Method and system for analyzing sample material

Abstract

The present invention relates to a method and a system for the simultaneous acquisition of spatially resolved measurement data of different modalities of a sample material arranged on a sample carrier, in particular the partial simultaneous acquisition of these measurement data, and in particular the simplified and improved co-registration of the different modalities measurement data, i.e. their spatial arrangement and orientation in the same coordinate system. The method and system according to the present disclosure can advantageously be applied to imaging ion spectrometry, for example, imaging time-of-flight mass analysis of tissue sections, in particular using matrix-assisted ionization, more particularly using matrix-assisted laser desorption and ionization (MALDI).

Description

Technical Field

[0001] This disclosure relates to methods and systems for acquiring spatially resolved measurement data of different modalities of sample material arranged on a sample carrier, particularly to the partial simultaneous acquisition of these measurement data, and to facilitating and improving co-registration of measurement data of different modalities, i.e., their spatial arrangement and orientation in the same coordinate system. The methods and systems according to this disclosure can be advantageously applied to imaging ion spectroscopy, for example, imaging time-of-flight quality analysis of tissue sections, particularly using matrix-assisted ionization, and further particularly using matrix-assisted laser desorption and ionization (MALDI). Background Technology

[0002] The prior art will be described below with reference to a specific aspect. However, this should not be construed as limiting. Useful extensions and modifications known from the prior art may also be applied beyond the relatively narrow scope of this introduction, and such extensions and modifications will be apparent to those skilled in the art upon reading the following disclosure.

[0003] Matrix-assisted laser desorption / ionization imaging mass spectrometry (MSI) is a rapidly developing field for spatial visualization of molecular distributions, such as in tissue sections and cell cultures. To enable correlation analysis with other imaging methods, such as optical microscopy, the modalities used need to be co-registered as precisely as possible. According to current techniques, this co-registration is achieved through images acquired independently of each other. At the micrometer scale, these methods typically exhibit significant inaccuracies as a result.

[0004] To couple fluorescence microscopy, a specific implementation of optical microscopy, with MALDI-MSI, measurements acquired independently are currently used. Normally, these measurements are performed using two completely independent instruments. Therefore, microscopic examination can be performed before, after, or both before and after MALDI-MSI measurements. To co-register optical imaging and mass spectrometry-based imaging, a transformation is defined that combines the two modalities based on a predefined metric (see Reference 1). For this purpose, ablation pits generated in the sample material during MALDI-MSI measurements can also be used (see Reference 2). In specific applications, the two modalities can be recorded using technically correlated but separate instruments (see Reference 3), or partially using the same complete instrument (see Reference 4). Here, co-registration is achieved through precise knowledge of the measurement locations of the two modalities, which can be designed to be consistent. In the context of other research, ionized fluorescence has been used to elucidate the MALDI process (see Reference 5) and to determine the size of laser spot on sample material (see Reference 6).

[0005] References: (1) Balluff, B.; Heeren, RMA; Race, AM: An Overview of Image Registration for Aligning Mass Spectrometry Imaging with Clinically Relevant Imaging Modalities. J. MassSpectrom. Adv. Clin. Lab 2022, 23, 26–38. https: / / doi.org / 10.1016 / j.jmsacl.2021.12.006.

[0006] (2) Patterson, NH; Tuck, M.; Van de Plas, R.; Caprioli, RM: Advanced Registration and Analysis of MALDI Imaging Mass Spectrometry Measurements through Autofluorescence Microscopy. Anal. Chem. 2018, 90 (21), 12395–12403. https: / / doi.org / 10.1021 / acs.analchem.8b02884.

[0007] (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. https: / / doi.org / 10.1007 / s00216-016-9594-9.

[0008] (4) Soltwisch, J.; Potthoff, A.; Schwenzfeier, J.; Bien, T.; Niehaus, M.; Dreisewerd, 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.

[0009] (5) Jaskolla, TW; Karas, M.: Using Fluorescence Dyes as a Tool for Analyzing the MALDI Process. J. Am. Soc. Mass Spectrom. 2008, 19(8), 1054–1061. https: / / doi.org / 10.1016 / j.jasms.2008.04.032.

[0010] (6) Steven, RT; Palmer, AD; Bunch, J.: Fluorometric Beam Profiling of UV MALDI Lasers. J. Am. Soc. Mass Spectrom. 2013, 24(7), 1146–1152. https: / / doi.org / 10.1007 / s13361-013-0650-9.

[0011] Patent publication WO 2023 / 205280 A1 describes a method for enhancing sample fluorescence intensity by applying aromatic compounds. It also describes a workflow for a combined fluorescence-MALDI microscope / imager, in which MALDI imaging and fluorescence imaging of the same sample are combined during the sample preparation step. The described workflow aims to reduce sample acquisition time to one working day and minimize sample degradation. In the scientific literature, the technical teachings disclosed in this patent are referred to by scientists as "FluoMALDI".

[0012] Existing techniques in standard applications require the acquisition of multiple microscope images, making them complex to implement and resulting in relatively high discrepancies in co-registration between optical and ion-spectrum-based images. Strategies for correlating machine parts to achieve direct modal connectivity are complex, not feasible on all devices, and require significant technological investment.

[0013] Alexander Fengler et al. presented a poster at the 54th Annual Meeting of the German Society for Mass Spectrometry (DGMS) in Dortmund from May 14 to 17, 2023, entitled "Duo-modal mass spectrometric and emission spectroscopic recordings of 2,5-dihydroxybenzoic acid on cerebellar artissue," which described parallel measurements of mass spectrometric and fluorescence spectrometric data from brain tissue, particularly for comparing the spatial distribution of the m / z mass signal with the tissue's fluorescence data. To the inventors' knowledge, this work is the first to utilize the fluorescence response generated during ionization in confocal microscopy.

