Methods for analyzing sample material

Abstract

The invention relates to methods and systems for detecting spatially resolved measurement data of different modalities, said data relating to a sample material which is provided on a sample support, in particular to the partly simultaneous detection of such measurement data, said methods and systems specifically facilitating and improving the co-registration of measurement data of different modalities, i.e. the spatial arrangement and alignment thereof in the same coordinate system. The methods and systems according to the invention can be advantageously applied to the area of ion spectrometry imaging, e.g. time-of-flight mass analysis imaging of tissue sections, in particular using matrix-assisted ionization and also in particular ionization with matrix-assisted laser desorption (matrix-assisted laser desorption and ionization, MALDI).

Description

Method and system for analyzing sample material Field of the invention

[0001] The disclosure relates to methods and systems for acquiring spatially resolved measurement data from different modalities of sample material arranged on a sample carrier, in particular the partially simultaneous acquisition of such measurement data, and specifically facilitates and improves the co-registration of the measurement data from the different modalities, i.e., their spatial arrangement and alignment in the same coordinate system. The methods and systems according to the disclosure can find advantageous applications in imaging ion spectrometry, e.g., imaging time-of-flight mass analysis of tissue sections, in particular using matrix-assisted ionization and, furthermore, in particular, ionization with matrix-assisted laser desorption (matrix-assisted laser desorption and ionization, MALDI). Background of the invention

[0002] The prior art is explained below with reference to a specific aspect. However, this should not be construed as a limitation. Useful developments and modifications of what is known from the prior art 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 following disclosure.

[0003] Mass spectrometry imaging (MSI) using matrix-assisted laser desorption ionization represents a rapidly growing technical field for the spatial visualization of molecular distributions, e.g., in tissue sections and cell cultures. Correlative analysis with other imaging techniques such as light microscopy requires the most accurate co-registration of the modalities used. Currently, this co-registration is achieved through independently acquired images. As a result, these techniques typically exhibit significant inaccuracies at the micrometer level.

[0004] Currently, fluorescence microscopy, a special type of light microscopy, and MALDI-MSI are coupled using independently acquired measurements. Normally, these measurements are performed with two completely independent devices. For this purpose, microscopy can take place before, after, or before and after the MALDI-MSI measurement. For the co-registration of optical and mass spectrometry-based imaging, a transformation is defined that combines both modalities using a previously defined metric (see Reference 1). For this purpose, the ablation craters in the sample material that arise during the MALDI-MSI measurement can be used (see Reference 2). In special applications, the two modalities can be recorded with technically linked but separate devices (see Reference 3) or partially using the same device (see Reference 4). Here, co-registration is achieved using precise knowledge of the measurement location of both modalities, which can be identical in design.In other studies, ionization-accompanying fluorescence was used to elucidate the MALDI process (see reference 5) and to determine laser spot sizes on the sample material (see reference 6).

[0005] Literature references:

[0006] (1) Balluff, B.; Heeren, R.M.A.; Race, AM: An Overview of Image Registration for Aligning Mass Spectrometry Imaging with Clinically Relevant Imaging Modalities. J. Mass Spectrom. Adv. Clin. Lab 2022, 23, 26-38. https: / / doi.org / 10.1016 / j.jmsacl.202L 12,006.

[0007] (2) Patterson, N.H.; Tuck, M.; Van de Pias, R.; Caprioli, RM: Advanced Registration and Analysis of MALDI Imaging Mass Spectrometry Measurements through Autofluorescence Microscopy. Anal. Chem. 2018, 90 (21), 12395-12403. http s: / / doi. or g / 10.1021 / acs . anal chem, 8 b 02884.

[0008] (3) Shimma, S.; Kumada, H.-O.; Taniguchi, H.; Konno, A.; Yao, I.; Furuta, K.; Matsuda, T.; Ito, S.: Microscopic Visualization of Testosterone in Mouse Testis by Use of Imaging Mass Spectrometry . Anal. Bioanal. Chem. 2016, 408 (27), 7607-7615. htt s: / / d0i.0rg / T 0, 1007 / s00216-016-9594-9.

[0009] (4) Soltwisch, J.; Potthoff, A.; Schwenzfeier, J.; Bien, T.; Niehaus, M.; Dreisew- erd, K. : Transmission-Mode MALDI -2 Ion Source for Online Co-Registration of MS and Optical Images with Sub-Micrometer Precision,' Uppsala, Sweden, 2023; Regional Mass Spectrometry Imaging Spring Workshop 2023.

[0010] (5) Jaskolla, T. W.; Karas, M.: Using Fluorescence Dyes as a Tool for Analyzing the MALDI Process. J. Am. Soc. Mass Spectrom. 2008, 19 (8), 1054-1061. http s : / / doi . org / 10, 1016 / j . j asms .2008.04.032.

[0011] (6) Steven, R. T.; Palmer, A. D.; Bunch, J.: Fluorometric Beam Profiling of UV MALDI Lasers. J. Am. Soc. Mass Spectrom. 2013, 24 (7), 1146-1152. https: / / doi.org / 10.1007 / sl3361-013-0650-9.

[0012] Patent publication WO 2023 / 205280 A1 describes methods for enhancing the fluorescence intensity of a sample by applying an aromatic compound. It also describes workflows for a combined fluorescence MALDI microscopy / imaging instrument, combining MALDI imaging and fluorescence imaging of the same sample in a single sample preparation step. The described workflow is intended to reduce sampling time to one working day and minimize sample degradation. In the scientific literature, the technical teaching of the patent publication is referred to by the scientists as "FluoMALDI."

[0013] The state of the art in standard applications requires the acquisition of multiple microscopy images, making it complex to implement and leading to a relatively high variance in the co-registration of optical and ion spectrometry-based images. Strategies for linking the machine components for a direct connection of the modalities are complex and cannot be implemented on all devices, and they require considerable technical effort.

[0014] The poster presentation by Alexander Fengier et al. at the 54th Annual Meeting of the German Society for Mass Spectrometry (DGMS), held from May 14-17, 2023, in Dortmund, entitled "Duo-modal mass spectrometric and emission spectroscopic recordings of 2,5-dihydroxybenzoic acid on cerebellar tissue," discusses the parallel measurement of mass spectrometric and fluorescence spectroscopic data from brain tissue, particularly to compare the spatial distribution of m / z mass signals with fluorescence data from the tissue. To the best of the inventors' knowledge, this work is the first to utilize a fluorescence response generated during the ionization process for confocal microscopy.

