Device for multi-modal analysis of sample material

Abstract

The invention relates to a device for multi-modal analysis of a sample material, e.g. From tissue, which detects molecular image information from the sample material in a spatially resolved manner, e.g. Using a MALDI time-of-flight mass analyzer, and records optical microscopic image information from the sample material in a spatially resolved manner, and the two images are associated with improved precision to give overall image information of spatially resolved common registration.

Description

Technical Field

[0001] The present invention relates to a device for performing multimodal analysis on sample material, for example from tissue, which uses, for example, a time-of-flight mass analyzer to detect molecular image information from the sample material in a spatially resolved manner, records optical microscopic image information from the sample material in a spatially resolved manner, and associates the two with improved accuracy to give spatially resolved, co-registered overall image information. Background Art

[0002] The prior art will be described below with reference to a specific aspect. However, this should not be considered limiting. Beneficial advances and variations of the present invention may also be applicable outside the relatively narrow scope of this introduction, and this will be apparent to a trained practitioner in the art after reading the disclosure of the present invention below the introduction.

[0003] In imaging MALDI mass spectrometry (mass spectrometry imaging = MSI; MALDI = matrix-assisted laser desorption ionization), the co-registration of ion images with high-resolution optical microscopy images is an important source of information. The methods used to date for this purpose can be very time-consuming and prone to errors.

[0004] In addition to the exact correlation of the plane coordinates xy of each pixel for co-registration, MALDI-MSI also requires information about the sample topography for each pixel, if possible. On this basis, material ablation can be optimized, thereby optimizing the data quality. This is especially true for high-resolution and ultra-high-resolution MALDI-MSI. The so-called transmission geometry technique, tMALDI, can be used here. The low depth resolution of the microscope objective used (for example, the depth resolution is 1.6 microns in the case of a 50X Mitutoyo Plan Apo NUV with an infinity-corrected objective) requires precise determination of the topography with submicron accuracy.

[0005] In the current state of the art, the two objects of spatially accurate overlay and topography determination are handled separately and are both independently affected by inaccuracies, which limits the information evaluation.

[0006] Various commercially available slide scanners are used to record optical images, such as Olympus VS200, Hamamatsu, etc. Slide refers here to the object carrier. These slide scanners are separate components and mainly work with individual images obtained by scanning the sample surface, and then combine these images by stitching to form an overall image. This combination is based on processed image data (including the discovery of consistent image features), rather than on raw data.

[0007] It is also known to co-register an externally recorded optical image with the ion image by adding teach marks during sample preparation. Various mass spectrometer manufacturers sell software programs for such multimodal optical / mass spectrometry imaging. Examples are Bruker: flexImaging, or Waters / Micromass: High Definition Imaging.

[0008] The co-registration of optical images in MALDI-MSI has two functions. First, before the measurement, the measurement range is limited by means of the co-registered optical scan. Secondly, in the multimodal method, the microscope image recorded before or after the MALDI measurement is used to combine the optical information and the mass spectrum information. These two functions can only be achieved in a very inaccurate and time-consuming manner by the known methods for this purpose. In order to perform the initial co-registration of the optical image (scan) and the ablation position in the mass spectrometer, the user or the automatic program must first associate the optical features of the externally recorded image with the real-time image of the camera inside the mass spectrometer. Here, the first error occurs when positioning the mark in the two images. Especially in the case of a pixel size of less than 10 microns, the optical imaging quality inside the mass spectrometer cannot usually guarantee accurate allocation. After marking, the optical image is processed, including stretching, rotation and tilt correction of the non-fixed aspect ratio. These image corrections also always lead to artifacts, which often lead to inaccurate co-registration. This systematic uncertainty in the initial co-registration has the direct consequence that the measurement range must always be selected with a certain safety margin. This prevents the measurement of directly adjacent areas, leaves parts of the sample area unchecked, and prolongs the measurement time.

[0009] In the case of a multimodal interpretation of the acquired data, very precise co-registration is important. For example, only precisely correlated images allow a direct comparison of morphological and molecular information. Especially in the case of high-resolution and ultra-high-resolution MALDI-MSI (pixel size <10 micrometers), systematic errors are often several times the pixel size. This requires a subsequent correction of the co-registration between the microscopic image on the one hand and the ion distribution image on the other hand. Since the two modalities generate their contrast in very different ways, systematic errors often remain on the order of magnitude of the pixel size.

[0010] As explained in two articles by Nathan Heath Patterson et al., accurate co-registration using existing methods also requires time-consuming and computationally intensive methods based on multiple microscopy images before and after MALDI-MSI. They describe a procedure for registering and analyzing MALDI MSI relative microscopy data using non-destructive MSI-compatible wide-field autofluorescence (AF) microscopy combined with computer-assisted image registration techniques (Anal. Chem. 2018, 90, 12395-12403), as well as a further developed histology-led platform that uses MSI-compatible AF microscopy prior to histological staining or MSI measurement (Anal. Chem. 2018, 90, 12404-12413).

[0011] The paper by Michael J. Taylor et al. (Metabol ites 2021, 11, 200) involves laser ablation electrospray ionization mass spectrometry (LAESI-MS) for single-cell metabolomics. According to the report, a microscope is integrated into the optical path of the LAESI source to enable visually monitored in situ single-cell analysis to facilitate the understanding of cell-to-cell differences within large cell populations under environmental conditions. The microscope records the optical image of the sample surface in individual tiles, which are then "stitched together" by an image processing program to give an overall image.

[0012] In the method proposed by Michael J. Taylor et al., it is described that in LAESI experiments, illumination and observation are coaxial to the ablative laser beam path. Here, too, an automatic focusing procedure based on image sharpness is used. Since the matrix coating is mostly opaque, this frontal observation in reflected light is not suitable for MALDI samples. In addition, observation along the sample normal is not possible or is very difficult to achieve, since this space is required for ion extraction in MALDI. Observation at angles greater than 0° to the sample normal leads to image distortions, which must be corrected. The required offset of the individual images relative to each other means that a sufficiently accurate co-registration cannot be achieved by this method.

[0013] In addition to co-registration, pixel-clear determination of topography may be an important prerequisite for ultra-high resolution MALDI-MSI, for example when examining highly contoured tissues such as the retina. Current methods for tissue samples or other mass spectrometry samples perform this topography determination independently. Therefore, the accuracy of the topographic information available in the mass spectrometer depends in part on the error-prone co-registration. In addition, the commonly used methods are not accurate enough for the low depth resolution in the low micron range used in tMALDI, and are limited to relatively large areas, so the pixels are not clear enough. For example, in Bruker's timsTOF fleX, an auxiliary laser is used to project a stripe pattern on the sample surface. Using this pattern, the distance between the laser focal plane and the sample surface can be accurately determined to about 5 microns. For low tMALDI depth resolution (such as 1.6 microns calculated for a 50x objective lens by the above example), this accuracy is not high enough. In addition, with the help of this method, averaging is performed on an area of ​​about 1 mm × 1 mm. Therefore, smaller structures are averaged out and escape evaluation.