[0014] In light of the foregoing, there is a need to improve the correlation between ion spectral modes and high-resolution fluorescence-based modes in the imaging analysis of sample materials, particularly when these measurements are performed in different sample chambers with different sample carriers. Other problems to be solved by the present invention will become apparent to those skilled in the art after reading the following disclosure. Summary of the Invention

[0015] This invention enables, for example, the simultaneous and spatially co-located acquisition of fluorescence response data, such as fluorescence spectral data, using MALDI, generated during ion spectral analysis of each studied pixel or image element during pulsed ablation. Specifically, this can include spatially and temporally synchronized acquisition of confocal hyperspectral fluorescence response images and MALDI-MSI measurements.

[0016] In the methods described herein, fluorescence generated by the localized action of energy injection on the sample material during MALDI analysis, for example as a pulsed laser, within the framework of ion spectroscopy, can be used to form an optical image with substantially the same positional coordinates and spatial resolution as ion spectroscopy. For this purpose, specific fluorescent dyes can also be used, which can be added to the sample material prior to measurement.

[0017] This invention utilizes the spatially and temporally correlated fluorescence response generated during ion spectroscopy measurements of the molecular content of a sample material, such as MALDI ionization and subsequent time-of-flight mass analysis. Here, the intensity of the fluorescence spectrum or emission over a specific wavelength range can be recorded at each illuminated image element or pixel, for example, in a so-called fluorescence channel. Limiting to a defined wavelength range offers the advantage of eliminating or at least reducing interference from the excitation light and, possibly, the intrinsic fluorescence emission of the matrix material itself, from the fluorescence response acquisition.

[0018] Based on this data, confocal hyperspectral images that are co-located and spatially precisely correlated with ion spectroscopy measurements can be generated. The ablation of materials and the excitation of fluorescence responses used in ion spectroscopy (e.g., MALDI time-of-flight mass spectrometry) are induced at the same location and by the same energy injection, such as by laser bombardment as in MALDI, and are therefore inherently correlated spatially and temporally. In this context, spatial lateral refers to the localization of sample material sampling in the xy-plane unfolded by the sample carrier or in a plane parallel to it (e.g., a plane formed by tissue sections on the sample carrier).

[0019] To generate fluorescence signals on sample materials (e.g., tissue or cell cultures), fluorescent dyes introduced during sample preparation can be used, whose excitation wavelength range coincides with or overlaps with the wavelength of the energy-injected laser. For lasers used in UV-MALDI, such as 337 nm or 355 nm lasers, UV-active fluorescent dyes such as Hoechst 33342, DAPI (4',6-diamidinyl-2-phenylindole), or Calcein Blue can be used. The fluorescence generated during ionization can be purified and detected by appropriate filters, or used for spectroscopic studies and evaluation. The positional coordinates of image elements or pixels in the resulting confocal microscopy image precisely correspond to the positional coordinates of the rasterized ion spectroscopy measurements.

[0020] It should be mentioned here that the image elements or pixels in imaging ion spectroscopy represent the smallest area units of the sample material whose molecular information is spatially resolved and can be displayed. The typical size of these area units, in terms of diameter (e.g., in the case of a circular laser spot) or side length (e.g., in the case of a rectangular pattern), can be selected from a range including or consisting of the following values: 1 to 10 micrometers, 10 to 100 micrometers, 100 to 1000 micrometers, 1 to 1000 micrometers, 10 to 1000 micrometers, or any other suitable range between 1 and 1000 micrometers. The size of the laser spot can be selected to be smaller than the size of the image element or pixel, and molecular information can be acquired from such image elements or pixels by repeatedly bombarding the sample material with a pulsed laser beam at slightly different locations within the boundaries of the image element or pixel. The molecular information from each sample is then accumulated or summed for the image element or pixel. It is understood that, in principle, this method can generate fluorescence response data with a finer spatial grid than the ion spectroscopy measurements, which contain molecular information from multiple closely adjacent sampling locations. However, fluorescence response data can also be mapped onto an image element grid or pixel grid for ion spectroscopy measurements using simple mathematical operations (such as addition). Alternatively, the fluorescence response data acquisition process can be designed so that data from these simultaneously acquired modalities are not recorded in different spatial grids.

[0021] The image elements or pixels generated using the aforementioned setup are typically significantly larger than those achievable using modern fluorescence microscopy. Therefore, the fluorescence response image generated simultaneously with ion spectroscopy measurements using the aforementioned invention should primarily serve as a link between the MSI mode and the high-resolution optical mode. Co-registration of different modes acquired at different resolutions involves a greater degree of error propagation, while the co-registration criterion is significantly simpler and more accurate for the same acquisition mode but with different image element or pixel sizes. The transformation determined here, which correlates and maps the positional coordinates of different modes, can also be directly applied to the MSI results based on the inherent coupling, thereby achieving accurate and simple co-registration between the high-resolution fluorescence image and the MSI. Here, high-resolution fluorescence microscopy measurements are typically performed before the sample material is prepared with the matrix material. However, it is also possible to acquire high-resolution fluorescence images after ion spectroscopy measurements, or additionally in addition to high-resolution fluorescence images acquired before ion spectroscopy measurements, for example, for process control and / or quality assurance reasons.

[0022] Fluorescence intensity measurements, whether using specific filters or spectroscopic methods, can be performed on most existing MSI systems with relatively low technical investment. This measurement is performed at the same location and time as ion spectroscopy, requiring no additional measurement time or energy injection, such as laser emission. It is more accurate, faster, and more resource-efficient than existing techniques.

[0023] In view of the above description, according to a first aspect, the present invention relates to a method for analyzing sample material arranged on a sample carrier and having at least one fluorescent substance, comprising the following steps: (a) acquiring spatially resolved ion spectrum data of the sample material using energy injection, the energy injection being designed and adapted to locally trigger the fluorescence response of the at least one fluorescent substance on the sample material, while acquiring spatially resolved fluorescence response data of the sample material having a first spatial resolution; (b) acquiring fluorescence image data of the sample material having a second spatial resolution, the second spatial resolution being substantially greater than the first spatial resolution, and wherein step (b) may be performed in particular before and / or after step (a); and (c) comparing and registering the spatially resolved fluorescence response data and the fluorescence image data.