[0015] In view of the above, there is a need to combine an ion spectrometric modality with a high-resolution fluorescence-based Modality in the imaging analysis of sample material, especially when these measurements are performed in different sample wells with different sample carrier clampings. 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

[0016] The invention enables the simultaneous and spatially identical or co-localized acquisition of fluorescence response data, e.g., fluorescence spectroscopic data generated during pulsed material ablation for the ion spectrometric analysis of each pixel or image element under investigation, e.g., using MALDI. In particular, this can include the spatially and temporally simultaneous acquisition of confocal hyperspectral fluorescence response images and a MALDI-MSI measurement.

[0017] In the method described here, fluorescent light generated by the local application of energy bursts to the sample material during ion spectrometric analysis, e.g., as pulsed laser light during MALDI analysis, can be used to create optical imaging that has substantially identical spatial coordinates and spatial resolution to ion spectrometric analysis. Specific fluorescent dyes can also be used for this purpose, which can be added to the sample material prior to measurement.

[0018] The invention utilizes the fluorescence response that arises in a spatially and temporally dependent manner during an ion spectrometric measurement aimed at the molecular content of the sample material, e.g., a MALDI ionization followed by time-of-flight mass analysis. A fluorescence spectrum or the intensity of a defined wavelength range of emission can be recorded for each irradiated image element or pixel, e.g., in a so-called fluorescence channel. Restricting the measurement to a limited wavelength range can provide the advantage of removing or at least attenuating interfering influences of the excitation light as well as any intrinsic fluorescence emission of a matrix substance from the fluorescence response detection.

[0019] Based on these data, co-localized and spatially precisely linked confocal hyperspectral images can be generated with the ion spectrometric measurement. Material removal for ion spectrometry, e.g., MALDI time-of-flight mass spectrometry, and excitation of the fluorescence response are performed at the same location and by the same Initiated by an energy burst, e.g., by laser irradiation as in MALDI, they are thus inherently linked both spatially and temporally. Spatially lateral in this context means the location of the local sampling of the sample material in the xy plane spanned by the sample carrier or a plane parallel to it, e.g., formed by a tissue section on the sample carrier.

[0020] To generate a fluorescence signal in the sample material, e.g., tissue or cell culture, fluorescent dyes can be used, particularly during sample preparation, whose excitation wavelength range coincides or overlaps with the wavelength of the energy-boosting laser. For lasers used in UV-MALDI, e.g., at 337 nanometers or 355 nanometers, UV-active fluorescent dyes such as Hoechst 33342, DAPI (4',6-diamidine-2-phenylindole), or Calcein Blue are suitable. The fluorescent light generated during the ionization process can either be purified and detected using suitable filters or examined and evaluated spectroscopically. The spatial coordinates of the image elements or pixels of the confocal microscopy image generated in this way correspond exactly to those of the scanned ion spectrometric measurement.

[0021] It should be noted here that an image element or pixel in an imaging ion spectrometric analysis represents the smallest area unit of the sample material whose molecular information is spatially resolved and can be displayed. Typical dimensions of these area units in terms of diameter (e.g., for a circular laser spot) or edge length (e.g., for a rectangular cut) can be selected from the group comprising or consisting of: 1 to 10 micrometers, 10 to 100 micrometers, 100 to 1000 micrometers, 1 to 100 micrometers, 1 to 1000 micrometers, 10 to 1000 micrometers, or any other suitable range between 1 and 1000 micrometers.The size of a laser spot can be selected to be smaller than the size of the image element or pixel, and the molecular information can be captured from such an image element or pixel by repeatedly exposing the sample material to the pulsed laser beam at slightly different locations within the boundaries of the image element or pixel. The molecular information from the individual samples is then accumulated or summed for the image element or pixel. It is understood that such an approach can, in principle, generate fluorescence response data with a tighter spatial grid than that of ion spectrometric measurement data, which contain the molecular information from several, closely adjacent sampling locations. However, the Fluorescence response data can be mapped to the image element or pixel grid of the ion spectrometric measurement using simple mathematical operations such as addition. It is also possible to design the fluorescence response data acquisition itself so that data from the simultaneously acquired modalities are not recorded in different spatial grids.

[0022] The image elements or pixels generated with the described setup are generally significantly larger than those that can be produced with a modern fluorescence microscope. The fluorescence response image generated with the described invention simultaneously with the ion spectrometric measurement is therefore primarily intended to serve as a link between MSI and high-resolution optical modalities. While co-registration of different modalities acquired at different resolutions involves a higher degree of error propagation, it can be performed significantly more easily and precisely using the same acquisition modality but with different image element or pixel sizes.The resulting transformation, which relates and maps the positional coordinates of the various modalities to one another, can be directly applied to the MSI results due to the inherent coupling described above, thus enabling precise and simple co-registration of high-resolution fluorescence images and MSI. High-resolution fluorescence microscopy is usually performed before the sample material is prepared with matrix substance. However, it is also possible to acquire a high-resolution fluorescence image after the ion spectrometric measurement or in addition to a high-resolution fluorescence image acquired before the ion spectrometric measurement, e.g., for process control and / or quality assurance purposes.

[0023] The measurement of fluorescent light intensity, either using specific filters or spectroscopically, can be retrofitted to most existing MSI systems with comparatively little technical effort. The measurement is performed simultaneously and at the same time as the ion spectrometric measurement and requires no additional measurement time or additional energy bursts, such as laser pulses. It is more accurate, faster, and more resource-efficient than existing technical solutions.

[0024] In view of the above explanations, the invention relates, according to a first aspect, to a method for analyzing sample material arranged on a sample carrier and comprising at least one fluorescence-capable substance, comprising the steps of: (a) acquiring spatially resolved, ion spectrometric data from the sample material using energy bursts that are designed and suitable to locally To trigger a fluorescence response of the at least one fluorescence-capable substance on the sample material, and simultaneously acquire spatially resolved fluorescence response data from the sample material having a first spatial resolution; (b) acquire fluorescence image data from the sample material having a second spatial resolution that is substantially greater than the first spatial resolution, and wherein step (b) can be carried out in particular before and / or after step (a); and (c) comparing and aligning the spatially resolved fluorescence response data and the fluorescence image data.