[0014] Mario Kompauer et al. (Nat. Methods 2017, 14, 1156-1158) proposes to use a point light source for each pixel in the height profile determination to perform a time-consuming autofocus procedure to resolve small structures. Currently, the pixel size here is limited to about 20 microns. Related to this is the patent publication EP 3 306 639 A1 (corresponding to US10,964,519B2), the purpose of which is to provide a device capable of simultaneously analyzing the two-dimensional spatial chemical composition and morphology of a sample.

[0015] Some prior art documents that may be relevant to the subject matter of the present disclosure are also briefly identified below:

[0016] The paper by F.Hillenkamp et al. (Appl.Phys.8,341-348(1975)) introduces a highly sensitive laser microprobe mass analyzer (LAMMA) designed for examining biological sample materials. Based on this pioneering research, the article by H.Vogt et al. (Fresenius Z.Anal.Chem.308,195-200(1981)) describes the principle and technical features of a laser microprobe mass analyzer (LAMMA) 500 equipped with an optical microscope for visual observation of samples in reflection through a coated electron microscope grating used as an object carrier.

[0017] The paper by Bernhard Spengler et al. (J Am Soc Mass Spectrom 2002, 13, 735–748) relates to a scanning microprobe matrix-assisted laser desorption ionization (SMALDI) mass spectrometer for analytically resolved LDI and MALDI surfaces in the submicron range. For an overview, the sample can be imaged using a standard optical microscope with a magnification of about 400 times. An area of ​​about 500 μm × 400 μm is displayed on a video monitor. Due to geometric constraints, the sample is not illuminated and exposed from the side, but through the objective lens. Since the objective lens is not chromatically corrected, the sample can only be imaged using monochromatic light (i.e., a helium-neon laser with an output light power of 15 mW).

[0018] Patent publication US2003 / 0222212 A1 discloses a method and system for creating a correlated optical image of the ion desorption region of a sample matrix using light focused down to the micrometer or submicrometer range for matrix-assisted laser desorption / ionization ( FIG. 3 ).

[0019] Patent publication US2006 / 0289734 A1 proposes a method for generating a clear image of an area on a sample plate for a matrix-based ion source, comprising: positioning the area in the field of view of an imaging device; using the imaging device to create a first image having an in-focus area and an out-of-focus area; using the imaging device to create a second image having an in-focus area and an out-of-focus area; and using the in-focus areas of the first and second images to create a final in-focus image.

[0020] Patent US7,180,058B1 protects an ion source for a mass spectrometer, comprising: a radiation source for generating a beam of radiation; a beam focusing optical device, which is configured so that the optical device focuses the beam of radiation onto a sample arranged on the front surface of a sample carrier. The beam focusing optical device has a focal length of less than 25 mm and is positioned adjacent to the rear surface of the sample carrier. The sample carrier is transparent to the wavelength of the beam of radiation so as to transmit the beam of radiation. It also includes an ion optical device, which is positioned adjacent to the front surface of the sample carrier and is configured so that it transmits ions generated by irradiation of the sample. It also includes an observation optical device for detecting an image of the sample, which is arranged adjacent to the rear surface of the sample carrier.

[0021] Patent publication DE102007006933A1 (corresponding to US2008 / 0191131A1 and GB2446699A) proposes a method by which, in a MALDI axial time-of-flight analyzer, the distance between the sample surface and the first accelerating electrode in the flight path can be adjusted with the aid of position information of the sample surface relative to a digital camera determined by evaluating an image from the digital camera.

[0022] Patent publication US2009 / 0146053A1 describes a mass spectrometer for performing mass analysis while microscopically observing a two-dimensional area of ​​a sample, and separating an observation position for selecting a target area from an analysis position for performing mass analysis, wherein an image of the sample recorded by a CCD camera is simultaneously observed at the observation position, and laser light is released onto the sample in the analysis position. The sample is positioned on a stage, which can be precisely moved between the observation position and the analysis position by a stage drive mechanism.

[0023] Patent publication US 2011 / 0315874 A1 discloses a mass spectrometer which is said to be capable of efficiently performing imaging mass spectrometry over a spatial region extending beyond the observation field of a microscope observation unit.

[0024] Patent publication US2011 / 0266438A1 discloses a mass spectrometer which is said to be capable of obtaining a microscopic observation image with high spatial resolution in real time during mass analysis without damaging the analysis. An opening is formed on a stage, and a transparent sample plate is located on the opening. A microscope observation unit including an optical observation system and a CCD camera is provided below the stage to observe the reverse side of the sample through the opening on the stage and the transparent sample plate. The observed image is displayed on a screen of a display unit.

[0025] In a study by Andre Zavalin et al. (J Mass Spectrom. 2012 November; 47(11), 1473-1481), the authors concluded that the transmission geometry configuration allows the laser beam in the ion source of a MALDI mass spectrometer to be focused to a size of less than 1 micron, which is said to enable direct imaging of heterogeneous tissue sections and single cells with subcellular resolution. Subcellular MS images were verified by co-registration of single images obtained by microscopic methods, such as optical phase contrast, DIC (differential interference contrast) and bright field images. Single optical microscope images were recorded before and after the MS imaging experiment. Figure 1 of the present disclosure shows a modification of Figure 2 of the publication. The reference numerals therein have the following meanings: 1-chamber; 2-ultraviolet laser; 3-aperture; 4-beam attenuator; 5-white light source; 6-CCD camera; 7-xy translation stage; 8-reverse observation window; 9-microscope objective; 10-time-of-flight analyzer tube.

[0026] The study by Marcel Niehaus et al. (Nat. Methods 2019, 16, 925-931) involves MALDI-2 mass spectrometry in transition mode for imaging cells and tissues with subcellular resolution. The same beam path, beam splitter, and CCD camera (charge-coupled device) as the MALDI laser using a single LED as the light source allow microscopic observation of the sample in transmitted light.