[0024] On the one hand, the conditions for exciting the fluorescence response differ from those for confocal fluorescence detection during ion spectroscopy measurements, and on the other hand, from those for separate high-resolution image acquisition. For example, energy injection ablates the material, naturally limiting the time between excitation and fluorescence response quenching, while excitation, for example, in high-resolution microscopy, remains below the ablation threshold, thus preserving the sample material, and therefore the fluorescence response can theoretically be excited for a longer period, for example, to enhance the accumulated fluorescence response. Nevertheless, it has been unexpectedly found that even under significantly different excitation conditions, the fluorescence response is sufficiently uniform and reliably detectable, allowing for spatial mapping with greater precision and automation.

[0025] Because measurement data for different modalities, such as ion spectroscopy and simultaneous confocal fluorescence detection, are acquired under the same clamp on the sample carrier, the positional coordinates of the measurement signals are co-located and precisely consistent. Furthermore, measurement data for the same modality, such as confocal fluorescence detection during ion spectroscopy and separately acquired high-resolution fluorescence images, have the same response characteristics. Therefore, confocal fluorescence response data can be used as an intermediary to automatically establish spatial correlation between ion spectroscopy data and high-resolution fluorescence images with higher precision. This method eliminates the need for directional markers on the sample material, such as cracks or ablation pits in the tissue, for co-registration, as the positioning of these markers in different modalities often cannot be performed with the same precision, which may lead to or even exacerbate co-registration errors. Moreover, especially when the user performs targeted staining, such as when cell nuclei are correspondingly labeled throughout the tissue, the distribution of the at least one fluorescent substance across the entire sample material enables data acquisition in every potential target area to be analyzed within the sample material, thereby ensuring high-quality co-registration across the entire sample material.

[0026] In principle, ion spectrum data, fluorescence response data, and high-resolution fluorescence image data can be acquired under the same clamping of the sample carrier carrying the sample material. This possibility exists, for example, when a high-resolution fluorescence signal receiver is coupled to or integrated into the ion generation region and used to acquire both fluorescence response data and fluorescence image data. An example of such a structure is shown in the applicant's patent publication WO 2024 / 041681 A1, where only the device for exciting fluorescence emission of the protective material needs to be added. The measurement procedure can be designed such that an actuator moves the sample carrier carrying the sample material to a specific ablation location, then first excites fluorescence emission of the protective material in a defined surface segment of the sample material and performs spatial high-resolution acquisition, subsequently applying energy injection to the corresponding surface region of the sample material for material ablation, acquiring the resulting ionized sample material by ion spectroscopy, and simultaneously detecting the fluorescence response from that surface region of the sample material. The measurement sequence then continues in such a way that the actuator moves the sample carrier carrying the sample material to the next ablation location and repeats the aforementioned cycle on another surface region until, for example, all available surface regions have been sampled. In this embodiment, fluorescence response data can be used for process control and quality assurance.

[0027] In various embodiments, the spatially resolved fluorescence response data may include a first spatial frequency distribution of the at least one fluorescent substance across the entire sample material, and the fluorescence image data may include a second spatial frequency distribution of the at least one fluorescent substance across the entire sample material, wherein the objective for the comparison and registration is to minimize the deviation between the first and second spatial frequency distributions. The frequency, intensity, or abundance distributions of the fluorescence response of the at least one fluorescent substance measured at different spatial resolutions using the same measurement modality can be adapted to the location coordinates of datasets obtained, for example, under different sample carrier clamping conditions. The evaluation can uniformly consider the overall frequency distribution of both the fluorescence image data and the fluorescence response data. Alternatively, the total area of ​​each data source can be divided into multiple facets, and the frequency distribution can be evaluated separately for each facet. Subsequently, the results from these facets can be stitched together into a unified overall image using an appropriate algorithm, particularly considering continuity.

[0028] The first spatial resolution can be in the micrometer range, for example selected from or consisting of the following ranges: 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 nanometer range, for example selected from or consisting of the following ranges: between 1 and 10 nanometers, between 10 and 100 nanometers, between 100 and 1000 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 differs from the first spatial resolution in length units by a factor of 1 / n, where n is selected from or a group consisting of the following ranges: ≥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 2. For the explanation of this factor: if the first spatial resolution is 10 micrometers in length units and the second spatial resolution is 10 nanometers, then n = 1000, i.e., the factor is one-thousandth.

[0029] In various embodiments, tissue sections or cell cultures can be used as sample materials. The sample material on the sample carrier may include tissue sections, provided, for example, from frozen blocks or as blocks prepared as FFPE (formalin fixed paraffin embedded), particularly using microtome. Tissue sections can be cut to a thickness of approximately 7-20 micrometers, and subsequent drying of the moist tissue can result in a significantly thinner final preparation. The sample material may also have an array arrangement or multiple individual preparations or spot preparations on the sample carrier, such as a cell array or multiple single cells. Individual preparations or spot preparations may in particular contain dried liquid spots or reagent spots containing sample material, for example, in the form of cells or analyte molecules. Similarly, single cells can be set as sample material on the sample carrier. These single cells can be cultured or grown directly on the sample carrier. Single cells may belong to prokaryotes (e.g., human, animal, plant, protist cells, or fungal cells) or eukaryotes (e.g., bacterial or archaea cells).

[0030] The sample carrier can be plate-shaped. It can have the dimensions of a standard microtiter plate, such as 127.71 mm long, 85.43 mm wide, and 14.10 mm thick. The material of the sample carrier can be conductive. For example, it can have a glass-, ceramic-, or plastic-based substrate, such that a conductive coating is applied to the surface carrying the sample material. As an example, an indium tin oxide-coated glass slide may be mentioned. The partial conductivity of the sample carrier allows for the generation of a reference potential, which is particularly useful for treating ions formed during a series of ablation events.

[0031] In various embodiments, the spatially resolved fluorescence response data and the fluorescence image data may originate from at least one exogenous fluorescent substance, such as Hoechst 33342, DAPI, or Calcein Blue, which is added to the sample material prior to the acquisition of ion spectral data, fluorescence response data, and fluorescence image data. Preferably, the fluorescent substance has good excitation characteristics in the ultraviolet spectral range to be compatible, particularly with UV-MALDI, for example, at wavelengths between 300 and 400 nm, particularly between 330 and 360 nm, and emits fluorescence after excitation with a wavelength range shifted to lower energies, such as in the visible light wavelength range, for example, between 400 and 800 nm, particularly between 450 and 650 nm.