[0025] The conditions for exciting a fluorescence response in confocal fluorescence detection during ion spectrometric measurement, on the one hand, and in separate, high-resolution image acquisition, on the other, differ. For example, a burst of energy ablates material, which naturally limits the time between excitation and quenching of the fluorescence response. In contrast, in a high-resolution microscope, for example, the excitation remains below an ablation threshold and thus preserves the sample material. A fluorescence response can, in principle, be excited over a longer period, e.g., to enhance a cumulative fluorescence response.Nevertheless, it has unexpectedly been shown that the fluorescence response is sufficiently uniform and reliably detectable even under strongly differing excitation conditions, so that spatial mapping with increased precision and in an automated manner becomes possible.

[0026] Since the measurement data are acquired with respect to the different modalities, such as ion spectrometry and simultaneous confocal fluorescence detection, in the same clamping of the sample carrier, so that the spatial coordinates of the measured signals are co-localized and exactly match, and since the same response behavior is observed with respect to the same modalities, such as confocal fluorescence detection during ion spectrometry and the separately acquired, high-resolution fluorescence image, the ion spectrometric data and the high-resolution fluorescence image can be spatially related to each other automatically with increased precision using the confocal fluorescence response data. This procedure allows for reference marks on the sample material, such asFor example, cracks in tissue or craters in the ablation zone are not suitable for co-registration, as their localization often cannot be performed with the same accuracy in the different modalities, which can cause or even worsen co-registration errors. Furthermore, a distribution of at least one fluorescent substance across the entire sample material allows for targeted detection. Staining by a user, for example if cell nuclei are appropriately labelled throughout a tissue, the acquisition of data in potentially every analytically interesting surface area of ​​the sample material, so that a high quality of co-registration can be ensured across the entire sample material.

[0027] In principle, it is possible to acquire the ion spectrometric data, the fluorescence response data, and the high-resolution fluorescence image data using the same clamping of the sample carrier carrying the sample material. This is possible, for example, if a high-resolution fluorescence signal receiver is coupled to or integrated into the ion generation region and used to acquire both the fluorescence response data and the fluorescence image data. An example of such a setup is illustrated in the applicant's patent publication WO 2024 / 041681 A1; only devices for material-friendly excitation of fluorescence emission would have to be added.A measurement sequence can be designed such that an actuator moves the sample carrier carrying the sample material into a specific ablation position, then first excites material-protective fluorescence emission in a limited surface section of the sample material and records it with high spatial resolution, whereupon the corresponding surface area of ​​the sample material is subjected to energy bursts for material ablation, the resulting ionized sample material is recorded by ion spectrometry, and simultaneously the fluorescence response from the surface area of ​​the sample material is detected. The measurement series can then be continued by the actuator moving the sample carrier carrying the sample material to the next ablation position and the previously described cycle being repeated on a different surface area, for example, until all available surface areas have been sampled.In this version, the fluorescence response data can be used for process control and quality assurance.

[0028] In various embodiments, the spatially resolved fluorescence response data can contain a first spatial frequency distribution of the at least one fluorescing substance across the sample material, and the fluorescence image data can contain a second spatial frequency distribution of the at least one fluorescing substance across the sample material, wherein the smallest possible deviation between the first spatial frequency distribution and the second spatial frequency distribution is sought for the comparison and alignment. The frequency, intensity, or abundance distribution of the fluorescence response of the at least one fluorescing substance, which is measured with the same measurement modality at different The measurement with spatial resolution allows the alignment of position coordinates from data sets obtained, for example, with different sample holder clamping arrangements. The evaluation can consider the frequency distribution uniformly across the entire fluorescence image data and the fluorescence response data. Alternatively, it is possible to divide the total area from which the respective data originate into area sections, for which the frequency distributions are then evaluated individually. The results from the area sections can subsequently be combined into a unified overall picture using suitable algorithms, with particular attention to continuity.

[0029] The first spatial resolution can be in the range of micrometers, e.g. selected from the group comprising or consisting of: between 1 and 10 micrometers, between 10 and 100 micrometers, between 100 and 1000 micrometers, between 1 and 100 micrometers, between 1 and 1000 micrometers, between 10 and 1000 micrometers, and any other range between 1 and 1000 micrometers. The second spatial resolution can be in the range of nanometers, e.g. selected from the group comprising or consisting of: between 1 and 10 nanometers, between 10 and 100 nanometers, between 100 and 1000 nanometers, between 1 and 100 nanometers, between 1 and 1000 nanometers, between 10 and 1000 nanometers, and any other range between 1 and 1000 nanometers.Preferably, the second spatial resolution in units of length differs from the first spatial resolution by a factor of 1 / n, where n is selected from the group comprising or consisting of: >2, >3, >4, >5, >6, >7, >8, >9, >10, >15, >20, >30, >40, >50, >100, >1000, >10000, >100000, 1000000, and any other integer or real number greater than two. To illustrate the factor: if the first spatial resolution in units of length is 10 micrometers and the second spatial resolution is 10 nanometers, this corresponds to n=1000, i.e. a factor of one thousandth.

[0030] In various embodiments, a tissue section or a cell culture can be used as sample material. The sample material on the sample carrier can comprise a tissue section, e.g., provided from a frozen block or as an FFPE (formalin fixed paraffin embedded) prepared block, in particular using microtomy. The tissue section can be cut to a thickness of approximately 7-20 micrometers, whereby subsequent drying of the moist tissue can make the final preparation significantly thinner. The sample material can also comprise a field or a plurality of single or spot preparations on the sample carrier, e.g., a field or a multitude of individual cells. A single or spot preparation can, in particular, comprise a dried spot of liquid or reagent containing sample material, e.g., in the form of cells or analyte molecules. It is also possible to provide individual cells on the sample carrier as sample material. The individual cells can be cultured or grown directly on the sample carrier. Individual cells can, for example, belong to prokaryotes (e.g., human, animal, plant, protist cells, or fungal cells) or eukaryotes (e.g., bacterial or archaeal cells).