[0027] In view of the above observations, there is a need to improve the co-registration of spatially resolved molecular image information and spatially resolved microscopy image information, particularly with respect to its accuracy and the complexity involved in the configuration of optical components. Other objects to be achieved by the present invention will be immediately apparent to those skilled in the art after reading the following disclosure. Summary of the invention

[0028] In a first aspect, the present disclosure relates to an apparatus for multimodal analysis of sample materials, comprising: - a desorption optical system, which is arranged and designed to subject a sample material arranged on one side of a sample carrier to a first radiation and to desorb the sample material into a gas phase, wherein the desorbed sample material is ionized, - an analyzer, which is arranged at a distance from the sample carrier and is designed to receive the desorbed and ionized sample material and process it into spatially resolved molecular image information, - a transmission and reflection light optical system, which is arranged and designed to record in a spatially resolved manner using a second radiation reflected through a transparent sample carrier Optical microscopic image information from a sample carrier and a sample material, wherein a 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 so that the second radiation, when incident on the sample carrier, does not pass through any optical component through which the first radiation passes when incident on the sample carrier, and, - a computing unit which is arranged and designed to communicate with the desorption optical system, the analyzer and the transmitted and reflected light optical system and to associate the spatially resolved molecular image information with the spatially resolved optical microscopic image information to give spatially resolved, co-registered overall image information.

[0029] The sample material used can be, in particular, a microscopically sectioned tissue section. Examples thereof include brain tissue and retinal tissue. The sample material can be cut, in particular, from frozen tissue blocks or formalin-fixed, paraffin-embedded (FFPE) tissues, which may require further processing steps before analysis, such as "dewaxing" and "de-crosslinking", also known as antigen repair. The thickness of the tissue section to be analyzed can be 2-20 microns, and in particular 2-15 microns for tMALDI applications. For reflected light or reflected MALDI, the section can also be thicker, for example 2-40 microns. Multimodal analysis of tissue sections is becoming increasingly important, particularly in the field of clinical applications, for determining the pathological state of the tissue and distinguishing it from a non-pathological state, or for determining the cellular response to the administration of a drug substance.

[0030] The desorption optical system may comprise a laser desorption ion source (LDI), which may be particularly designed as a MALDI source. Useful ionization methods are MALDI methods in reflected light or transmitted light, as desired. The MALDI method requires a specific sample preparation using a light absorbing matrix substance, such as sinapinic acid, 2,5-dihydroxybenzoic acid, α-cyano-4-hydroxycinnamic acid or 2,5-dihydroxyacetophenone, all of which strongly absorb in the ultraviolet spectral region, for example, laser light from a nitrogen laser at a wavelength of about 337 nanometers, or laser light from a frequency tripled solid-state Nd:YAG laser at a wavelength of about 355 nanometers.

[0031] The sample material may be subjected to the first radiation in the form of pulses. The frequency of the pulse sequence may be from a few Hertz (e.g. 1-20 pulses per second) to up to 10 3 or 10 4 Hertz range.

[0032] The analyser may be a mobility analyser, a mass analyser or a combined mobility-mass analyser. In general, ion mass spectrometry and testing methods may be said to involve mobility separation, mass separation or a combination of the two.

[0033] Ion mobility analyzers separate charged molecules or molecular ions according to the ratio of collision cross section to charge (sometimes referred to as Ω / z or σ / z). The basis is the interaction of ion species with the electric field coupled to the charge of the ions while a buffer gas acts on the average cross-sectional area of ​​the ions. In particular, drift tube mobility separators with static electric field gradients are known, which drive ions through a substantially stationary gas, thereby determining the drift velocity of the ion species according to the driving force of the electric field and the deceleration force of collision with gas particles. Also familiar are trapped ion mobility separators (TIMS), which use a constant laminar gas flow to drive ions forward, and a gradually changing electric field gradient resists the gas flow with a corresponding variable deceleration force. Traveling wave mobility separators may also be mentioned.

[0034] The mass analyzer, in turn, separates charged molecules or molecular ions according to the mass-to-charge ratio (commonly referred to as m / z). A time-of-flight analyzer can be used, for which a linear or reflectron setup and / or those with axial or orthogonal acceleration into the time-of-flight region can be provided. Other types of mass dispersive separators can also be used, such as quadrupole mass filters ("single quadrupole"), triple quadrupole analyzers ("triple quadrupole"), ion cyclotron resonance cells (ICR), Kingdon type analyzers such as (Thermo Fisher Scientific) etc. Obviously, separators of the above type can be coupled so as to be able to separate ion species in multiple dimensions, ie according to more than one physicochemical property, such as m / z and Ω / z or σ / z.

[0035] Correlating spatially resolved molecular image information and spatially resolved optical microscopy image information may mean precisely superimposing these two types of image information in space and creating an image or graphical representation thereof, for example on a computer screen. In this way, a user may be empowered to visually identify corresponding and / or different feature intensities or structures in image information from various modalities in a spatially resolved manner. Correlating in the context of the present disclosure may alternatively include further processing of the image information, such as creating an overview of the sample material on which an index is plotted in grayscale or color scale, the index being obtained by calculating the spatially resolved intensity of one or more ion species of interest m / z and the spatially resolved intensity of one or more wavelengths λ in the electromagnetic spectrum. The ion species of interest may 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 in two dimensions, such as the entire area of ​​the analyzed tissue section. The image information patterns of the various modalities are typically of different sizes; optical modalities allow finer patterns, i.e., image elements with smaller sizes, compared to mass analysis modalities. Depending on the configuration of the optical microscopy optics, the optical resolution is typically in the sub-micrometer range, such as 500-700 nanometers, while the pixel size (or lateral resolution) used for mass analysis ablation is typically in the low micrometer range, such as 1-10 microns. In certain embodiments, the association can include adjusting the image information data from a modality of higher spatial resolution to a pattern of a modality with a lower resolution.

[0036] In various embodiments, the desorption optical system may have a transmitted light optical system, which is arranged and designed so that the first radiation is incident on the sample material after passing through the sample carrier. Configuration as a transmitted light optical system allows the ion formation area to be moved away from beam guiding elements that may interfere with ion extraction. In addition, the transmitted light optical system enables a stronger focusing of the first radiation so that a local and very limited ablation of the sample material can be achieved, so that a significantly higher spatial resolution can be achieved than with reflected light optical systems (such as reflective MALDI). With a laser beam, ablation areas can be achieved, thereby achieving image elements or pixel areas with diameters in the single-digit micrometer range, and with particularly careful fine-tuning, image elements or pixel areas with diameters in the sub-micrometer range can be achieved. The terms "image element" and "pixel" have the same meaning and are used synonymously in the present disclosure.

[0037] In various embodiments, the observation axis of the transflective light optical system and the optical axis of the first radiation incident on the sample carrier can be coordinated one above the other by using a dichroic mirror and / or a dielectric mirror. This allows some optical components (such as lenses and / or mirrors) to be used dually, not only to guide the first light beam to be incident on the sample carrier, but also to guide the second light beam through the sample carrier to be reflected by the sample material.

[0038] In various embodiments, the device can be designed to use a conductively coated glass plate as a sample carrier. Indium tin oxide (ITO) coated glass plates are particularly suitable because, through their conductivity, the glass plate allows the generation of an electrical reference potential on its surface carrying the sample material, which facilitates further processing of the generated ionized sample material based on the potential and electric field.