[0032] The fluorescent dye Hoechst 33342 possesses several advantageous properties. It is specific for DNA (deoxyribonucleic acid) because it preferentially binds to the adenine-thymine (AT) region of DNA. This allows for specific staining of cell nuclei in both living and fixed cells and tissues. Its fluorescence properties ensure that it is excited by ultraviolet light and emits light in a spectral range from blue to cyan. This enables visualization of DNA using fluorescence spectroscopy or fluorescence microscopy. Hoechst 33342 is also compatible with antibodies and other samples labeled with fluorescein and rhodamine dyes. This allows for the simultaneous visualization of DNA and specific cellular targets.

[0033] One advantageous feature of DAPI is that its fading can be delayed by using so-called anti-quenching agents, thereby extending the optical observation time, such as when acquiring high-resolution fluorescence image data.

[0034] In various implementations, the energy injection can be triggered by a pulsed laser, such as one that emits laser pulses in the ultraviolet spectral range. The clock frequency of the pulse sequence can be in the range of a few hertz, for example, 1-20 pulses per second, up to 10. 3 Or 10 4 Hertz. Preferably, the laser pulse can be locally applied to the sample material in either incident or transmitted light. Incident light irradiation allows for the use of opaque sample carrier supports and analysis of thick sample materials. Transmitted light irradiation allows the ion generation region to be designed as optically free as possible for acquiring fluorescence response, and allows the focused diameter of the laser spot to be set in the low micrometer range, for example, in the single-digit micrometer range.

[0035] In various embodiments, sample materials can be prepared prior to step (a) using a matrix material suitable for matrix-assisted ionization, particularly matrix-assisted laser desorption / ionization (MALDI). Examples of UV-sensitive MALDI matrix materials include 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 primarily imparts a charge state z=1, which simplifies ion spectroscopy processing and analysis. To apply the matrix material to the sample material, methods that produce uniform layer thickness and minimal crystal irregularities are preferred. An example of this approach is (re)sublimation, especially see the paper by Joseph A. Hankin et al. (J Am Soc Mass Spectrom. Sep 2007; 18(9): 1646–1652, Sublimation as a Method of Matrix Application for Mass Spectrometric Imaging). If method step (b) is performed before method step (a), the matrix material can be applied before step (b), for example, when the acquisition of ion spectrum data, fluorescence response data, and fluorescence image data is performed in the same clamping of the sample carrier and / or in the same sample chamber, or it can be applied between step (b) and step (a).

[0036] In various embodiments, the fluorescence response may include fluorescence emission from the at least one fluorescent substance, detected optically, for example, using a microscope. For this embodiment, the microscope is preferably coupled to or integrated into the ion-generating region, such as a MALDI source. Fluorescence emission may be detected optically, for example, in the form of a broadband spectrum or a narrow wavelength / frequency range, after filtering. A broad wavelength range provides greater sensitivity to changes in the fluorescence response. The advantage of a narrow bandgap is that the optical components used to collect and detect fluorescence can be optimized for reception at that wavelength, and it also allows for a clearer distinction from the energy injection used to excite the fluorescence response, and from intrinsic fluorescence emission that may occur in some matrix materials, such as during laser bombardment. Narrow bandgap Δλ = λ max -λ min It can be selected from the following values ​​or groups of the following values: 5 nanometers, 10 nanometers, 20 nanometers, 30 nanometers, 40 nanometers, 50 nanometers, and any other suitable range between 5 and 50 nanometers.

[0037] In various embodiments, the ion spectroscopy data may include the third spatial frequency distribution of at least one target molecule in the sample material, particularly selected from the group consisting of or including proteins, peptides, lipids, carbohydrates, nucleotides, metabolites, and biopolymers. The third spatial frequency distribution may be in the form of an ion distribution map, reflecting the intensity or abundance of the analyte molecule from the sample material throughout the entire sample material. Such a distribution map can be generated for multiple analyte molecules in the sample material. For example, the distribution map can be used to study physiological, biological, and / or metabolic processes in the sample material.

[0038] In various implementations, to perform the comparison and registration, the second spatial resolution of the fluorescence image data and the first spatial resolution of the fluorescence response data can be adapted to each other, for example, by reducing the second spatial resolution. In a simple example, image data from different image elements or pixels can be summarized by wavelength-resolved summation of fluorescence-specific intensity information across multiple planar regions of the fluorescence image data, corresponding to an image element or pixel of the fluorescence response data, using mathematical operators of interpolation, extrapolation, and / or regression as necessary.

[0039] In various embodiments, the results of the comparison and mutual registration can be used to spatially co-register the ion spectral data and the fluorescence image data, for example, using transformation matrices, polynomial fitting, or vector fields. Images of different modalities, such as optical or microscopic acquisitions of fluorescence and ion distribution maps, can be computationally fused (image fusion) to provide more information about the sample material than a single-modal image or spectrum. Examples of this can be found in the applicant's patent publication DE 10 2008 023 438 A1 and the research of Raf Van de Plas et al. (Nature Methods, Vol. 12 No. 4, April 2015, 366-372).

[0040] In various embodiments, the spatially resolved fluorescence response data can be acquired in incident light from the sample carrier and / or transmitted light through the sample carrier. Acquisition in incident light can be achieved using an opaque sample carrier support and a thick sample material. Acquisition in transmitted light eliminates the need for optical components in the ion-generating region to collect and guide fluorescence, and allows for optical observation with a high aperture value and along an observation axis perpendicular to the sample carrier orientation, thus enabling fluorescence response observation with less geometric influence.

[0041] According to a second aspect, the present invention relates to a system for analyzing sample material disposed on a sample carrier and having at least one fluorescent substance, comprising: an ion analyzer arranged and designed to acquire spatially resolved ion spectrum data of the sample material; a fluorescence response receiver arranged and designed to acquire spatially resolved fluorescence response data of the sample material; a fluorescence image receiver arranged and designed to acquire spatially high-resolution fluorescence image data of the sample material; and a processing device in data communication with the ion analyzer, the fluorescence response receiver, and the fluorescence image receiver, and arranged and programmed to perform the method described above.

