmospheric pressure), e.g. 0.1-10 hectopascals. In the case of embodiments using In vacuum MALDI, local desorption and ionization of the sample material take place in a gas-tight and continuously pumped vacuum chamber, which is in fluid communication with the analyzer to transport the locally desorbed and ionized sample material.

[0044] In various embodiments, the operation of the computing unit, the desorption optics system, and the transmission-illumination optics system can include recording the spatially resolved light microscopic image information before and / or after the sample material is exposed to the first radiation. The special design of the device with a transmission-illumination optics system connected to the rear of the ion source region for observing and recording spatially resolved light microscopic image information of the sample material makes it possible to capture light images and molecular images in the same sample carrier holder, in the same clamping, so to speak, and in the same absolute position coordinate system. This not only ensures that the position coordinates of the corresponding image elements or pixels are known very precisely, but they also correspond for the different modalities: light microscopy and ion analysis.The only variance that could occur is any missteps in the motion mechanics when repeatedly approaching a specific position. However, translation stages are available that, according to manufacturer specifications, have a positioning accuracy in the tens of nanometers range, e.g., piezoelectrically driven translation stages from SmarAct (Oldenburg, Germany), particularly the CLL, CLS, and SLC series, with a positioning accuracy of 40 nanometers. Furthermore, any positioning inaccuracies that may occur can be reliably corrected using known monitoring methods and, if necessary, by correcting the position of the motion mechanics.

[0045] In various embodiments, the device can further comprise an imaging objective which is arranged such that the light source lies along an observation axis of the transmitted-light optical system between the sample carrier and the objective. The imaging objective can be designed to image the first radiation upon incidence on the sample carrier and the second radiation after passing through the sample carrier and being reflected from the sample material. The objective can in particular have dual imaging properties if a transmitted-light desorption optical system is used, in that on the one hand it directs the light reflected by the sample material through the sample carrier, which light is used to determine the spatially resolved light microscopic image information, to a camera used for recording and, if necessary.conditioned and on the other hand the first radiation is conditioned for the back incidence onto the sample carrier and the sample material arranged on the other side, e.g. to focus sizes with diameters in the range of a few micrometers or even less.

[0046] In various embodiments, the light source and the imaging lens can be designed as an integral assembly. Preferably, the light source is integrated into a part of a lens body facing the back of the sample carrier. Such a space-saving design reduces the distance between the light source and the sample carrier and allows the sample material to be irradiated with the light emitted by the light source without additional beam-guiding and beam-shaping optical components.

[0047] In various embodiments, an operation of the computing unit and the transmission-illuminated optical system can include recording the spatially resolved light microscopic image information by sequentially scanning a plurality of individual xy image areas on the sample material and computationally merging the isolated xy image information obtained in this way. The individual image information can be combined based on the acquired raw data to form an overall image covering the entire sample material. Prior image data processing, which could impose further inaccuracies on the light microscopic data that would then be passed through subsequent processing steps, is not required. In this way, the image quality of the overall optical image can be improved.In addition, no overlapping areas of the individual images with subsequent feature comparison in the overlapping areas need to be provided for merging, since the position of the individual image areas is known very precisely due to the high positioning accuracy of the movement mechanics.

[0048] According to a second aspect, the present disclosure relates to a device for the multimodal analysis of sample material, comprising: - a desorption optics system arranged and configured to expose the sample material arranged on one side of a sample carrier to a first radiation and to cause desorption of the sample material into the gas phase, wherein the desorbed sample material is ionized; - an analyzer arranged away from the sample carrier and configured to receive the desorbed and ionized sample material and process it into spatially resolved molecular image information; - a transmission-incident light optics system arranged and configured to record spatially resolved light microscopic image information of the sample carrier and sample material using a second radiation reflected through the transparent sample carrier;wherein the second radiation is emitted by a light source which is arranged on a side of the sample carrier facing away from the sample material and is designed such that the spatially resolved light microscopic image information is recorded substantially shadow-free, and - a computing unit which is arranged and designed such that it communicates with the desorption optical system, the analyzer and the transmission incident light optical system and processes the spatially resolved molecular image information as well as the spatially resolved light microscopic, To combine image information into spatially resolved co-registered overall image information, wherein one mode of operation of the computing unit and the transmission incident light optical system includes recording the spatially resolved light microscopic image information by sequentially scanning a plurality of xy individual image areas on the sample material and computationally combining the isolated xy individual image information obtained in this way.