[0039] In various embodiments, the light source may be arranged and designed such that spatially resolved optical microscopy image information is recorded in a substantially shadow-free manner. In a heavily magnified image, the surface of the sample material, which may be covered by the matrix substance and which faces away from the sample carrier, may have a highly cracked appearance. Strong directional illumination of such a clearly structured surface may confuse automatic image evaluation algorithms due to shadows, since the actual topography can only be clearly identified on the side of the structure facing the incident light, while the areas in the shadow provide hardly any reference points for image-based feature recognition due to the small intensity differences and thus uniform appearance. The light source may be multi-part in that it has more than one light generator, the radiation from which is then combined for observing spatially resolved optical microscopy image information in the sample material, or it may have one light generator, in which case the light from which is spatially dispersed using one or more diffusers before being radiated onto the sample material.

[0040] In various embodiments, the light source may be annular and the observation axis of the transflective light optical system may be in a circumferential form. In particular, by being annular around the observation axis, the light may be incident on the reverse side of the sample carrier, so that observation points on a possibly highly structured sample material (e.g., image elements or pixels having an area of ​​0.01-1 square micrometers) may be illuminated by the light from an extended solid angle range and thus may be illuminated at a plurality of different angles of incidence. Thus, this configuration is able to avoid shadows and the associated adverse consequences described above.

[0041] In various embodiments, the light source may include a plurality of light emitting diodes. The light from each individual light emitting diode may include a mixture of different wavelengths that together form a white color effect. However, one or more light emitting diodes may also be configured so that they emit light with different non-white properties, for example, one or more of these complementary colors, red-cyan, green-magenta and blue-yellow. Monochromatic light is advantageous for optical configurations used for beam guidance because the optical components used do not need to handle an extended wavelength range, but can be optimized for narrowband use. Using light of different colors to record spatially resolved microscope image information may be advantageous for perceiving and automatically evaluating specific sample features. For example, using monochromatic light may make it easier to locate contrast or image clarity maxima in an optical image. All light emitting diodes may have the same design, for example, with white light properties. In a variant, light emitting diodes of different colors may also be juxtaposed, and if all light emitting diodes are activated and emit light, these light emitting diodes together produce light of a first color, for example, white, and if some light emitting diodes are not activated, these light emitting diodes together produce light of different colors, for example, green. The user can choose the color characteristics that are best suited to the planned experiment and the sample material used by selective switching, without having to perform complex modification operations on the device.

[0042] In various embodiments, the light source can be arranged and designed so that the second radiation does not pass through any imaging and / or deflecting optical components when it is incident on the sample carrier. This simplifies the setting of the transmissive and reflective light optical devices and enables the light source to be positioned directly close to the sample carrier or its support. The optical components here refer in particular to refractive and reflective (mirror) components, such as lenses, mirrors, optical fibers, etc. The microscope objective should also be considered as an optical component as a whole. In the context of the present disclosure, optical components should not be considered to include, for example, purely transparent plates made of glass, through which light or electromagnetic waves largely pass under paraxial conditions (i.e., at an angle that is only slightly different from the surface normal of the plate). In particular, in the context of the present disclosure, a sample carrier support that supports a sample carrier (for example in a transmissive light desorption optical system, where the first light beam is incident on the reverse side of the sample carrier) should not be considered as an optical component. A transparent sample carrier should also not be considered as an optical component in the context of the present disclosure.

[0043] In various embodiments, the device may further include a moving mechanism for the sample carrier, the moving mechanism being arranged and designed to move the sample carrier in at least one spatial direction relative to the incident direction of the first radiation and / or the second radiation. The moving mechanism may specifically include an xy translation stage, on which the sample carrier is placed, and the xy translation stage spatially displaces the sample carrier and the sample material placed thereon in two spatial directions x and y, the two spatial directions x and y extending substantially at right angles to the surface normal of the sample carrier and substantially at right angles to the direction of observation of the microscope. The moving mechanism is preferably also designed to spatially displace the sample carrier in a third spatial direction z that is at right angles to the above-mentioned spatial directions x and y. In particular, the moving mechanism may be designed and arranged to operate under reduced pressure. For example, the typical pressure that can be used for vacuum MALDI is substantially greater than high vacuum (>10 -3 hPa) and less than about 10 2 Hectopascals (<atmospheric pressure), which is, for example, 0.1-10 hectopascals. In the case of embodiments using vacuum MALDI, local desorption and ionization of sample material is performed in a gas-tight and continuously evacuated reduced pressure chamber, which is fluidly connected to the analyzer for forwarding the locally desorbed and ionized sample material.

[0044] In various embodiments, the operating mode of the computing unit, the desorption optical system and the transflected light optical system may include recording spatially resolved optical microscopic image information before and / or after the sample material is exposed to the first radiation. By particularly implementing a device with a transflected light optical system, which is adjacent to the reverse side of the ion source area and is used to observe and record spatially resolved optical microscopic image information from the sample material, optical images and molecular images can be detected efficiently in the same holder of the sample carrier, in the same installation, and in the same absolute position coordinate system. As a result, not only are the position coordinates of the corresponding image elements or pixels known very accurately, but they also correspond to each other for different modalities: optical microscopy and ion analysis. The only difference that may occur here is any erroneous steps caused by the movement mechanism in the case of repeated addressing of specific positions. However, there are available displacement stages that have a positioning accuracy in the double-digit nanometer range according to the manufacturer's specifications, such as the piezoelectrically operated displacement stages from the manufacturer SmarAct (Oldenburg, Germany), in particular the CLL, CLS and SLC series, which have a positioning accuracy of 40 nanometers. Furthermore, any errors that occur in the positioning can be reliably corrected by known monitoring methods and optional position correction of the kinematic mechanism.

[0045] In various embodiments, the device may further comprise an imaging objective lens arranged such that the light source is located between the sample carrier and the objective lens along the observation axis of the transmissive-reflective light optical system. The imaging objective lens may be designed to image a first radiation incident on the sample carrier and a second radiation after passing through the sample carrier and reflecting from the sample material. If a transmissive-light desorption optical system is used, the objective lens may particularly have dual imaging properties, since the objective lens first directs the light reflected back from the sample material through the sample carrier (which light is used to determine spatially resolved optical microscopy image information) to a camera for recording, and optionally adjusts the light, and secondly the objective lens adjusts the first radiation incident on the reverse side of the sample carrier and the sample material arranged on the other side, for example so that the focal spot size has a diameter in the range of a few micrometers or even lower.

[0046] In various embodiments, the light source and the imaging objective can be designed as an integral component. The light source is preferably integrated into a portion of the objective body facing the reverse side of the sample carrier. This space-saving configuration reduces the distance between the light source and the sample carrier and allows irradiation of the sample material with light emitted by the light source without additional beam-guiding and beam-forming optical components.