[0031] The sample carrier can be plate-shaped. The sample carrier can have the dimensions of a standard microtitration plate, e.g., 127.71 millimeters long, 85.43 millimeters wide, and 14.10 millimeters thick. The material of the sample carrier can be conductive. For example, it can comprise a glass, ceramic, or plastic substrate with a conductive coating on a surface supporting the sample material. Examples include indium tin oxide-coated glass slides. The partial electrical conductivity of the sample carrier allows the generation of an electrical reference potential, which is particularly useful when handling the ions formed during ablation events.

[0032] In various embodiments, the spatially resolved fluorescence response data and the fluorescence image data can originate from at least one extrinsic fluorescent substance, e.g., Hoechst 33342, DAPI, or Calcein Blue, which is added to the sample material prior to acquiring the ion spectrometric data, the fluorescence response data, and the fluorescence image data. Preferably, the fluorescent substance exhibits good excitation properties in the ultraviolet spectral range, particularly to be compatible with UV-MALDI, e.g., between 300 and 400 nanometers, and especially between 330 and 360 nanometers, and upon excitation, emits fluorescent light in a wavelength range shifted to lower energies, such as the visible wavelength range, e.g., between 400 and 800 nanometers, especially between 450 and 650 nanometers.

[0033] The fluorescent dye Hoechst 33342 has several advantageous properties. It is specific for DNA (deoxyribonucleic acid) because it binds preferentially to adenine-thymine (AT) regions of DNA. This enables specific staining of nuclei in living or fixed cells and tissues. Its fluorescence properties ensure that it is excited by UV light and emits light in the spectrum from blue to cyan. This enables the visualization of DNA using fluorescence spectroscopic or microscopic means. Hoechst 33342 is also compatible with antibodies and other probes that labeled with fluorescein and rhodamine dyes. This allows for the simultaneous visualization of DNA and specific cellular targets.

[0034] An advantageous property of DAPI is that its bleaching can be delayed by the use of so-called anti-fading agents, which extends the light-optical observation time, e.g., when acquiring high-resolution fluorescence image data.

[0035] In various embodiments, the energy bursts can be triggered by a pulsed laser, which, for example, emits laser pulses in the ultraviolet spectral range. The clock 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 4Hertz. The laser pulses can preferably be applied locally to the sample material in reflected or transmitted light. Irradiation in reflected light allows the use of opaque sample holders and the analysis of thick sample material. Irradiation in transmitted light allows the ion generation region to be designed largely free of light-optical components for detecting a fluorescence response, and the laser spot can be focused to diameters in the low, e.g., single-digit, micrometer range.

[0036] In various embodiments, the sample material can be prepared prior to step (a) with a matrix substance suitable for matrix-assisted ionization, in particular matrix-assisted laser desorption and ionization (MALDI). Examples of UV-sensitive MALDI matrix substances are 2,5-dihydroxyacetophenone (DHAP), 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (HCCA), or sinapic acid (SA). MALDI has the advantage of ionizing analyte molecules in their native state and predominantly imparts the charge state z=1, which simplifies ion spectrometric handling and analysis. For applying the matrix substance to the sample material, methods that produce a uniform layer thickness and few crystal irregularities are preferred. One example of such a method is (re-)sublimation; see in particular the work of Joseph A. Hankin et al. (J Am Soc Mass Spectrom.2007 September; 18(9): 1646-1652, Sublimation as a Method of Matrix Application for Mass Spectrometric Imaging). If step (b) of the method is carried out before step (a) of the method, the matrix substance can be applied before step (b), e.g., if the acquisition of the ion spectrometric data, the fluorescence response data, and the fluorescence image data takes place in the same clamping of the sample carrier and / or in the same sample well, or between step (b) and step (a).

[0037] In various embodiments, the fluorescence response can comprise fluorescent light emission from the at least one fluorescing substance, which is detected light-optically, e.g., using a microscope. For this embodiment, a microscope is preferably coupled to or integrated into the ion generation region, e.g., a MALDI source. The fluorescent light emission can be detected light-optically in the form of a broadband spectrum or narrowband wavelength-Z frequency range, e.g., after filtering. Covering an extended wavelength range provides greater sensitivity to changes in the fluorescence response.A narrowband range, in turn, has the advantage that light-optical assemblies used for collecting and detecting fluorescent light can be optimized for reception of this wavelength. It also enables sharper discrimination against energy bursts for excitation of the fluorescence response, as well as against any intrinsic fluorescence emission that can occur in some matrix substances upon laser irradiation. A narrowband range AA = Amax - Amin can be selected from the group comprising or consisting of: 5 nanometers, 10 nanometers, 20 nanometers, 30 nanometers, 40 nanometers, 50 nanometers, and any other suitable range between 5 and 50 nanometers.

[0038] In various embodiments, the ion spectrometric data can include a third spatial frequency distribution of at least one molecule of interest in the sample material, in particular selected from the group comprising or consisting of: proteins, peptides, lipids, saccharides, nucleotides, metabolites, and biopolymers. The third spatial frequency distribution can take the form of an ion distribution map that represents the intensity or abundance of an analyte molecule from the sample material across the sample material. Such distribution maps can be created for a multitude of analyte molecules in the sample material. The distribution maps allow, for example, the investigation of physiological, biological, and / or metabolic processes in the sample material.

[0039] In various embodiments, the second spatial resolution of the fluorescence image data and the first spatial resolution of the fluorescence response data can be adjusted to each other for the purpose of matching and aligning, e.g., by downscaling the second spatial resolution. In a simple example, the image data from different image elements or pixels can be aligned by summing the wavelength-resolved and fluorescence-specific intensity information in surface areas of the fluorescence image data. corresponding to a picture element or pixel of the fluorescence response data, optionally using mathematical operators applying interpolation, extrapolation and / or regression.