[0049] In various embodiments, an operating mode of the computing unit and the transmission-incident optical system may include scanning a plurality of xy individual image areas in a third spatial direction z for a contrast and / or image sharpness maximum position. A preferred operating mode of the computing unit and the transmission-incident optical system may include using the contrast and / or image sharpness maximum position to (i) determine spatially resolved height profile information of the sample material above the sample carrier and / or (ii) compose an overall optical image that has, in each image element, an image component from a z-position of the respective contrast and / or image sharpness maximum position (focus stacking).

[0050] For sample material that exhibits pronounced height differences above the sample carrier, it may be useful to adjust the ablation position when the sample material is exposed to the first radiation in order to maintain uniform desorption conditions over an extended area, e.g., on the order of square millimeters to square centimeters for microtomed tissue sections. One mode of operation of the computing unit and the desorption optics system may therefore involve using the contrast and / or image sharpness maximum position when the sample material is exposed to the radiation for adjusting a position of (i) the focus of the first radiation and / or (ii) the sample carrier along the third spatial direction z.

[0051] According to a third aspect, the present disclosure relates to a method for the multimodal analysis of sample material, comprising: - locally exposing the sample material, which is arranged on one side of a sample carrier, to a first radiation and causing local desorption of the sample material into the gas phase, wherein the locally desorbed sample material is ionized, - receiving and processing the locally desorbed and ionized sample material to spatially resolved molecular image information using an analyzer arranged away from the sample carrier, - recording spatially resolved light microscopic image information from the sample carrier and sample material using a second radiation in reflection through the light-transmissive sample carrier, wherein the second radiation is emitted by a light source arranged on a side of the sample material arranged on the side facing away from the sample carrier and is designed such that the second radiation, when incident on the sample carrier, does not pass through an optical component which is passed through by the first radiation when incident on the sample carrier, and - linking the spatially resolved molecular image information and the spatially resolved light microscopic image information to form spatially resolved co-registered overall image information.

[0052] According to a fourth aspect, the present disclosure relates to a method for the multimodal analysis of sample material, comprising: - locally exposing the sample material arranged on one side of a sample carrier to a first radiation and causing local desorption of the sample material into the gas phase, wherein the locally desorbed sample material is ionized, - receiving and processing the locally desorbed and ionized sample material to spatially resolved molecular image information using an analyzer arranged away from the sample carrier, - recording spatially resolved light microscopic image information of the sample carrier and sample material using a second radiation reflected by the light-transmissive sample carrier, wherein the second radiation is emitted by a light source,which is arranged on a side of the sample carrier facing away from the sample material and is designed such that the spatially resolved light microscopic image information is recorded substantially shadow-free, and - linking the spatially resolved molecular image information and the spatially resolved light microscopic image information to form spatially resolved co-registered overall image information, wherein the spatially resolved light microscopic image information is recorded by sequentially scanning a plurality of xy individual image areas on the sample material and computationally combining the isolated xy individual image information obtained thereby.

[0053] In various embodiments, the method according to the third and / or fourth aspect of the present disclosure may be carried out using an apparatus as previously explained. Short description of the figures

[0054] For a better understanding of the invention, reference is made to the following figures. The elements in the figures are not necessarily drawn to scale, but are primarily intended to illustrate the principles of the invention (largely schematically). In the figures, corresponding elements are designated by like reference numerals throughout the different views.

[0055] Figure 1 shows a state-of-the-art setup of a tMALDI ion source including an integrated light microscopic observation modality.

[0056] Figure 2A schematically illustrates the desorption and ion generation region including connected optics systems of a device according to principles of the present disclosure in an isometric view.

[0057] Figure 2B shows a cross-section of the desorption and ion generation region from Figure 2A in a first orientation.

[0058] Figure 2C shows the cross-section of the desorption and ion generation region from Figure 2B in a second different orientation.

[0059] Figure 3 A shows an isometric view of a microscope objective in which a multi-component light source is integrated for recording light microscopic image information.

[0060] Figure 3B shows a cross-section of the microscope objective from Figure 3A.

[0061] Figure 4 shows the creation of an overall light microscopic image from a plurality of single-image area light microscopic images on the sample material according to principles of the present disclosure.