[0047] In various embodiments, the operation mode of the computing unit and the transmissive-reflective light optical system may include recording spatially resolved optical microscopic image information by sequentially scanning a plurality of xy single image areas on the sample material and by computationally combining the isolated xy single image information thus obtained. The single image information may be combined together based on the detected raw data to form an overall image covering the entire sample material. No prior image data processing is required, which may cause further inaccuracies in the optical microscopic data, which are then propagated through subsequent processing steps. In this way, the imaging quality of the entire optical image may be improved. Furthermore, for the combination, it is not necessary to provide overlapping areas of the single images and to perform subsequent feature matching in the overlapping areas, because the positions of the single image areas can be known very accurately by means of the high positioning accuracy of the motion mechanism.

[0048] In a second aspect, the present disclosure relates to an apparatus for multimodal analysis of sample materials, comprising: - a desorption optical system, which is arranged and designed to subject a sample material arranged on one side of a sample carrier to a first radiation and desorb the sample material into a gas phase, wherein the desorbed sample material is ionized; - an analyzer, which is arranged at a certain distance from the sample carrier and is designed to receive the desorbed and ionized sample material and process it into spatially resolved molecular image information; - a transmission and reflection light optical system, which is arranged and designed to record optical microscopic image information from the sample carrier and the sample material in a spatially resolved manner using a second radiation reflected through a transparent sample carrier, wherein the second radiation is emitted by a light source arranged on the sample carrier, a light source emitting on a side facing away from the sample material and designed to record spatially resolved optical microscopy image information in a substantially shadow-free manner, and - a computing unit arranged and designed to communicate with the desorption optical system, the analyzer and the transmissive reflection light optical system and to associate the spatially resolved molecular image information and the spatially resolved optical microscopy image information to give spatially resolved co-registered overall image information, wherein an operating mode of the computing unit and the transmissive reflection light optical system comprises recording the spatially resolved optical microscopy image information by sequentially scanning a plurality of xy single image areas on the sample material and by computationally combining the separated xy single image information obtained thereby.

[0049] In various embodiments, the operating mode of the computing unit and the transflective light optical system may include scanning a plurality of xy single image areas in a third spatial direction z to obtain a maximum contrast and / or image sharpness position. A preferred operating mode of the computing unit and the transflective light optical system may include using the maximum contrast and / or image sharpness position to (i) determine spatially resolved height profile information of the sample material above the sample carrier, and / or (ii) to combine an optical integral image having an image component from the z position of the respective maximum contrast and / or image sharpness position in each image element (focal stacking).

[0050] If the height of the sample material on the sample carrier varies significantly, it may be advantageous to adjust the ablation position during exposure of the sample material to the first radiation in order to maintain uniform desorption conditions over an extended area, e.g. in the case of microtomed tissue sections, of the order of square millimeters to square centimeters. The operating mode of the computing unit and the desorption optical system may therefore comprise adjusting (i) the focus of the first radiation and / or (ii) the position of the sample carrier in the third spatial direction z during exposure of the sample material using a maximum contrast and / or image sharpness position.

[0051] In a third aspect, the present disclosure relates to a method for multimodal analysis of sample materials, comprising: - locally exposing sample material arranged on one side of a sample carrier to a first radiation, and locally desorbing the sample material into a gas phase, wherein the locally desorbed sample material is ionized, - using an analyzer arranged at a certain distance from the sample carrier to receive and process the locally desorbed and ionized sample material to provide spatially resolved molecular image information, - using a second radiation reflected through a transparent sample carrier to record spatially resolved optical microscopic image information from the sample carrier and the sample material, wherein the second radiation is emitted by a light source, which is arranged on a side of the sample carrier away from the sample material and is designed so that the second radiation, when incident on the sample carrier, does not pass through any optical component through which the first radiation passes when incident on the sample carrier, and, - associating the spatially resolved molecular image information and the spatially resolved optical microscopic image information to provide spatially resolved, co-registered overall image information.

[0052] In a fourth aspect, the present disclosure relates to a method for multimodal analysis of sample materials, comprising: - locally exposing sample material arranged on one side of a sample carrier to a first radiation, and locally desorbing the sample material into a gas phase, wherein the locally desorbed sample material is ionized, - using an analyzer arranged at a certain distance from the sample carrier to receive and process the locally desorbed and ionized sample material to provide spatially resolved molecular image information, - using a second radiation reflected through a transparent sample carrier to record spatially resolved optical microscopy image information from the sample carrier and the sample material, wherein the second radiation is emitted by a light source, which is arranged on a side of the sample carrier away from the sample material and is designed to record the spatially resolved optical microscopy image information in a substantially shadow-free manner, and - associating the spatially resolved molecular image information and the spatially resolved optical microscopy image information to provide spatially resolved, co-registered overall image information, wherein the spatially resolved optical microscopy image information is recorded by sequentially scanning multiple xy single image areas on the sample material and by computationally combining the separated xy single image information obtained thereby.

[0053] In various embodiments, the apparatus described above may be used to perform the method according to the third and / or fourth aspect of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] For a better understanding of the present invention, reference is made to the following drawings. The elements in the drawings are not necessarily shown in true proportion, but are mainly intended to illustrate the principles of the present invention (mostly schematic). In the drawings, elements corresponding to each other in different views are marked with the same reference numerals.

[0055] FIG. 1 shows a setup of a tMALDI ion source with an integrated optical microscopy modality according to the prior art.

[0056] Figure 2A The desorption and ion generation regions (including connected optics) of a device according to the principles of the present disclosure are schematically shown in an isometric view.

[0057] Figure 2B Shown according to Figure 2A Cross-section of the desorption and ion generation regions in the first viewing direction.

[0058] Figure 2C Shown according to Figure 2B Cross-section of the desorption and ion generation regions at a second different viewing direction.

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

[0060] Figure 3B Shown according to Figure 3A Cross section of a microscope objective.

[0061] Figure 4 The creation of an overall optical microscopic image from multiple individual optical microscopic area images on a sample material is shown in accordance with the principles of the present disclosure.

[0062] Figure 5 A method based on focal stacking and determining a depth or profile map of a profiled sample material on a sample carrier is shown in accordance with the principles of the present disclosure.

[0063] Figure 6 It is shown that spatially resolved optical microscopy image information is correlated with spatially resolved molecular image information to give spatially resolved co-registered overall image information in accordance with the principles of the present disclosure.

[0064] Figure 7 A MALDI time-of-flight mass analyser is shown with a conventional setup suitable for implementing the principles of the present disclosure. DETAILED DESCRIPTION

[0065] While the present invention has been described and illustrated using several embodiments, those skilled in the art will recognize that various changes in form and details may be made without departing from the scope of the technical teaching defined in the following claims.