[0040] In various embodiments, a result of the alignment and aligning can be used to spatially co-register the ion spectrometric data and the fluorescence image data, e.g., using a transformation matrix, polynomial fitting, or a vector field. Images from different modalities, e.g., a light-optical or microscopic image of fluorescent light and an ion distribution map, can be computationally merged (image fusion), thus providing more information about the sample material than an image or map from a single modality could. Examples of this can be found in the applicant's patent publication DE 10 2008 023 438 A1 and in the study by Raf Van de Pias et al. (Nature Methods, Vol. 12 No. 4, April 2015, pp. 366-372).

[0041] In various embodiments, the spatially resolved fluorescence response data can be acquired in reflected light from and / or transmitted light through the sample carrier. Acquisition in reflected light allows the use of opaque sample carrier holders and the examination of thick sample material. Acquisition in transmitted light keeps the ion generation region free of light-optical assemblies for collecting and guiding fluorescent light and enables optical observation with a high numerical aperture and along an observation axis oriented perpendicular to the sample carrier, thus allowing observation of the fluorescence response with fewer geometric influences.

[0042] According to a second aspect, the invention relates to a system for analyzing sample material arranged on a sample carrier and comprising at least one fluorescence-capable substance, comprising: - an ion analyzer arranged and configured to acquire spatially resolved, ion spectrometric data from the sample material; - a fluorescence response receiver arranged and configured to acquire spatially resolved fluorescence response data from the sample material; - a fluorescence image receiver arranged and configured to acquire spatially highly resolved fluorescence image data from the sample material; and - a processing device that is in data communication with the ion analyzer, fluorescence response receiver, and fluorescence image receiver and is arranged and programmed to carry out a method as described above.

[0043] In various embodiments, the ion analyzer can comprise a mass analyzer, a mobility analyzer, or a combined mobility-mass analyzer. Mass analysis can be the final analytical step to which the ionized sample material is subjected. Ion-conducting intermediate stages can be arranged upstream of the actual mass analyzer or the multiple analyzers connected in series, and also in various sections between such analyzers connected in series. These can be high-frequency voltage ion guides such as rod multipoles, ring-stack ion guides, or even RF funnel arrangements. Likewise, different analyzers and intermediate stages can be operated at different vacuum levels.

[0044] A mass analyzer separates charged molecules or molecular ions according to their mass-to-charge ratio (m / z). Time-of-flight analyzers can be used, which can be linear or reflector-type, and / or those with axial or orthogonal acceleration into the path of flight. Other types of mass-dispersive analyzers can also be used, such as 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.

[0045] 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 G / Z. This is based on the interaction of the ion species with an electric field that couples to the ion's charge, with the simultaneous exposure of a buffer gas acting on the ion's mean cross-sectional area. Particularly well-known are drift tube mobility analyzers with a static electric field gradient that drive ions through an essentially static gas, with the drift velocity of an ion species resulting from the propulsive 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 stopping force. Traveling wave mobility analyzers are also worth mentioning. It is understood that analyzers 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 q / z or o / z.

[0046] In various embodiments, the fluorescence response receiver can comprise a camera, a photomultiplier, and / or a photospectrometer, which are illuminated, in particular, directly or using an optical waveguide, such as an optical fiber. A flexible optical waveguide, in particular, increases the options for arranging individual optical components. In the case of non-wavelength-resolved observation, the measurement range can be restricted to the emission attributable to a fluorescent substance. Narrowband optical filters can be used for this purpose, which can be individually adapted and used to the emission behavior of the respective fluorescent substance used.In this way, the proportion of the total signal that can be generated by a broadband emission of a matrix substance or by autofluorescence of the sample material is significantly reduced and the specificity of the measured fluorescence response with respect to the fluorescent substance used is increased.

[0047] The processing device can contain an arithmetic unit, which in particular has one or more circuits or one or more microprocessors. The processing device can serve as a control center for the operation of the communicatively connected components, e.g. by receiving data from these components, processing and outputting it and / or deriving control commands from it, which in turn are sent to the communicatively connected components in order to initiate, terminate and / or control workflows of these components. The processing device can be designed to be centralized or decentralized. Decentralized can mean that a plurality of arithmetic unit subunits are present, each of which is arranged close to the communicatively connected components and communicates with a central arithmetic unit where all information converges. Short description of the figures

[0048] 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.

[0049] Figure 1 schematically illustrates an embodiment of a method and system according to principles of the present disclosure operating in reflected light.

[0050] Figure 2 schematically illustrates an embodiment of a method and system according to principles of the present disclosure, operating partly in incident light and partly in transmitted light.

[0051] Figure 3 schematically illustrates a process of comparing and aligning measurement data of the same modality acquired at different spatial resolutions, according to principles of the present disclosure.

[0052] Figure 4 shows measurement data of a fluorescence modality acquired at different spatial resolutions and measurement data of an ion spectrometric modality whose spatial coordinates and spatial resolution match one of the fluorescence modalities. Detailed description

[0053] 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.

[0054] Figure 1 schematically shows an ion source 2 in which, by applying energy bursts 4 to a sample material (not shown) deposited on a sample carrier 6, molecules are locally removed from the sample material, transferred into the gas phase, and ionized (schematically shown at arrow 8). The energy burst 4 can be a pulsed laser beam and, for example, comprise laser light with a wavelength from the ultraviolet spectral range, e.g., 337 nanometers or 355 nanometers. In the example shown, the pulsed laser beam falls onto the sample material in reflected light. The sample carrier 6 can be a flat indium tin oxide-coated glass slide. The sample material can comprise individual cells, which, for example, are grown on the sample carrier or applied to it after cultivation, or a composite of cells, as in a tissue section.The sample material may be prepared with a matrix substance to facilitate the absorption of an energy burst and promote gentle ionization.

[0055] In various embodiments, the sample material and sample carrier 6 can be kept in a negative pressure area. 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 Hectopascal (< atmospheric pressure), e.g. 0.1-10 hectopascal. The Material removal can be further supplemented by post-desorption ionization, for example by the MALDI-2 method, see the work of Jens Soltwisch et al. (Science, 10 April 2015 • Vol 348 Issue 6231, 211-215).