[0042] In various embodiments, the ion analyzer may include a mass analyzer, an ion mobility analyzer, or a combination of ion mobility and mass analyzers. Mass analysis may be the last analytical step performed on ionized sample material. Intermediate stages for guiding ions, such as radio frequency voltage ion directors, bar multipole ion directors, ring stacked ion directors, or RF funnel devices, may be arranged before the actual mass analyzer or multiple sequentially connected analyzers, and in different sections between such sequentially connected analyzers. Similarly, different analyzers and intermediate stages may operate at different low-voltage levels.

[0043] Mass analyzers separate charged molecules or molecular ions based on their mass-to-charge ratio (m / z). Time-of-flight analyzers can be used, and can be configured with linear structures, reflector structures, and / or structures with axial or orthogonal accelerations into the flight path. Other types of mass dispersive analyzers can also be used, such as single quadrupole mass filters, triple quadrupole analyzers, ion cyclotron resonance (ICR) cells, and Kingdon-type analyzers such as Orbitrap® (Thermo Fisher Scientific).

[0044] Ion mobility analyzers separate charged molecules or molecular ions based on the ratio of their collision cross-section to charge (sometimes expressed as Ω / z or σ / z). This is based on the interaction between the ion species and an electric field coupled to the ion charge, with a buffer gas acting on the average cross-sectional area of ​​the ions. In particular, drift tube ion mobility analyzers with a static electric field gradient are known, driving ions through a substantially stationary gas, where the drift velocity of the ion species is derived from the driving force of the electric field and the braking force from collisions with gas particles. Trapping ion mobility separators (TIMS) are also common, featuring a stable laminar gas flow driving the ions, with a gradually varying electric field gradient acting in the opposite direction to the gas flow. Traveling wave ion mobility analyzers can also be mentioned. It is understood that analyzers of these types can be coupled to achieve multidimensional separation of ion species based on more than one physicochemical property (such as m / z and Ω / z or σ / z).

[0045] In various embodiments, the fluorescence response receiver may include a camera, photomultiplier tube, and / or spectrometer, which are particularly illuminated directly or using an optical waveguide (e.g., optical fiber). Flexible optical waveguides, in particular, increase the options for arranging the various optical components. In non-wavelength-resolved observations, the measurement range can be limited to correspond to the emission of the fluorescent substance. For this purpose, narrowband optical filters can be used, which can be personalized and applied to the emission behavior of the corresponding fluorescent substance used. In this way, components in the total signal that may arise from the broadband emission of the matrix material or from the autofluorescence of the sample material are significantly reduced, and the specificity of the measured fluorescence response relative to the fluorescent substance used is improved.

[0046] The processing device may include an arithmetic logic unit (ALU), which in particular has one or more circuits or one or more microprocessors. The processing device can serve as a control center for operating components connected to the communication network, for example, by receiving data from these components, processing and outputting that data, and / or deriving control commands from that data, which are then sent to the communication-connected components to trigger, terminate, and / or control the workflow of those components. The processing device can be configured as centralized or distributed. A distributed configuration may mean that multiple ALU subunits exist, each located close to the communication-connected components and communicating with a central ALU unit where all information is aggregated. Attached Figure Description

[0047] To better understand the present invention, refer to the following drawings. The elements in the drawings are not necessarily drawn to scale, but are primarily (mostly schematic) used to illustrate the principles of the invention. In the drawings, corresponding elements in different views are indicated by the same reference numerals.

[0048] Figure 1 An embodiment of a method and system for operating in incident light according to the principles of this disclosure is illustrated schematically.

[0049] Figure 2 An embodiment of a method and system that operates partly in incident light and partly in transmitted light (in a transmission manner) according to the principles of this disclosure is illustrated schematically.

[0050] Figure 3 The process of comparing and registering measurement data of the same mode acquired at different spatial resolutions according to the principles of this disclosure is illustrated schematically.

[0051] Figure 4 Measurement data for fluorescence modes acquired at different spatial resolutions are shown, as well as measurement data for ion spectroscopy modes, whose position coordinates and spatial resolutions are consistent with one of the fluorescence modes. Detailed Implementation

[0052] Although the invention has been shown and illustrated by means of several embodiments, those skilled in the art will recognize that various modifications to its form and details may be made without departing from the scope of the technical teachings defined by the appended claims.

[0053] Figure 1 An ion source 2 is schematically shown, in which molecules are locally ablated, converted into a gaseous phase, and ionized by applying energy injection 4 to sample material (not shown) disposed on a sample carrier 6 (schematically indicated by arrow 8). Energy injection 4 can be a pulsed laser beam and, for example, includes lasers with wavelengths in the ultraviolet spectral range, such as 337 nm or 355 nm. In the illustrated example, the pulsed laser beam is incident on the sample material as incident light. Sample carrier 6 can be a planar glass slide coated with indium tin oxide. The sample material can include single cells, such as those cultured on or applied to the sample carrier after culture, or can include cell populations, such as those in tissue sections. The sample material can be prepared using a matrix material to simplify energy absorption during energy injection and promote gentle ionization.

[0054] In various embodiments, the sample material and sample carrier 6 can be maintained in a low-pressure region. For example, typical pressures suitable for vacuum MALDI are substantially higher than high vacuum (>10). -3 (hPa) and below about 10 2hPa (<atmospheric pressure), for example, 0.1 to 10 hPa. Material ablation can also be supplemented by post-desorption ionization, for example by the MALDI-2 method, see Jens Soltwisch et al. (Science, April 10, 2015, Vol. 348, No. 6231, pp. 211-215).