[0062] Figure 5 illustrates a method based on focus stacking and a determination of a depth or profile map of profiled sample material over a sample support according to principles of the present disclosure.

[0063] Figure 6 illustrates the linking of spatially resolved light microscopic image information with spatially resolved molecular image information to form spatially resolved co-registered overall image information according to principles of the present disclosure.

[0064] Figure 7 shows a MALDI time-of-flight mass analyzer of conventional design suitable for implementing principles of the present disclosure. Detailed description

[0065] While the invention has been shown and explained in terms of a number of embodiments, it will be appreciated by those skilled in the art that various changes in form and detail may be made therein without departing from the scope of the technical teachings defined in the appended claims.

[0066] Various key terms are used in this description, which are briefly explained below, particularly with regard to optical or microscopic observation. The smallest unit of an image is referred to as a pixel / image element. Pixel sizes in the optical / microscopic modality are generally significantly smaller than in the corresponding molecular images. The size of the sample material area imaged on an optical pixel depends on the sensor size of the camera used and the optical imaging properties and can, for example, be 100 x 100 square nanometers. A single image is an optical image recorded of a single image area using a camera with a single exposure through the observation optics. Typical edge lengths of such an area are 30-200 micrometers.The overall optical image is the mosaic-like composition of several individual images to obtain an overview and can encompass the entire sample material with edge lengths of a few millimeters or even centimeters. A detailed description of the terms is provided in the following sections.

[0067] The invention makes it possible to acquire high-resolution optical images directly in the ion source of a mass analyzer. This means that the absolute position coordinates for the spatially resolved light microscopic image information and the spatially resolved molecular image information correspond to the laboratory position coordinate system and are thus identical or congruent. Intrinsically, this enables almost perfect co-registration with the ion image acquired in the same source. Errors can be kept on the order of a few 100 nanometers, which corresponds to an accuracy smaller than the commonly used mass analysis pixel edge length (e.g., ~0.5-10 micrometers for tMALDI, ~5-100 micrometers for incident or reflected light MALDI). In addition, the newly introduced optical observation allows the sample topography to be determined with pixel sharpness and high resolution.

[0068] Figures 2A-C schematically illustrate an embodiment of a device according to the principles of the present disclosure in various views and focus on an ion source region. However, devices for further processing of ionized sample material are not shown in Figures 2A-C. For contextualization, reference is made to Figure 7, which will be explained further below.

[0069] Figure 2A shows a vacuum chamber 20 which is hermetically sealed to the surrounding atmosphere and which, using suitable vacuum generators, e.g. pumps, can be maintained at a pressure between 10' 3 and 10 2 Hectopascals; a pressure range in which vacuum MALDI can be performed. For the sake of clarity, some side walls of the chamber 20 are omitted from the illustration, only the rear wall 20-1 and upper and lower walls 20-2, 20-3 are visible. An xyz translation stage 22 is arranged in the chamber 20, onto which a sample carrier 24 can be placed. The translation stage 22 is mounted on the lower wall 20-3 of the chamber 20 and is designed to have a large opening 22-1 through which radiation is directed onto the sample carrier 24 from the rear side, i.e., away from the surface on which the sample material 26 is deposited. In this arrangement, the sample carrier 24 rests on the edges around the opening 22-1.

[0070] Between the rear wall 20-1 of the chamber 20 and the translation stage 22, a microscope objective 28 is mounted on the rear wall 20-1 and extends to just before the opening 22-1 in the translation stage 22. Details of the beam guidance and optical components in the objective 28 are not shown for the sake of clarity.

[0071] Beyond the rear wall 20-1 of the chamber 20, two beam paths 30, 32 are indicated, one associated with a transmitted-light desorption optical system and the other with a transmitted-light reflected-light optical system. The beam paths 30, 32 contain several beam-guiding and beam-shaping components, such as lenses and deflecting mirrors, which ensure that the first radiation 30 required for the desorption of sample material 26 is directed to the sample carrier 24, and the second radiation 32 required for recording the spatially resolved light microscopic image information, reflected by the sample material 26 through the sample carrier 24, is directed to a camera 34.