[0056] The locally ablated and ionized sample material is guided using electromagnetic fields and, if necessary, assisted by the flow of an inert gas such as molecular nitrogen and transferred to an ion processing device, ultimately comprising a mass analyzer. Schematically indicated by an extraction electrode 10, which is located opposite the point of impact of the pulsed laser beam on the sample material, is used. The sample carrier 6 and the sample material deposited thereon can be spatially aligned to this extraction electrode 10 by an actuator coupled to the sample carrier 6 (not shown), e.g., a translation stage.Repeatedly incident energy bursts 4 on the same impact point as well as on spatially displaced impact points on the sample material (schematically at 12) generate ion packets that are sorted by the mass analyzer and ultimately recorded in histograms (schematically at 14) that plot the abundance or intensity of the ions against a mass-related value such as the mass-to-charge ratio m / z.

[0057] The sample material on the sample carrier 6 comprises a substance capable of fluorescence; for example, the sample material can be treated with an extrinsic fluorescent dye before measurement. The fluorescent dye can, for example, have good excitation properties in a wavelength range that corresponds to a preferred excitation wavelength of a matrix substance for matrix-assisted ionization, particularly preferably in the MALDI version, with which the sample material has been prepared. This excitation wavelength range can, for example, be between 300 and 400 nanometers, in particular between 330 and 360 nanometers. The fluorescent dye can preferentially bind to certain molecules of the sample material, whereas it is less sensitive to other molecules of the sample material. In cells, for example, the fluorescent dye can preferentially bind to certain features of a cell, e.g.the cell walls or the cell nucleus, whereas it has little tendency to bind to surrounding parts of the cell. Thus, the application of the fluorescent dye results in a high-contrast or contrast-variable frequency distribution across the entire sample material, which can be used for precise co-registration in the further course of the procedure. For the purposes of co-registration, this comprehensive distribution of at least one fluorescent substance across the entire sample material is essential. This is advantageous across the entire sample material, as it can be ensured that all surface areas of the sample material have enough support points or anchor points for efficient execution of mathematical operations of spatial mapping.

[0058] In addition to the local ablation of sample material, an energy burst 4 simultaneously excites a local fluorescence response in the sample material. This fluorescence response can manifest itself in the emission of fluorescent light (schematically shown at 16). The fluorescent light will usually have lower energy than the light used for excitation, i.e., a lower frequency and longer wavelength. In the case of a pulsed UV laser, for example, the fluorescent light can be emitted in the visible spectral range. The fluorescent light can, in principle, be emitted in all directions. Regarding a sample carrier for simultaneous ion spectrometric analysis, there are basically two ways to detect fluorescent light: in reflected light or in transmitted light (in transmission). The latter, of course, only when a correspondingly transparent and translucent sample carrier is used, for example, an indium-tin oxide glass slide.Figure 1 shows a schematic illustration of detection in reflected light.

[0059] Since the energy burst 4 in the form of the pulsed laser beam in Figure 1 is incident on the sample material in reflected light at an angle to a surface normal of the sample carrier 6, and the ion extraction 8 occurs substantially along the surface normal, the detection axis for the fluorescent light can also be at an angle to the surface normal and, for example, be substantially diametrically opposite the incidence axis of the pulsed laser beam. A collimating lens 18 or a corresponding lens system can be located along the detection axis, parallelizing the light entering the optical aperture. A filter 20 can remove light components that are not necessary for detecting the fluorescent light from the beam path. For example, light components from the excitation light of the pulsed laser that are reflected from the surface of the sample material can be filtered out.In the case of a pulsed UV laser, for example, a long-pass filter can be used because the fluorescent light components have a substantially lower frequency and longer wavelength.

[0060] Behind the filter 20, a further filter 22 can be arranged along the detection axis, which allows an optional light-optical observation of the processes in the sample material on the sample carrier 6 in real time. For this purpose, the further filter 22 can be designed as a bandpass filter, which redirects a portion of the fluorescent light to a photon receiver 24 for recording the fluorescent light response and another portion via an imaging lens 26 or a corresponding lens system passes through to a camera 28, whose captured image data can be displayed, for example, on a screen for monitoring and process control. The dashed outlines indicate that real-time monitoring by a camera 28 is optional.

[0061] On the path of the redirected light component, which, despite the spatial change in direction in this example, is still considered to be on the detection axis, an optional narrowband filter 30, which can be very precisely designed for a fluorescent light wavelength and filters out any interfering residual light components, an imaging lens 32, which focuses the incident parallel light beam onto the photon receiver 24, or a corresponding lens system, as well as an optional pinhole 34 for noise reduction can be arranged. The photon receiver 24 can have a camera, a photomultiplier, or a photospectrometer; the latter, of course, only makes sense if optical components in the previous beam path are not designed to narrow the transmitted wavelength or frequency range too much.A photomultiplier or a photospectrometer can be illuminated directly or via an intermediate optical waveguide (not shown), such as an optical fiber. A flexible optical waveguide increases the options for arranging the individual optical components.

[0062] Due to the simultaneous generation of molecular ions from the sample material and excitation of a fluorescence response from the sample material, the position coordinates and spatial resolution of the acquired fluorescence light data correspond to the position coordinates and spatial resolution of the ion spectrometric analysis data and are essentially determined by the dimension of the impact area of ​​the energy burst, e.g. the diameter of the light spot of a pulsed UV laser, and possibly by the spatial summary of the molecular information from the sample material in image elements or pixels, which are often added together to improve the signal-to-noise ratio of neighboring impact areas on the sample material, e.g. in imaging MALDI mass analysis.

[0063] In addition to the simultaneous acquisition of ion spectrometric data and fluorescence response data, the sample material is separately subjected to a high-resolution fluorescence analysis (schematically shown at arrow 36), which preferably takes place before the ion spectrometric analysis, but can in principle also be carried out after this, provided that the sample material and the at least one fluorescent substance on the sample material have not yet been consumed or rendered unusable by the ion spectrometric analysis. are made. Spatial resolutions of high-resolution fluorescence image data can be in a range of nanometers selected from the group comprising or consisting of: <1000 nanometers, <500 nanometers, <300 nanometers, <100 nanometers, <50 nanometers, or below any other upper limit of 1000 nanometers. An example of a high-resolution microscope is the Nikon Eclipse 90i (Nikon Instruments Inc., Melville, New York).