[0055] The locally ablated and ionized sample material is guided and transported under electromagnetic field conditions, and optionally with the assistance of an inert gas (such as molecular nitrogen) flow, to an ion processing apparatus that ultimately includes a mass analyzer. This is schematically represented by extraction electrode 10, which is positioned opposite the impact point of the pulsed laser beam on the sample material. The sample carrier 6 and the sample material placed on it can be spatially aligned relative to the extraction electrode 10 by an actuator (not shown), such as a displacement stage, coupled to the sample carrier 6. Repeated energy injection 4 into the same impact point and multiple spatially offset impact points (schematically shown in 12) on the sample material generates ion packets. These ion packets are classified by the mass analyzer and ultimately recorded in histograms (schematically shown in 14) that plot the abundance or intensity of ions against mass-related values ​​(such as mass-to-charge ratio m / z).

[0056] The sample material on sample carrier 6 contains a fluorescent substance; for example, the sample material can be treated with an exogenous fluorescent dye before measurement. The fluorescent dye, for example, can have good excitation characteristics within a certain wavelength range corresponding to the preferred excitation wavelength of the matrix material used for preparing the sample material, which is matrix-assisted ionization (particularly preferably implemented as MALDI). This excitation wavelength range can be, for example, between 300 nm and 400 nm, particularly between 330 nm and 360 nm. The fluorescent dye can preferentially bind to specific molecules of the sample material, while being relatively inert to other molecules of the sample material. For example, in cells, the fluorescent dye can preferentially attach to specific features of the cell, such as the cell wall or nucleus, while showing less tendency to bind to the pericellular portion. In this way, using the fluorescent dye, a high-contrast or variable-contrast frequency distribution can be obtained across the entire sample material span, which can be used for accurate co-registration in subsequent processes. For co-registration purposes, this widespread distribution of the at least one fluorescent substance across the entire sample material is advantageous because it ensures that all surface areas of the sample material have sufficient support points or anchors to effectively perform the mathematical operations of spatial mapping.

[0057] In addition to locally ablating the sample material, energy injection 4 also simultaneously excites a localized fluorescence response in the sample material. This fluorescence response can manifest as fluorescence emission (schematically shown in Figure 16). The fluorescence energy is typically lower than that of the excitation light, i.e., it has a lower frequency and a larger wavelength. In the case of pulsed UV lasers, fluorescence can, for example, be emitted in the visible spectrum. In principle, fluorescence can be emitted in all directions. Regarding the sample carrier used for simultaneous ion spectroscopy analysis, there are basically two schemes for detecting fluorescence: in incident light or (in the case of transmission) in transmitted light, the latter of course only possible when using a correspondingly transparent and translucent sample carrier, such as an indium tin oxide slide. Figure 1 The detection in incident light is illustrated schematically.

[0058] because Figure 1 Energy injection 4 in the form of a pulsed laser beam irradiates the sample material at an angle relative to the surface normal of the sample carrier 6, and ion extraction 8 proceeds substantially along the surface normal direction. Therefore, the fluorescence detection axis can also be angled relative to the surface normal, and for example, can be substantially aligned with the incident axis of the pulsed laser beam. A collimating lens 18 or a corresponding lens system can be placed along the detection axis to parallelize the light entering the optical aperture. A filter 20 removes light components from the optical path that are unnecessary for fluorescence detection. For example, light components from the pulsed laser excitation light that are reflected by the sample material surface can be filtered out. In the case of a pulsed UV laser, a long-pass filter can be used, for example, because the fluorescence component has a significantly lower frequency and a larger wavelength.

[0059] Following filter 20, another filter 22 can be arranged along the detection axis, enabling optional light microscopic observation of the process occurring on the sample material on the sample carrier 6 in real time. For this purpose, the additional filter 22 can be designed as a bandpass filter, deflecting a portion of the fluorescence to the photon receiver 24 for recording the fluorescence response, while allowing another portion to pass through the imaging lens 26 or a corresponding lens system to reach the camera 28. The image data acquired by the camera can be displayed on a screen, for example, for monitoring and process control. The dashed outline indicates that real-time monitoring via camera 28 is optional.

[0060] Along the path of the deflected light component (which, although spatially oriented, can still be considered to be on the detection axis in this example), the following can be arranged: an optional narrowband filter 30, which can be very precisely designed for the fluorescence wavelength and optionally filters out interfering residual light components; an imaging lens 32, which focuses the incident parallel beam onto the photon receiver 24; or a corresponding lens system; and an optional aperture plate 34 for noise reduction. The photon receiver 24 may include a camera, a photomultiplier, or a spectrometer, the latter of course only meaningful if the optical components in the preceding optical path are not designed to excessively narrowly limit the transmitted wavelength or frequency range. The photomultiplier or spectrometer can be illuminated directly or via an intermediately connected optical waveguide (not shown), such as an optical fiber. Flexible optical waveguides increase the options for arranging the various optical components.

[0061] Since the generation of molecular ions from the sample material and the excitation of the sample material's fluorescence response occur simultaneously, the position coordinates and spatial resolution of the acquired fluorescence data correspond to the position coordinates and spatial resolution of the ion spectroscopy analysis data. These are primarily determined by the size of the energy-injected impact surface, such as the spot diameter of the pulsed UV laser, and optionally by the spatial summarization of the molecular information of the sample material in image elements or pixels, such as in imaging MALDI quality analysis, where these image elements or pixels are typically superimposed to improve the signal-to-noise ratio of adjacent impact surfaces on the sample material.

[0062] In addition to simultaneously acquiring ion spectrum data and fluorescence response data, the sample material is also subjected to high-resolution fluorescence analysis separately (illustrated as arrow 36). This fluorescence analysis is preferably performed before ion spectrum analysis, but in principle, it can also be performed afterward, provided that the sample material and the at least one fluorescent substance on the sample material have not been consumed or rendered unusable by the ion spectrum analysis. The spatial resolution of the high-resolution fluorescence image data can be in the nanometer range, selected from or consisting of the following ranges: ≤1000 nm, ≤500 nm, ≤300 nm, ≤100 nm, ≤50 nm, or below any other upper limit of 1000 nm. An example of a high-resolution microscope is the Nikon Eclipse 90i (Nikon Instruments, Melville, NY).