[0072] In the upper and lower walls 20-2, 20-3 of the chamber 20, two windows 36', 36" are indicated, through which a third radiation, e.g. a laser beam for the post-ionization of neutrally desorbed sample material, can be coupled into or out of the vacuum chamber 20 and focused into a desorption cloud above the section of the exposed sample material 26. Details on this process, called MALDI-2, can be found, for example, in the article by Jens Soltwisch et al. (Science, 10 April 2015 • Vol. 348 Issue 6231, 211 ff.) or the study by M. Niehaus et al. mentioned in the introduction for a tMALDI setup.

[0073] The structure can be divided into two parts, or two beam paths 30, 32. The observation beam path 32 and the laser beam path 30 are aligned with the aid of a dichroic and / or dielectric mirror 38, see the views in Figures 2B and 2C, on their path from and to the sample carrier 26. The coupling and decoupling of the radiation into and out of the vacuum chamber 20 takes place via a corresponding window 40 in the rear wall 20-1 of the chamber 20. With the aid of the UV-transmitting objective 28 in the vacuum chamber 20, which both the observation beam path 32 and the laser beam path 30 On the one hand, a microscopic observation of the sample material 26 in transmission incident light is made possible and, on the other hand, the focusing of the laser beam 30 on the front side of the sample carrier 24 carrying the sample material 26 to a beam waist in the submicrometer range is realized.

[0074] Both beam paths 30, 32 pass through the sample carrier 24, with the observation beam path 32 originating from the interface of the sample material 26 with any matrix applied thereto and being imaged onto the light-sensitive chip of the observation camera 34. The image section imaged can have an edge length of 50-250 micrometers, in which almost no contrast differences appear, and an optical resolution of 0.5-2 micrometers, depending on the choice of lens 28. The laser beam path 30 strikes the same interface from the back and is focused on a small section of the sample material 26. This section corresponds to an image element or pixel for the MS measurement, e.g., with a size of 0.5-10 micrometers in diameter or edge length. The configuration is therefore a transmission MALDI (tMALDI) setup.

[0075] The illustrated embodiment is based on the dual use of a microscope objective 28 for both sample material observation and sample material ablation. This dualism enables the precise determination of the ablation point on the generated optical image. With the help of customized software, individual high-resolution microscope images can be combined into a large overview image. At the same time, the analysis of the contrast of microscopic images at different object distances allows the topography to be determined with high spatial precision.

[0076] Observation is realized by a camera 34, which is arranged along a surface normal of the sample carrier 26 and away from it in the rear area outside the vacuum chamber 20. The objective 28 is infinity corrected, which is why an imaging lens 42 is positioned at a distance corresponding to its focal length from the camera 34. Microscopic observation in transmitted incident light (reflective), i.e., reflected by the sample material 26 through the sample carrier, requires the most diffuse illumination possible to reduce shadowing effects. This illumination is realized here by an illumination ring consisting of several light-emitting diodes (LEDs) 44.

[0077] In the example shown, 15 LEDs 44 are used. The number of LEDs could also be smaller or larger. Light sources or LEDs with white light or monochromatic color characteristics, such as green, can be used. Light sources are also conceivable.or LEDs with variable color characteristics, which, for example, are tunable over a wavelength range or have mixed colors, in particular polychromatic but not white color characteristics. The light-emitting diodes 44 are arranged in a cylindrical, axially projecting attachment 46, which is joined to the lens body 28* to form an integral assembly, as shown in Figures 3A-B, and surround the optical observation axis 48 running through the lens 28 in a ring. The light-emitting diodes 44 themselves are each housed in cylindrical housings, which are embedded in the attachment 46 at an angle different from 0° and 180° to the optical observation axis 48 of the lens 28, so that the optical axes (dotted lines 50) of all diodes 44 converge or meet at a point 52 of maximum illumination, remote from the attachment end of the lens body 28* and lying on the optical observation axis 48.This point of maximum illumination 52 is located on the sample carrier 24 in order to be able to illuminate the sample material 26 placed on the front side thereof with high intensity and without shadows in transmitted incident light, and corresponds to the optimal working distance for the objective 28 and / or its focal point.

[0078] The laser beam 30 for ablating the sample material 26 is aligned coaxially with the observation beam path 32 through the lens 28 to the targeted section of the sample material 26 using the dichroic and / or dielectric mirror 38. This firmly couples the ablation position to a defined position on the observed image. Subsequent, error-prone co-registration of the camera image and the ablation position is eliminated.