[0066] In the context of this specification, various central terms are used, which are briefly explained below with particular reference to optical or microscopic observations. A pixel / picture element refers to the smallest unit of an image. The pixel size in an optical / microscopic modality is usually smaller than the pixel size in the corresponding molecular image. The size of the sample material area imaged in an optical pixel depends on the sensor size of the camera used and the optical image properties, and can be, for example, 100×

[0067] 100 square nanometers. A single image is an optical image recorded by a camera using the viewing optics with a single shot of a single image area. Typical side lengths of such an area are 30-200 micrometers. An overall optical image is a mosaic-like combination of several single images to get an overview and may contain the entire sample material with side lengths of several millimeters or even centimeters. The terms are described in detail in the following paragraphs.

[0068] By means of the present invention, high-resolution optical images can be directly recorded in the ion source of a mass analyzer. This means that the absolute position coordinates for the optical microscopic image information of spatial resolution and the molecular image information of spatial resolution correspond to the laboratory position coordinate system, and are therefore identical or congruent. This essentially enables the ion image recorded in the same source to achieve almost perfect co-registration. The error can remain on the order of magnitude of less than 100 nanometers, which corresponds to a smaller precision than the commonly used mass analysis pixel side length (e.g., about 0.5-10 micron for tMALDI, about 5-100 micron for reflected light or reflection MALDI). In addition, by means of the newly introduced optical observation technology, sample morphology can be determined in a pixel-clear manner and with high resolution.

[0069] Figures 2A-2C A working example of an apparatus according to the principles of the present disclosure is schematically shown in various views and focuses on an ion source region. Figure 2A-2C Equipment for further processing of the ionized sample material is not shown. For ease of understanding, see Figure 7 , which will be further elaborated below.

[0070] Figure 2A A reduced pressure chamber 20 is shown which is hermetically sealed from the surrounding atmosphere and which can be maintained at a pressure of 10 -3 Up to 10 2Between hPa and 1000 Pa: Vacuum MALDI can be performed within this pressure range. For the sake of clarity, some of the side walls of the chamber 20 are omitted in the figure; only the rear wall 20-1 and the upper wall 20-2 and the lower wall 20-3 are visible. An xyz translation stage 22 is arranged in the chamber 20, on 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 larger opening 22-1, through which radiation is guided to be incident on the sample carrier 24 from the reverse side (i.e., the back of the surface on which the sample material 26 is placed). In this arrangement, the sample carrier 24 is located on the edge surrounding the opening 22-1.

[0071] Microscope objective 28 is mounted on rear wall 20-1 of chamber 20 between the rear wall 20-1 and stage 22 and extends to just in front of opening 22-1 in stage 22. Details of beam guiding components and optical components in objective 28 are not shown for clarity.

[0072] Outside the rear wall 20-1 of the chamber 20, two beam paths 30, 32 are shown, which are assigned firstly to the transmitted light desorption optics and secondly to the transmitted reflected light optics. The beam paths 30, 32 comprise a plurality of beam guiding and beam forming components, such as lenses and deflection mirrors, which ensure that the first radiation 30 required for desorption of the sample material 26 is guided to the sample carrier 24 and the second radiation 32 which is reflected back by the sample material 26 through the sample carrier 24 and is required for recording spatially resolved optical microscopy image information is guided to the camera 34.

[0073] In the upper wall 20-2 and the lower wall 20-3 of the chamber 20, two windows 36', 36" are shown, through which third radiation (e.g. a laser beam for post-ionization of the neutrally desorbed sample material) can be coupled into and out of the reduced pressure chamber 20 and focused into the desorption cloud above the exposed portion of the sample material 26. Details of this method, called MALDI-2, can be found, for example, in the article by Jens Soltwisch et al. (Science, 10 April 2015·Vol 348 Issue 6231, 211ff.) or in the study by M. Niehaus et al. mentioned in the introduction to the tMALDI setup.

[0074] The arrangement can be divided into two parts, or two beam paths 30, 32. With the aid of a dichroic mirror and / or a dielectric mirror 38, the observation beam path 32 and the laser beam path 30 are coordinated one above the other when traveling to and from the sample carrier 26; see Figure 2B and 2C. The coupling of radiation into and out of the reduced pressure chamber 20 is effected via corresponding windows 40 in the rear wall 20-1 of the chamber 20. By means of a UV-transparent objective lens 28 in the reduced pressure chamber 20, through which the observation beam path 32 and the laser beam path 30 pass, firstly a microscopic observation of the sample material 26 in transmitted and reflected light is possible and secondly a focusing of the laser beam 30 onto a beam waist in the submicrometer range is achieved on the front side of the sample carrier 24 carrying the sample material 26.

[0075] Both beam paths 30, 32 pass through the sample carrier 24; the observation beam path 32 originates from the interface of the sample material 26 with any matrix applied thereto and is imaged onto the light-sensitive chip of the observation camera 34. The depicted image portion can have a side length of 50-250 microns, in which practically no differences in contrast occur, and can have an optical resolution of 0.5-2 microns depending on the choice of the objective lens 28. The laser beam path 30 hits the opposite side of the same interface and is focused onto a small portion of the sample material 26. This portion corresponds to an image element or pixel for MS measurement, which has a size of, for example, 0.5-10 microns in diameter or side length. This configuration is therefore that of a transmission MALDI (tMALDI) setup.

[0076] The embodiment shown is based on the dual utilization of the microscope objective 28, both for observing the sample material and for ablating it. This duality enables an accurate determination of the ablation points on the generated optical image. With the aid of custom software, individual high-resolution microscope images can be combined here to give a large overview image. At the same time, the contrast of the microscope images of different object widths is analyzed, enabling the topography to be evaluated with high spatial precision.

[0077] Observation is performed with the aid of a camera 34 arranged along the surface normal of the sample carrier 26 and located at a distance from the sample carrier 26 in a region outside the decompression chamber 20 behind the sample carrier. The objective 28 is infinity corrected, so that the imaging lens 42 is positioned at a distance from the camera 34 corresponding to its focal length. When microscopic observation is performed in transflected light (reflection mode), i.e. light that has passed through the sample carrier and has been reflected back by the sample material 26, highly diffuse illumination is required to reduce shadow effects. In the present context, this illumination is achieved by an illumination ring consisting of a plurality of light emitting diodes (LEDs) 44.