[0064] After the measurements, three data sets are generally available: ion spectrometric data, which contain molecular content information and molecular distribution information from the image elements or pixels on the sample material; fluorescence response data, which were acquired substantially simultaneously with the ion spectrometric data on the same spatial grid and contain information on the frequency distribution of at least one fluorescent substance on the sample material; and high-resolution fluorescence image data, which contain distribution information of the at least one fluorescent substance with a substantially higher spatial resolution and, if necessary, acquired on a different spatial grid.It is understood that for an integrated analysis of these data, it is best to co-register and process the high-resolution fluorescence image data, which provide highly detailed information about the arrangement of visible features in the sample material, and the ion spectrometric data, which reflect the molecular content of the sample material. Due to the different data acquisition modalities, the often significantly different spatial resolutions, and the potentially different clamping systems during data acquisition, co-registration can be challenging. In particular, the data quality, particularly with regard to positional fidelity, can suffer after merging due to the required computer-aided adjustment processes.

[0065] The fluorescence response data acquired simultaneously with the ion spectrometric data form a suitable link between the ion spectrometric data and the high-resolution fluorescence image data, which allows for a higher data quality when combining the different modalities: they were in each case acquired in the same clamping of the sample carrier and therefore intrinsically have the same position coordinates as the ion spectrometric data; they contain the frequency distribution of the at least one fluorescent substance, e.g. fluorescent light intensity in a narrow-band wavelength range per image element or pixel, which corresponds to the frequency distribution of the same at least one fluorescent substance in the high-resolution Fluorescence image data generated with the high-resolution fluorescence image receiver must match. Aligning and aligning the fluorescence response data and the fluorescence image data, which is possible with high accuracy, simultaneously aligns and maps the high-resolution fluorescence image data with the ion spectrometric data, which are inherently coupled to the fluorescence response data in terms of spatial resolution and positional coordinates.

[0066] Figure 2 illustrates an embodiment very similar to that shown in Figure 1. Therefore, only the differences from the first embodiment in Figure 1 will be discussed below. The same reference numerals identify the same components in both figures. The key difference is that fluorescent light, which is emitted in response to the local excitation of the sample material by an energy burst 4, is not detected in reflected light, but in transmitted light through a suitably transparent and translucent sample carrier 6. This design has the advantage that the area of ​​the ion source, in which sample material ablation, ion generation, and ion extraction take place, can be kept free of optical components required for detecting the fluorescence response.Furthermore, the transmitted-light design allows observation with a high numerical aperture and along an axis that is substantially perpendicular to the extension of the sample carrier 6 and therefore experiences less geometric distortion.

[0067] In continuation of the embodiments shown in Figures 1 and 2, other arrangements for exciting a fluorescence response and acquiring fluorescence response data simultaneously with the acquisition of ion spectrometric data can also be implemented. In an embodiment not illustrated, the energy burst for exciting a fluorescence response in the sample material can be incident on the sample material in transmitted light through the sample carrier (in transmission). This is a long-established method, particularly in MALDI imaging; see, for example, the study by Andre Zavalin et al. (J Mass Spectrom. 2012 Nov; 47(11): i). Excitation in transmitted light can be combined with fluorescence response data acquisition in reflected light, similar to the example shown in Figure 1, or also in transmitted light, as example shown in Figure 2.In the latter variant, where excitation and detection both occur in transmitted light, it is advantageous if the excitation beam and response beam are well separated. A necessary adaptation of the system shown in Figure 2. However, the beam path using beam splitters, appropriately selected filters and dichroic mirrors does not pose any major difficulties for an optics expert.

[0068] Figure 3 illustrates how fluorescence response data acquired simultaneously with an ion spectrometric measurement of the sample material and having a comparatively low spatial (lateral) resolution (in the figure on the right) can be co-registered with high-resolution fluorescence image data acquired separately from the same sample material using a different instrument with its own sample well (in the figure on the left) in an xy plane that is parallel to the sample support surface carrying the sample material. The different spatial resolution is indicated in the two grids by different image element or pixel sizes. The different clamping for the measurements can also lead to effects that amount to distortion and are therefore indicated accordingly in the figure.An algorithm for matching and aligning the different images of the same modality can aim to superimpose the two frequency distributions of the at least one fluorescent substance with the greatest possible match. To this end, a transformation can be created that results from the application of various mathematical operations of stretching, compressing, and / or rotating. These mathematical operations can be applied uniformly across the entire image data and response data (affine), or they can be calculated area-segment-by-area or area-section-by-area (elastic) to achieve the best possible match between the frequency distributions of the at least one fluorescent substance.

[0069] Examples of affine transformations include operations such as rotation, scaling, shearing, compression, or translation, as well as combinations of these operations. Affine transformations can be represented by a transformation matrix. Elastic transformations also allow for the compensation of distortions and deformations. The thin plate spline model is an example of an elastic transformation. Elastic transformations generally cannot be represented by a transformation matrix, but are solved using polynomial fits or vector fields.

[0070] Preferably, the different spatial resolutions of the spatially resolved fluorescence response data and the high-resolution fluorescence image data are aligned. This can be achieved by mapping the high-resolution image data to the lower spatial resolution of the response data, e.g., by combining Image information from individual picture elements or pixels, possibly supported by mathematical operations that apply interpolation, extrapolation and / or regression.

[0071] Measurement example: Figure 4 shows the results of an example measurement. The measurement was performed using a mass spectrometer with a setup similar to that described in the work by Marcel Niehaus et al. (Nature Methods 2019, 16, 925-931, Transmission-mode MALDI-2 mass spectrometry imaging of cells and tissues at subcellular resolution). The lateral spatial resolution of the mass spectrometric measurement was 5 micrometers pixel size. No MALDI-2 post-ionization was performed. The sample material was a mouse cerebellum section, and DHAP was used as the matrix substance. Before preparation, the sample material was stained with matrix substance using Hoechst 33342 fluorescent dye (bound to cell nuclei, UV excitation, emission maximum at 455 nanometers) and Green Actin Tracker (bound to actin filaments, excitation at 503 nanometers, emission at 512 nanometers).High-resolution fluorescence images were then acquired using a slide scanner in a first clamping position of the sample carrier (referred to as external high-resolution fluorescence microscopy in Figure 4 on the left). The data were sorted into two channels: the fluorescence data from the Hoechst 33342 (fluorescence channel 1) and the fluorescence data from the Green Actin Tracker (fluorescence channel 2). The matrix substance was then applied by (re-)sublimation, and the MALDI-MSI measurement was performed in a second clamping position of the sample carrier.