[0079] To generate a high-resolution optical overview image (scan), individual images 54 of defined areas 56 on the sample carrier 26 are captured at high magnification and stitched together in a mosaic-like manner to form an overall image 58. The corresponding principle is schematically illustrated in Figure 4. For this purpose, the camera 34 and the movement mechanism of the translation stage 22 are coordinated and synchronized by software. Mosaic images are then constructed using the X:Y coordinates stored for each individual image 54. Due to the precise positioning and minimal processing of the individual images 54, no distortion effects such as stretching, rotation, etc. are introduced. The positioning accuracy is limited by the accuracy of the movement mechanism of the translation stage 22 and the size of an optical pixel, i.e., depending on the scaling factor, on the order of 100 nanometers or even less.Since the coordinates of the traversing mechanism remain constant in the laboratory position coordinate system when scanning the sample material 26 for recording the optical image and during a preceding or subsequent mass analysis, a nearly perfect co-registration of the overall optical and molecular image is achieved without additional intervention or subsequent correction.

[0080] In order to determine the topography of the extensive sample material 26 in a preferred embodiment, a defined set of points X are first measured over the entire scanning area, e.g. the entire area of ​​a tissue section. n :Y mThese can be instrumentally fixed to a grid or manually set. An autofocus routine is performed at these points. The technique used to determine the focal plane at each individual point is similar to the method described by Michael J. Taylor et al., see Introduction. For any location on the sample material 26, by interpolation and / or extrapolation, starting from the support points of the selected points X n :Y m , the true sample material elevation above the sample carrier 24 is calculated and introduced as a software-based z-correction. This method is particularly suitable for correcting large-scale topographical variances, such as those that can arise when clamping the sample carrier 24 into the designated holder.

[0081] In a particular embodiment, a method based on focus stacking is used. This allows the topography of the sample material 26 to be determined laterally with high resolution, e.g., to within a few micrometers. Analogous to the above-mentioned method for generating an overall optical image, several overall optical images for defined z-values ​​are created using the movement range of the translation stage 22 in the z-direction with the settings of the transmission incident light optical system otherwise unchanged, schematically indicated in Figure 5 as z1, z2, z3. Depending on the expected sample topography, these z-values ​​can, for example, cover a range of ±10 micrometers with a step size of 1 micrometer around a starting value, e.g., an average or expected tissue section thickness.

[0082] A Laplace filter is applied to each of these images. The kernel size and the optional use of additional filters depends on the optical configuration and the observed sample material 26. The results from this step serve as a local measure of the contrast or image sharpness and are written into a three-dimensional matrix. The first and second dimensions correspond to the x and y position on the sample material 26, the third dimension corresponds to the z plane of the underlying image. For each x and y position, the maximum of the result matrix is ​​determined along the third dimension. The z index of the maximum is written for each x and y position into a two-dimensional matrix, which can be called a depth or profile map, see the far right in Figure 5. The depth or profile map therefore contains, if applicable,extended or supplemented by inter- and / or extrapolation, the z-plane of highest image sharpness for any x and y position.

[0083] The resulting depth or profile map can be used to keep the ablation laser in focus with pixel precision during a subsequent MALDI-MSI measurement, for example by changing the focus position or by moving the sample carrier along the z-axis. A Focus tacA image can also be constructed, referred to as a "stack image" in Figure 5. In this case, the real image pixel intensities of the recorded z-planes are combined pixel by pixel so that the resulting image contains only the image data of the sharpest z-plane at the respective xy position. This approach significantly facilitates the visual evaluation of the overall light microscopic image information and its visual comparison with the molecular image information, particularly due to the consistently sharp display.

[0084] The techniques described above significantly simplify and improve the workflow and / or accuracy of tMALDI-MSI measurements with small pixel sizes. Figure 6 illustrates the co-registration of the spatially resolved light microscopic image information, represented by an overall optical image 58* at the top left, whose individual image grid is indicated by black bars and the individual image center coordinates by crosses. The dimensions of each individual image and its location in the coordinate system are stored for a specific translation stage position. Also shown is the spatially resolved molecular image information, represented by the ion image 60 at the bottom left, which, for example, depicts the intensity distribution of a specific ion or molecular species of interest, e.g., a protein, peptide, lipid, polynucleotide, or polysaccharide.The information from both modalities can then be combined to form a single piece of image information, which—due to acquisition in the identical coordinate system—is co-registered with high spatial resolution in the submicrometer range, represented by the composite image 62 on the right. The co-registered single piece of image information, comprising a light microscopic image component and a molecular image component, can then be visually evaluated by a user or subjected to a computer-assisted automated evaluation routine, e.g., to find feature correlations in the data from the different modalities.