[0063] After measurement, essentially three datasets exist: ion spectrum data, containing molecular abundance and distribution information of image elements or pixels from the sample material; fluorescence response data, acquired substantially simultaneously with the ion spectrum data at the same spatial grid, and containing information about the frequency distribution of at least one fluorescent substance on the sample material; and high-resolution fluorescence image data, with distribution information of said at least one fluorescent substance, which has substantially higher spatial resolution and is optionally acquired at a different spatial grid. Understandably, for the comprehensive analysis of these data, it is most advantageous to co-register and process the high-resolution fluorescence image data (which has very detailed information about the arrangement of visible features of the sample material) and the ion spectrum data (which reflects the molecular abundance of the sample material). Co-registration can pose challenges due to different data acquisition modalities, often significantly different spatial resolutions, and potentially different clamping methods during data acquisition. In particular, the data quality, especially in terms of positional fidelity, may be affected after fusion due to the computationally assisted matching process required.

[0064] Fluorescence response data acquired simultaneously with ion spectrometry data forms a suitable link between ion spectrometry data and high-resolution fluorescence image data, allowing for higher data quality when fusing different modalities: They are acquired under the same clamping conditions on the sample carrier regardless, and therefore inherently possess the same positional coordinates as the ion spectrometry data; they contain the frequency distribution, e.g., fluorescence intensity, of the at least one fluorescent substance within a narrow band wavelength range for each image element or pixel, a frequency distribution necessarily consistent with the frequency distribution of the same at least one fluorescent substance in the high-resolution fluorescence image data generated by a high-resolution fluorescence image receiver. By comparing and registering the fluorescence response data with the high-precision fluorescence image data, and simultaneously registering and mapping the high-resolution fluorescence image data with the ion spectrometry data, the ion spectrometry data and fluorescence response data are inherently coupled in terms of spatial resolution and positional coordinates.

[0065] Figure 2 It shows a kind of Figure 1 The embodiments shown are very similar. Therefore, the following discussion will only focus on those similar to those shown. Figure 1The difference lies in the first embodiment. The same reference numerals denote the same components in both figures. The main difference is that the fluorescence emitted in response to the local excitation of the sample material by energy injection 4 is collected in transmitted light through a suitable transparent and light-transmitting sample carrier 6, rather than in incident light. The advantage of this embodiment is that the area in the ion source where sample material ablation, ion generation, and ion extraction occur can remain free of the optical components required for detecting the fluorescence response. Furthermore, the embodiment in transmitted light allows observation with a high aperture value and along an axis substantially perpendicular to the extension direction of the sample carrier 6, thus experiencing less geometric distortion.

[0066] As Figure 1 and Figure 2 The illustrated embodiment can be extended to employ other arrangements to excite fluorescence response and acquire fluorescence response data, while simultaneously acquiring ion spectrum data. In an embodiment not shown, the energy injection used to excite fluorescence response on the sample material can be transmitted through the sample carrier to the sample material in the transmitted light (transmission case). This is a method that has been established particularly well in MALDI imaging, see, for example, the study by Andre Zavalin et al. (J MassSpectrom., November 2012, 47(11):i). Excitation in transmitted light can be combined with the acquisition of fluorescence response data in incident light, similar to... Figure 1 As exemplified in the example, or in combination with fluorescence response data acquisition in transmitted light, such as Figure 2 As exemplified in the example. In the latter variant (where both excitation and acquisition occur in transmitted light), it is advantageous if the excitation beam and response beam are well separated from each other. However, the use of a beam splitter, appropriately selected filters, and dichroic mirrors is also beneficial. Figure 2 The necessary adjustments to the optical path shown do not pose a significant challenge for those skilled in optics.

[0067] Figure 3This illustrates how fluorescence response data (acquired simultaneously with ion spectroscopy measurements of the sample material and having relatively low spatial (lateral) resolution (right side of the figure)) can be co-registered with high-resolution fluorescence image data (acquired separately from the same sample material using a separate instrument with its own sample chamber (left side of the figure)) on the xy plane parallel to the surface of the sample carrier supporting the sample material. Different spatial resolutions are represented in the two grids by different image element sizes or pixel sizes. Different clamping methods used for measurement can also lead to effects equivalent to distortion, and are therefore illustrated accordingly in the figure. Algorithms for comparing and co-registering different records of the same modality can be designed to overlay the two frequency distributions of the at least one fluorescent substance with the greatest possible consistency. To this end, a transformation can be created derived from applying various stretching, compression, and / or rotation mathematical operations. These mathematical correlation operations can be uniformly applied to the entire image and response data (affine), or calculated on surface segments or surface regions (elastic), to achieve optimal consistency between the frequency distributions of the at least one fluorescent substance.

[0068] Examples of affine transformations can include operations such as rotation, scaling, shearing, compression, or translation, as well as combinations of these operations. Affine transformations can be represented using transformation matrices. Elastic transformations additionally allow for compensation for distortion and deformation. An example of an elastic transformation is the thin plate spline method. Elastic transformations are typically not represented using transformation matrices but are solved using polynomial fitting or vector fields.

[0069] Preferably, the different spatial resolutions of the spatially resolved fluorescence response data and the high-resolution fluorescence image data are adapted to each other. This can be achieved by mapping the high-resolution image data to the lower spatial resolution of the response data, for example, by summarizing image information from individual image elements or pixels, aided by applying mathematical operations such as interpolation, extrapolation, and / or regression, if necessary.

[0070] Measurement example: Figure 4An exemplary measurement result is shown. This measurement was performed using a mass spectrometer with a structure consistent with that described in the paper 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 spectrometry measurement was 5 micrometers in pixel size. No MALDI-2 post-ionization was performed. The sample material was a slice of mouse cerebellum, and DHAP was used as the matrix material. Prior to matrix material preparation, the sample material was stained with Hoechst 33342 fluorescent dye (bound to the cell nucleus, UV excitation, emission peak at 455 nm) and Green Actin Tracker (bound to actin filaments, excitation at 503 nm, emission at 512 nm). High-resolution fluorescence images were then created using a Slide Scanner during the first clamping of the sample carrier. Figure 4 The data on the left, labeled “External, High-Resolution Fluorescence Microscopy,” is categorized into two channels: one for Hoechst 33342 fluorescence data (fluorescence channel 1) and the other for GreenActin Tracker fluorescence data (fluorescence channel 2). Subsequently, matrix material was applied via (re)sublimation, and MALDI-MSI measurements were performed during a second clamping of the sample carrier.