[0072] The signal on the far right, referred to as "MALDI-accompanying fluorescence," represents the signal intensity of the fluorescence response of Hoechst33342 during the MALDI process between 450 and 460 nanometers. For clarity, this signal has been normalized to the total fluorescence intensity at this pixel (equivalent to a total ion count (TIC) normalization). The far right of Figure 4 shows examples of ion spectrometric MALDI image data acquired simultaneously with the accompanying fluorescence response. These can be combined and combined to form a segmentation representation (far right in the figure).

[0073] If the co-registration of the high-resolution image data and ion spectrometric data is carried out using at least one fluorescence-capable substance via the fluorescence response data, the transformation that maps the position coordinates of the ion spectrometric data to those of the high-resolution fluorescence image data can of course also be applied to images generated with other fluorescence-capable substances. Apply fluorescence image data acquired simultaneously with the data from the fluorescence images used to calculate the transformation. In the example shown, it is then also possible to co-register the ion spectrometric data with the fluorescence image data, for example, from fluorescence channel 2 (Green Actin Tracker). This can be useful when different fluorescent dyes bind to different molecules in the sample material, which in turn characterize different physiological, biological, or metabolic features in the tissue section.In this way, within the scope of this disclosure, the ion spectrometric data can be compared and aligned with one another and, in particular, co-registered with fluorescence image data of a number of fluorescence-capable substances, mediated by the simultaneously acquired fluorescence response data, which is selected from the group comprising or consisting of: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or any other suitable integer between 1 and 20.

[0074] The invention has been described above with reference to various specific embodiments. However, it is 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, provided this appears practical to a person skilled in the art. Furthermore, the above description serves only to illustrate the invention and does not limit the scope of protection, which is defined exclusively by the appended claims, taking into account any existing equivalents.

Claims

Claims 1. A method for analyzing sample material arranged on a sample carrier and containing at least one fluorescent substance, comprising the steps: (a) acquiring spatially resolved ion spectrometric data from the sample material using energy bursts designed and suitable to locally trigger a fluorescence response of the at least one fluorescent substance on the sample material, and simultaneously acquiring spatially resolved fluorescence response data from the sample material having a first spatial resolution; (b) acquiring fluorescence image data from the sample material having a second spatial resolution that is substantially greater than the first spatial resolution; and (c) Aligning and aligning the spatially resolved fluorescence response data and the fluorescence image data.

2. The method according to claim 1, wherein the spatially resolved fluorescence response data contain a first spatial frequency distribution of the at least one fluorescing substance across the sample material and the fluorescence image data contain a second spatial frequency distribution of the at least one fluorescing substance across the sample material, wherein the smallest possible deviation between the first spatial frequency distribution and the second spatial frequency distribution is sought for the matching and aligning.

3. A method according to claim 1 or claim 2, wherein step (b) is carried out before and / or after step (a).

4. Method according to one of claims 1 to 3, wherein a tissue section or a cell culture is used as sample material.

5. The method according to any one of claims 1 to 4, wherein the spatially resolved fluorescence response data and the fluorescence image data originate from at least one extrinsic fluorescence-capable substance, eg Hoechst33342, DAPI or Calcein Blue, which is added to the sample material prior to the acquisition of the ion spectrometric data, the fluorescence response data and the fluorescence image data.

6. Method according to one of claims 1 to 5, in which the energy bursts are triggered by a pulsed laser which, for example, emits laser pulses in the ultraviolet spectral range.

7. Method according to claim 6, wherein the laser pulses are incident locally on the sample material in incident light or transmitted light.

8. The method according to any one of claims 1 to 7, wherein the sample material is prepared before step (a) with a matrix substance suitable for matrix-assisted ionization, in particular matrix-assisted laser desorption and ionization, MALDI.

9. The method according to any one of claims 1 to 8, wherein the fluorescence response comprises fluorescent light emission from the at least one fluorescing substance, which is detected light-optically, e.g. using a microscope.

10. Method according to claim 9, wherein the fluorescent light emission is detected light-optically in the form of a broadband spectrum or narrowband wavelength-Z frequency range, e.g. after filtering.

11. Method according to one of claims 1 to 10, wherein the ion spectrometric data include a third spatial frequency distribution of at least one molecule of interest in the sample material, in particular selected from the group comprising or consisting of: proteins, peptides, lipids, saccharides, nucleotides, metabolites, biopolymers.

12. Method according to one of claims 1 to 11, wherein for the adjustment and alignment, the second spatial resolution of the fluorescence image data and the first spatial resolution of the fluorescence response data are adjusted to each other, e.g. by downscaling the second spatial resolution.

13. The method according to any one of claims 1 to 12, wherein a result of the matching and aligning is used to spatially co-register the ion spectrometric data and the fluorescence image data, e.g. using a transformation matrix, polynomial fitting or a vector field.

14. Method according to one of claims 1 to 13, wherein the spatially resolved fluorescence response data are acquired in reflected light from and / or in transmitted light through the sample carrier.

15. System for analyzing sample material arranged on a sample carrier and comprising at least one fluorescent substance, comprising: - an ion analyzer arranged and designed to measure spatially resolved, to collect ion spectrometric data from the sample material; - a fluorescence response receiver arranged and designed to acquire spatially resolved fluorescence response data from the sample material; - a fluorescence image receiver arranged and designed to acquire spatially high-resolution fluorescence image data from the sample material; and - a processing device which is in data communication with the ion analyzer, fluorescence response receiver and fluorescence image receiver and is arranged and programmed to carry out a method according to one of claims 1 to 14.

16. The system of claim 15, wherein the ion analyzer comprises a mass analyzer, mobility analyzer, or combined mobility mass analyzer.

17. System according to claim 15 or claim 16, wherein the fluorescence response receiver comprises a camera, a photomultiplier and / or a photospectrometer, which are illuminated in particular directly or using an optical fiber.

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