[0085] Figure 7 schematically shows a generally known MALDI time-of-flight mass analyzer in an axial reflector configuration, in which principles of the present disclosure can be applied. The sample material is located on the sample support plate 71 opposite the accelerating electrodes 72 and 73 and can be spatially resolved ionized by the laser light pulse beam 74, 74* delivered by the laser 75, 75* in incident or transmitted light. The ions are accelerated by the accelerating electrodes 72 and 73 to an ion beam 78, which forms a gas cell 79, which can be filled with collision gas if required. The ion passes through a precursor ion selector 80, a fragment ion post-acceleration unit 81, and the precursor ion suppressor 82, and is then reflected by the reflector 83 onto the ion detector 84. The mass analyzer housing is pumped by a powerful vacuum pump 85. The ion detector 84 may have a multi-channel plate as the ion receiver. A transmission-incident optical system according to the principles of the present disclosure may be integrated into the rear portion behind the sample carrier 71.

[0086] The invention has been described above with reference to various specific embodiments. However, it should be understood that various aspects or details of the described embodiments may be modified without departing from the scope of the invention. Furthermore, the features and measures disclosed in connection with different embodiments may be combined in any way that appears practical to a person skilled in the art. Furthermore, the above description serves only to illustrate the invention and not to limit the scope of protection, which is defined exclusively by the appended claims, taking into account any existing equivalents.

Claims

Claims 1. Device for multimodal analysis of sample material, comprising: - a desorption optics system arranged and designed to expose the sample material arranged on one side of a sample carrier to a first radiation and to cause desorption of the sample material into the gas phase, wherein the desorbed sample material is ionized, - an analyzer arranged away from the sample carrier and designed to receive the desorbed and ionized sample material and to process it into spatially resolved molecular image information, - a transmission-incident light optical system arranged and designed to record spatially resolved light microscopic image information of the sample carrier and sample material using a second radiation in reflection through the light-transmissive sample carrier, wherein the second radiation is emitted by a light source arranged on a side of the sample carrier facing away from the sample material and designed such that the second radiation, upon incidence onto the sample carrier, does not pass through an optical component which is passed through by the first radiation upon incidence onto the sample carrier, and - a computing unit arranged and designed to communicate with the desorption optics system, the analyzer and the transmission reflected light optics system and to combine the spatially resolved molecular image information and the spatially resolved light microscopic image information to form spatially resolved co-registered overall image information.

2. Device according to claim 1, wherein the desorption optical system comprises a transmitted light optical system arranged and designed such that the first radiation impinges on the sample material after passing through the sample carrier.

3. Device according to claim 2, wherein an observation axis of the transmission incident light optical system and an optical axis of the first radiation are superimposed upon incidence on the sample carrier using a dichroic and / or dielectric mirror.

4. Device according to one of claims 1 to 3, which is designed for the use of a conductively coated glass plate as a sample carrier.