[0078] In the example shown, 15 LEDs 44 are used. The number of LEDs can be less or greater. Light sources or LEDs with white light properties or monochromatic properties (e.g. green) can be used. Also conceivable are light sources or LEDs with variable color properties, which are, for example, adjustable over a wavelength range, or have mixed colors, in particular polychromatic but non-white color properties. The light-emitting diodes 44 are arranged in a cylindrical, axially protruding collar 46, which is attached to the objective body 28. * Combine to form Figure 3A-3B The overall assembly shown is annularly surrounding an optical observation axis 48 passing through the objective lens 28. The light emitting diodes 44 themselves are each accommodated in a cylindrical housing, which is embedded in a collar 46 in a manner that is not 0° and not 180° with respect to the optical observation axis 48 of the objective lens 28, so that the optical axes (dotted lines 50) of all diodes 44 are focused or converged on the optical observation axis 48, at a distance from the objective lens body 28. * The maximum illumination point 52 is located at a certain distance from the collar end of the objective lens 28. The maximum illumination point 52 is located on the sample carrier 24 so that the sample material 26 placed on its front side can be illuminated with high intensity and in a shadow-free manner in transmitted reflected light, and corresponds to the optimal working distance of the objective lens 28 and / or its focus.

[0079] The laser radiation 30 for ablating the sample material 26 is adjusted by means of a dichroic and / or dielectric mirror 38 to an imaginary portion of the sample material 26 coaxial with the observation beam path 32 through the objective 28. This means that the ablation position is fixedly coupled to a defined position on the observed image. A subsequent error-prone co-registration of the camera image and the ablation position is not necessary.

[0080] In order to create an optical overview image (scan) with high resolution, individual images 54 of a defined area 56 on the sample carrier 26 are recorded with high resolution and these images are combined in a mosaic-like manner to give an overall image 58. Figure 4 The corresponding principle is schematically illustrated in . To this end, the kinematic mechanisms of the camera 34 and the stage 22 are coordinated and synchronized on the software side. Subsequently, a mosaic image is constructed with the aid of the coordinates X:Y stored for each individual image 54. By precisely positioning and minimally processing the individual images 54, no distortion effects such as stretching, rotation, etc. are introduced. The positioning accuracy is limited by the accuracy of the kinematic mechanism of the stage 22 and the size of the optical pixels, i.e. depends on the scale factor, which is of the order of 100 nanometers or even lower. Since the coordinates of the kinematic mechanism remain constant in the laboratory position coordinate system used to record the optical image when scanning the sample material 26 and in the previous or subsequent quality analysis, an almost perfect co-registration of the optical and molecular overall images can be achieved without additional intervention or subsequent correction.

[0081] In a preferred embodiment, to determine the topography of a two-dimensionally extending sample material 26, a set of defined points X are first selected over the entire scan area (eg, the total area of ​​a tissue section). n :Y m These points can be fixed by the instrument using a pattern or can be set manually. An autofocus procedure is performed using these points. The technique used here 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 point on the sample material 26, the focal plane can be determined by moving from the selected point X to the selected point X. n :Y m The actual sample material height above the sample carrier 24 is calculated and introduced as a software-side z-value correction by interpolating and / or extrapolating from the reference point. This method is particularly suitable for correcting for topographic differences over large areas, which may occur when the sample carrier 24 is clamped in a dedicated holder.

[0082] In a specific embodiment, a method based on focus stacking is used. In this way, the topography of the sample material 26 can be determined with a high lateral resolution (e.g., several microns). Similar to the above-mentioned method of creating a whole optical image, a plurality of whole optical images are created for a defined z value by utilizing the movement space of the translation stage 22 in the z direction while keeping other settings of the transflective light optical system unchanged. Figure 5 Schematically indicated as z1, z2, z3 in . Depending on the expected sample morphology, these n values ​​may cover, for example, a range of ±10 micrometers around a starting value (eg, the average or expected value of the tissue section thickness) with a step size of 1 micrometer.

[0083] A Laplacian filter is applied to each image. The kernel size and the selective use of other filters depend on the optical configuration and the sample material 26 being observed. The results obtained from this step are used as local measures of contrast or image sharpness and are written into a three-dimensional matrix. In this case, the first and second dimensions correspond to the x and y positions on the sample material 26; the third dimension corresponds to the z plane of the base image. For each x and y position, the maximum of the resulting matrix is ​​determined in the third dimension. For each x and y position, the z index of the maximum is written into a two-dimensional matrix, which can be called a depth map or a contour map; see Figure 5 Thus, the depth map or contour map, optionally extended or supplemented by interpolation and / or extrapolation, contains the z-plane of highest image definition at any x and y position.

[0084] The resulting depth map or profile map can firstly be used to keep the ablation laser in focus in a pixel-precise manner during the subsequent MALDI-MSI analysis, for example by changing the focus position or by moving the sample carrier along the z-axis. On the other hand, a focus stack image can be constructed. Figure 5 In this case, the real pixel intensities of the recorded z-planes are combined pixel by pixel so that the resulting image only includes the image data of the respective sharpest z-plane at the corresponding xy position. This process, especially due to its continuous and sharp display, significantly facilitates the visual assessment of the overall optical microscopy image information and its visual matching with the molecular image information.

[0085] The above-described techniques significantly facilitate and improve the process and / or accuracy of tMALDI-MSI analysis with small pixel size. Figure 6 shows the co-registration of spatially resolved optical microscopy image information, which is represented by the overall optical image 58 in the upper left corner. * , where the individual image grid is identified by black bars and the individual image center coordinates are identified by X. For a particular translation stage position, the dimensions of each individual image and its position in the coordinate system are stored. Also shown is the spatially resolved molecular image information, represented by the ion image 60 in the lower left corner, which shows, for example, the intensity distribution of a particular ion or molecular species of interest, such as a protein, peptide, lipid, polynucleotide or polysaccharide. The information from the two modalities can then be correlated to give overall image information, represented by the composite image 62 on the right, which has been co-registered with high spatial resolution in the sub-micrometer range due to detection in the same coordinate system. The user can then visually evaluate the co-registered overall image information with the optical microscopy image component and the molecular image component, or subject it to a computer-assisted automatic evaluation procedure, for example for discovering feature correlations in data from various modalities.

[0086] Figure 7 A schematic diagram of a substantially known MALDI time-of-flight mass analyzer in an axial reflectron setup is shown, in which the principles of the present disclosure can be used. The sample material is present on a sample carrier plate 71 opposite accelerating electrodes 72 and 73 and can be accelerated by lasers 75, 75 * The laser pulse beam 74, 74 provided *Ionization is performed in a spatially resolved manner in reflected light or transmitted light. Ions are accelerated by accelerating electrodes 72 and 73 to produce an ion beam 78, which passes through a gas chamber 79 (which can be filled with collision gas as needed), a precursor ion selector 80, a fragment ion post-acceleration unit 81 and a precursor ion suppressor 82, and is then reflected by a reflector 83 onto an ion detector 84. The housing of the mass analyzer is evacuated by a high-power vacuum pump 85. The ion detector 84 can have a multi-channel plate as an ion receiver. The transmission-reflection optical system according to the principles of the present disclosure can be integrated into the rear portion behind the sample carrier 71.