[0071] The signal labeled "Fluorescence Accompanying MALDI" in the right half represents the fluorescence response signal intensity of Hoechst 33342 between 450 and 460 nm during the MALDI process. For better illustration, this signal has been normalized to the total fluorescence intensity on that pixel (equivalent to total ion count (TIC) normalization). Figure 4 The far right shows an example of MALDI ion spectroscopy image data acquired simultaneously with the accompanying fluorescence response. These MALDI image data can be summarized and correlated into a segmented display (far right of the figure).

[0072] If the co-registration of high-resolution image data and ion spectrum data is performed based on at least one fluorescent substance via fluorescence response data as an intermediary, then the transformation mapping the positional coordinates of the ion spectrum data to the positional coordinates of the high-resolution fluorescence image data can also be applied to fluorescence image data generated using other fluorescent substances, which are acquired simultaneously with the fluorescence image data used to calculate the transformation. In the example shown, the ion spectrum data can also be co-registered with, for example, fluorescence image data from fluorescence channel 2 (Green Actin Tracker). It may be advantageous that different fluorescent dyes bind to different molecules in the sample material, which in turn mark different physiological, biological, or metabolic characteristics in the tissue sections. In this way, within the framework of this disclosure, the ion spectrum data can be compared and mutually registered, particularly co-registered, with fluorescence image data of several fluorescent substances, mediated by simultaneously acquired fluorescence response data, the number of which is selected from the group consisting of or including: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or any other suitable integer between 1 and 20.

[0073] The invention has been described above with reference to various specific embodiments. However, it is understood that modifications can be made to various aspects or details of the embodiments without departing from the scope of the invention. Furthermore, the features and measures disclosed in connection with different embodiments can be combined arbitrarily, provided that this is feasible to those skilled in the art. Moreover, the above description is for illustrative purposes only and is not intended to limit the scope of protection, which is defined solely by the appended claims and any possible equivalents.

Claims

1. A method for analyzing sample material arranged on a sample carrier and having at least one fluorescent substance, comprising the following steps: (a) Using energy injection to acquire spatially resolved ion spectrum data of a sample material and simultaneously acquiring spatially resolved fluorescence response data of the sample material with a first spatial resolution, wherein the energy injection is designed and adapted to locally trigger the fluorescence response of the at least one fluorescent substance on the sample material; (b) Acquire fluorescence image data of the sample material with a second spatial resolution, the second spatial resolution being substantially greater than the first spatial resolution; as well as (c) The spatially resolved fluorescence response data and the fluorescence image data are compared and registered together.

2. The method according to claim 1, wherein, The spatially resolved fluorescence response data includes a first spatial frequency distribution of the at least one fluorescent substance over the entire sample material, and the fluorescence image data includes a second spatial frequency distribution of the at least one fluorescent substance over the entire sample material, wherein, for the comparison and mutual registration, the deviation between the first spatial frequency distribution and the second spatial frequency distribution is minimized.

3. The method according to claim 1 or 2, wherein, Step (b) is performed before and / or after step (a).

4. The method according to any one of claims 1 to 3, wherein, Use tissue sections or cell cultures as sample materials.

5. The method according to any one of claims 1 to 4, wherein, The spatially resolved fluorescence response data and fluorescence image data are derived from at least one exogenous fluorescent substance, such as Hoechst33342, DAPI, or CalceinBlue, which is added to the sample material before the acquisition of the ion spectrum data, the fluorescence response data, and the fluorescence image data.

6. The method according to any one of claims 1 to 5, wherein, The energy injection is triggered by a pulsed laser, which, for example, emits laser pulses in the ultraviolet spectral range.

7. The method according to claim 6, wherein, The laser pulse is applied locally to the sample material as either incident or transmitted light.

8. The method according to any one of claims 1 to 7, wherein, Prior to step (a), the sample material is prepared using a matrix material suitable for matrix-assisted ionization, particularly for matrix-assisted laser desorption / ionization (MALDI).

9. The method according to any one of claims 1 to 8, wherein, The fluorescence response includes the fluorescence emission of the at least one fluorescent substance, which is detected optically, for example, using a microscope.

10. The method according to claim 9, wherein, The fluorescence emission is optically detected in the form of a broadband spectrum, or, for example, in a filtered narrow-band wavelength / frequency range.

11. The method according to any one of claims 1 to 10, wherein, The ion spectroscopy data includes the third spatial frequency distribution of at least one target molecule in the sample material, particularly selected from the group consisting of or composed of the following: proteins, peptides, lipids, carbohydrates, nucleotides, metabolites, and biopolymers.

12. The method according to any one of claims 1 to 11, wherein, To perform the comparison and registration, the second spatial resolution of the fluorescence image data and the first spatial resolution of the fluorescence response data are adapted to each other, for example, by reducing the second spatial resolution.

13. The method according to any one of claims 1 to 12, wherein, The results of the comparison and mutual registration are used to perform spatial co-registration of the ion spectrum data and the fluorescence image data, for example, using a transformation matrix, polynomial fitting, or vector field.

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

15. A system for analyzing sample material disposed on a sample carrier and having at least one fluorescent substance, comprising: An ion analyzer, which is arranged and designed to acquire spatially resolved ion spectrum data of sample materials; A fluorescence-response receiver, which is arranged and designed to acquire spatially resolved fluorescence response data of sample material; A fluorescence image receiver, which is arranged and designed to acquire high spatial resolution fluorescence image data of sample materials; as well as A processing apparatus, which is in data communication with the ion analyzer, the fluorescence response receiver and the fluorescence image receiver, and is arranged and programmed to perform the method according to any one of claims 1 to 14.

16. The system according to claim 15, wherein, The ion analyzer includes a mass analyzer, an ion mobility analyzer, or a combination of ion mobility and mass analyzers.

17. The system according to claim 15 or 16, wherein, The fluorescence-response receiver has a camera, a photomultiplier, and / or a spectrometer, which are particularly directly irradiated or, in the case of using an optical waveguide.

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