5. Device according to one of claims 1 to 4, wherein the light source is arranged and designed such that the spatially resolved light microscopic image information is recorded substantially shadow-free. Device according to one of claims 1 to 5, wherein the light source is annular and surrounds an observation axis of the transmitted-light optical system. Device according to one of claims 1 to 6, wherein the light source comprises a plurality of light-emitting diodes. Device according to one of claims 1 to 7, wherein the light source is arranged and designed such that the second radiation does not pass through any imaging and / or deflecting optical components upon incidence onto the sample carrier. Device according to one of claims 1 to 8, further comprising a movement mechanism for the sample carrier, which is arranged and designed to move the sample carrier along at least one spatial direction relative to a direction of incidence of the first radiation and / or the second radiation.Device according to one of claims 1 to 9, wherein an operating mode of the computing unit, the desorption optical system, and the transmission-incident optical system includes recording the spatially resolved light microscopic image information before and / or after the sample material is exposed to the first radiation. Device according to one of claims 1 to 10, further comprising an imaging objective arranged such that the light source lies between the sample carrier and the objective along an observation axis of the transmission-incident optical system. Device according to claim 11, wherein the imaging objective is designed to image the first radiation upon incidence onto the sample carrier and the second radiation after passing through the sample carrier and reflection from the sample material. Device according to claim 11 or claim 12, wherein the light source and the imaging objective are formed as an integral assembly.Device according to one of claims 1 to 13, wherein an operating mode of the computing unit and the transmission-illuminated optical system includes recording the spatially resolved light microscopic image information by sequentially scanning a plurality of xy individual image areas on the sample material and computationally combining the isolated xy individual image information obtained thereby. Device for multimodal analysis of sample material, comprising: - a desorption optics system arranged and designed to detect the sample material, which is arranged on one side of a sample carrier, with a first radiation and to cause desorption of the sample material into the gas phase, whereby the desorbed sample material is ionized, - an analyzer arranged away from the sample carrier and designed to receive the desorbed and ionized sample material and to process it into spatially resolved molecular image information, - a transmission incident light optical system arranged and designed to record spatially resolved light microscopic image information of the sample carrier and sample material using a second radiation reflected through the light-transmitting sample carrier, wherein the second radiation is emitted by a light source arranged on a side of the sample carrier facing away from the sample material and designed such that the spatially resolved light microscopic image information is recorded substantially shadow-free, and - a computing unit arranged and configured to communicate with the desorption optics system, the analyzer, and the transmission-illuminated optical system, and to combine the spatially resolved molecular image information and the spatially resolved light microscopic image information to form spatially resolved, co-registered overall image information. An operating mode of the computing unit and the transmission-illuminated optical system includes recording the spatially resolved light microscopic image information by sequentially scanning a plurality of xy individual image areas on the sample material and computationally combining the individual xy individual image information obtained thereby. Device according to claim 14 or claim 15, wherein an operating mode of the computing unit and the transmission-illuminated optical system includes scanning a plurality of xy individual image areas in a third spatial direction z to a maximum contrast and / or image sharpness position.Apparatus according to claim 16, wherein an operation of the computing unit and the transmission incident light optical system includes using the contrast and / or image sharpness maximum position to (i) determine spatially resolved height profile information of the sample material above the sample carrier and / or (ii) assemble an overall optical image which has an image component from a z-position of the respective contrast and / or maximum position in each image element (focus stacking). The device according to claim 16 or claim 17, wherein an operating mode of the computing unit and the desorption optics system includes using the contrast and / or image sharpness maximum position upon exposure to the sample material for adjusting a position of (i) the focus of the first radiation and / or (ii) the sample carrier along the third spatial direction z. A method for multimodal analysis of sample material, comprising: - locally exposing the sample material, which is arranged on one side of a sample carrier, to a first radiation and causing local desorption of the sample material into the gas phase, whereby the locally desorbed sample material is ionized, - Receiving and processing the locally desorbed and ionized sample material to obtain spatially resolved molecular image information using an analyzer located away from the sample carrier, - recording spatially resolved light microscopic image information of the sample carrier and sample material using a second radiation reflected through the light-transmitting sample carrier, wherein the second radiation is emitted by a light source arranged on a side of the sample carrier facing away from the sample material and designed such that the second radiation, when incident on the sample carrier, does not pass through an optical component which is passed through by the first radiation when incident on the sample carrier, and - Combining the spatially resolved molecular image information and the spatially resolved light microscopic image information to spatially resolved co-registered overall image information. Method for multimodal analysis of sample material, comprising: - locally exposing the sample material, which is arranged on one side of a sample carrier, to a first radiation and causing local desorption of the sample material into the gas phase, whereby the locally desorbed sample material is ionized, - Receiving and processing the locally desorbed and ionized sample material to obtain spatially resolved molecular image information using an analyzer located away from the sample carrier, - recording spatially resolved light microscopic image information of the sample carrier and sample material using a second radiation reflected through the light-transmitting sample carrier, wherein the second radiation is emitted by a light source arranged on a side of the sample carrier facing away from the sample material and designed such that the spatially resolved light microscopic image information is recorded substantially shadow-free, and - Combining the spatially resolved molecular image information and the spatially resolved light microscopic image information to form spatially resolved co-registered overall image information, wherein the spatially resolved light microscopic image information is recorded by sequentially scanning a plurality of xy individual image areas on the sample material and computationally combining the individual xy individual image information obtained thereby. Method according to claim 19 or claim 20, which is carried out using a device according to any one of claims 1 to 18.

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