[0087] The present invention has been described above with reference to various specific working examples. However, it is apparent that various features or details of the embodiments described may be changed without departing from the scope of the present invention. In addition, if a person skilled in the art considers it feasible, the features and measures disclosed in association with different embodiments may be combined as needed. In addition, this specification is intended only to illustrate the present invention and is not intended to limit the scope of protection, which is determined solely by the appended claims for any existing equivalents.

Claims

1. A device for multimodal analysis of sample materials, comprising: a desorption optical system arranged and designed to subject a sample material arranged on one side of the sample carrier to a first radiation and to desorb the sample material into a gas phase, wherein The desorbed sample material is ionized, - an analyzer, which is arranged at a distance from the sample carrier and is designed to receive the desorbed and ionized sample material and process it into spatially resolved molecular image information, a transflective light optical system arranged and designed to record optical microscopic image information from the sample carrier and the sample material in a spatially resolved manner 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 any optical component through which the first radiation passed when incident on the sample carrier, and - A computing unit arranged and designed to communicate with the desorption optical system, the analyzer and the transflected light optical system and to correlate the spatially resolved molecular image information and the spatially resolved optical microscopy image information to give spatially resolved co-registered overall image information.

2. The device according to claim 1, wherein: The desorption optical system has a transmitted light optical system which is arranged and designed in such a way that the first radiation is incident on the sample material after passing through the sample carrier.

3. The device according to claim 2, wherein: Using dichroic mirrors and / or dielectric mirrors, the observation axis of the transflective light optical system and the optical axis of the first radiation incident on the sample carrier are coordinated one above the other. 4 . The device according to claim 1 , which is designed to use a conductively coated glass plate as sample carrier.

5. The device according to any one of claims 1 to 4, wherein: The light source is arranged and designed such that spatially resolved optical microscopic image information is recorded in a substantially shadow-free manner.

6. The device according to any one of claims 1 to 5, wherein: The light source is annular, and the observation axis of the transflective light optical system is in a surrounding form.

7. The device according to any one of claims 1 to 6, wherein: The light source includes a plurality of light emitting diodes.

8. The device according to any one of claims 1 to 7, wherein: The light source is arranged and designed such that the second radiation, when incident on the sample carrier, does not pass through any imaging and / or deflection optical components.

9. The device according to any one of claims 1 to 8, further comprising a movement mechanism for a sample carrier, wherein the movement mechanism is arranged and designed to move the sample carrier in at least one spatial direction relative to an incident direction of the first radiation and / or the second radiation.

10. The device according to any one of claims 1 to 9, wherein: The operating mode of the computing unit, the desorption optical system and the transflective light optical system comprises recording spatially resolved optical microscopic image information before and / or after exposure of the sample material to the first radiation.

11. A device according to any one of claims 1 to 10, further comprising an imaging objective which is arranged in such a way that the light source is located between the sample carrier and the objective along the observation axis of the transflective light optical system.

12. The device according to claim 11, wherein The imaging objective is designed to image a first radiation incident on the sample carrier and a second radiation after passing through the sample carrier and reflecting from the sample material.

13. The device according to claim 11 or 12, wherein: The light source and the imaging objective are designed as an integral component.

14. The device according to any one of claims 1 to 13, wherein: The operating mode of the computing unit and the transflective light optical system comprises recording spatially resolved optical microscopic image information by sequentially scanning a plurality of xy individual image areas on a sample material and computationally combining the separate xy individual image information obtained thereby.

15. An apparatus for multimodal analysis of a sample material, comprising: a desorption optical system arranged and designed to subject a sample material arranged on one side of the sample carrier to a first radiation and to desorb the sample material into a gas phase, wherein The desorbed sample material is ionized, - an analyzer, which is arranged at a distance from the sample carrier and is designed to receive the desorbed and ionized sample material and process it into spatially resolved molecular image information, a transflective light optical system arranged and designed to record optical microscopic image information from the sample carrier and the sample material in a spatially resolved manner using a second radiation reflected through the transparent 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 the light source is designed to record the spatially resolved optical microscopic image information in a substantially shadow-free manner, and a computing unit arranged and designed to communicate with the desorption optical system, the analyzer and the transflected light optical system and to correlate the spatially resolved molecular image information and the spatially resolved optical microscopy image information to give spatially resolved co-registered overall image information, Therein, the operation mode of the computing unit and the transflective light optical system includes recording spatially resolved optical microscopic image information by sequentially scanning a plurality of xy single image areas on the sample material and computationally combining the separated xy single image information obtained thereby.

16. The device according to claim 14 or 15, wherein: The operating mode of the computing unit and of the transflective light optical system comprises scanning a plurality of xy individual image areas in a third spatial direction z to obtain a position of maximum contrast and / or image sharpness.

17. The device according to claim 16, wherein: The operating modes of the computing unit and the transmitted-reflected light optical system include using the maximum contrast and / or image clarity positions (i) to determine spatially resolved height profile information of the sample material above the sample carrier, and / or (ii) to combine an optical integral image having in each image element an image component from the z position of the respective maximum contrast and / or image clarity position (focal stacking).

18. The device according to claim 16 or 17, wherein: The operating mode of the computing unit and the desorption optical system comprises using the maximum contrast and / or image sharpness position to adjust (i) the position of the focus of the first radiation, and / or (ii) the position of the sample carrier in a third spatial direction z during exposure of the sample material.

19. A multimodal analysis method for a sample material, comprising: - locally exposing a sample material arranged on one side of the sample carrier to a first radiation and locally desorbing the sample material into a gas phase, wherein the locally desorbed sample material is ionized, - receiving and processing the locally desorbed and ionized sample material using an analyzer arranged at a distance from the sample carrier to give spatially resolved molecular image information, - recording spatially resolved optical microscopic image information from the sample carrier and the sample material using 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 any optical components through which the first radiation passed when incident on the sample carrier, and - Correlating the spatially resolved molecular image information and the spatially resolved optical microscopy image information to give spatially resolved co-registered overall image information.

20. A multimodal analysis method for a sample material, comprising: - locally exposing a sample material arranged on one side of the sample carrier to a first radiation and locally desorbing the sample material into a gas phase, wherein the locally desorbed sample material is ionized, - receiving and processing the locally desorbed and ionized sample material using an analyzer arranged at a distance from the sample carrier to give spatially resolved molecular image information, - recording spatially resolved optical microscopy image information from the sample carrier and the sample material using 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 optical microscopy image information is recorded in a substantially shadow-free manner, and - correlating the spatially resolved molecular image information and the spatially resolved optical microscopy image information to give spatially resolved co-registered overall image information, in, Spatially resolved optical microscopy image information is recorded by sequentially scanning a plurality of xy individual image areas on the sample material and computationally combining the separate xy individual image information obtained thereby.

21. The method according to claim 19 or 20, which is carried out using a device according to any one of claims 1 to 18.

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