Methods for the analysis of sample material

Abstract

The invention relates to methods for the analysis of sample material which is arranged on a sample carrier, comprising: - providing the sample carrier with sample material, - exciting fluorescent light emission from the sample material, - detecting emitted fluorescent light, - determining location data for the sample material along a z axis which is substantially perpendicular to a plane of the sample carrier, using the detected fluorescent light, - making a focus plane, object plane and / or image plane of an imaging optical assembly, which is used during scanning of the sample material, substantially congruent with the sample material using the determined location data, and - ion-spectrometrically analysing the sample material. The invention also relates to devices for carrying out such methods.

Description

Method for analyzing sample material Field of the invention

[0001] The invention relates to methods for analyzing sample material arranged on a sample carrier, and to devices for carrying out such methods. Principles of the present disclosure can be advantageously used, particularly in mass spectrometric imaging, e.g., of tissue sections, but they can also be advantageous when sampling fields from spot preparations. 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] In laterally high-resolution imaging mass spectrometry using matrix-assisted laser desorption and ionization (MALDI-MSI, Mass Spectrometry Imaging), the size of the ablation laser's light spot on the sample material's surface significantly influences the achievable image element or pixel size. For small laser spot sizes with diameters of approximately 1 micrometer, for example, lenses or more complex lens systems such as objectives (hereinafter referred to as lenses) with a short focal length of a few tens of millimeters are required, depending on the beam parameter product. See, for example, patent US 7,180,058 B1, according to which an upper limit for the focal length of 25 millimeters must be observed for a transmission MALDI setup. At the same time, the depth of field of such short-focal-length lenses is very shallow, at just a few micrometers.Consequently, the distance between the focusing lens and the sample material is a critical parameter.

[0004] To keep the sample material in the laser's focal plane during a MALDI-MSI measurement with high lateral resolution, it is often necessary to track the sample material in the z-axis direction, which is substantially perpendicular to the surface of the sample carrier. It is necessary to determine the plane in which the sample material is located in space with the necessary accuracy before ablation. The focal point of the ablation beam is Typically, this is the location of smallest spatial extension, sometimes referred to as the beam waist. If the ablation beam hits the sample material outside its focal plane, where it is broadened, the best possible lateral resolution is not achieved. Furthermore, an unnecessarily enlarged light spot on the sample material can lead to a critical energy density or fluence for the ablation process not being achieved, which can result in suboptimal ionization.

[0005] Sample tracking along the z-axis during a MALDI-MSI measurement requires automatic detection of the focal or image plane of the ablation laser. This can be performed individually for each pixel of a MALDI-MSI measurement. Alternatively, points distributed across the sample surface can be measured. The z-correction for additional points can then be calculated by interpolation.

[0006] The detection of the focal plane can basically be realized by two different approaches:

[0007] 1. A camera lens is aligned coaxially to the beam path of an ablation laser in transmission, which acquires a bright-field microscope image. The focal planes of the camera and laser are adjusted so that they match as precisely as possible. Subsequently, the focal plane of the camera also serves as a measure for the focal plane of the ablation laser, as described in the recently filed patent application PCT / DE2022 / 100627. The focal plane of the camera can be found using various autofocus approaches. This procedure requires that the camera delivers a high-contrast image in the focal plane. However, circumstances may arise under which this cannot be guaranteed depending on the sample material. In particular, if the sample material is already coated with MALDI matrix substance, this approach may not fully meet the requirements.

[0008] 2a. Furthermore, a light pattern can be projected onto the sample material and optically recorded in incident light, from which the height can be determined by triangulation, see the applicant's patent publication DE 10 2007 006 933 A1 (corresponding to US 2008 / 0191131 A1 and GB 2 446 699 A). The resolution in the z-axis direction that can be achieved with this approach is limited to a few micrometers and therefore does not meet the requirements for more demanding accuracies, e.g., approximately 1 micrometer. Furthermore, the resolution in the xy-plane (sample support plane) is limited to the size of the projected pattern, e.g., approximately 100 x 100 square micrometers, which ultimately amounts to an averaging over the area of ​​the sample material covered by the projected pattern.

[0009] 2b. In further development of approach 2a, an additional laser can be used for triangulation, see the study by Mario Kompauer et al. (Nat. Methods 2017, 14, 1156-1158) and the related patent US 10,964,519 B2. In this case, an additional laser must be purchased and adjusted, making the method expensive and technically complex. Furthermore, the additional laser is irradiated at an angle, which, in unfavorable cases, can cause shadowing effects and thus inaccuracies in the optical-visual analysis. Furthermore, this method is not optimally compatible with transmission MALDI. The lateral resolution of the method in the xy plane is limited to the diameter of the laser used, e.g., < 10 micrometers in Mario Kompauer et al.

[0010] In the following, some prior art documents that may be relevant to the subject matter of the present disclosure are briefly reviewed:

[0011] Patent publication US 2006 / 0289734 A1 presents a method for generating a sharp image of a region on a sample plate for a matrix-based ion source, comprising: positioning the region in a field of view of an imaging device; generating a first image having an in-focus region and an out-of-focus region using the imaging device; generating a second image having an in-focus region and an out-of-focus region using the imaging device; and generating a final sharp image using the in-focus regions of the first and second images.

[0012] Patent publication US 2019 / 0113448 A1 discloses a mass spectrometer using near-infrared (NIR) fluorescence, comprising: a plate configured to be loaded with a sample containing a matrix for ionization and a fluorescent material with an excitation wavelength of an NIR wavelength band; a fluorescence detection unit configured to acquire a fluorescence image of the sample on the plate using the fluorescent material; a light-emitting unit configured to emit a laser beam to ionize the sample on the plate; and an ion detection unit configured to detect ions generated by the sample using the laser beam. By using NFR fluorescence, fluorescence interference due to a matrix used for MALDL imaging is said to be eliminated, even after coating the matrix.Since fluorescence interference due to autofluorescence in tissue is low, fluorescence should be measured even in a thick sample, thus enabling accurate analysis by performing MALDI imaging on a portion of interest in a sample determined based on a fluorescence image.

[0013] The authors of the study "FluoMALDI microscopy: matrix co-crystallization simultaneously enhances fluorescence and MALDI imaging" by Ethan Yang et al. (bioRxiv preprint doi: https: / / doi.Org / l0.1101 / 2023.05.27.542340; May 27, 2023) report that co-crystallization of fluorophores with matrix substances for MALDI imaging can increase the brightness of fluorophores by up to 79-fold. This should facilitate the imaging of the same biological sample using both fluorescence microscopy and MALDI imaging. The high spatial resolution and specific labeling capabilities of fluorescence microscopy are combined with the multiplexed, versatile imaging capabilities of MALDI imaging.According to this report, MALDI matrix substances do not obscure or hinder optical microscopy approaches, and it should be possible to image exactly the same cells in tissues without physical changes between fluorescence and MALDI imaging, which should minimize data registration processes. Co-crystallization of matrix and fluorophore should also facilitate applications with insufficient fluorescence brightness.

[0014] There is therefore a need to improve and optimize the conditions for analysis, and in particular, scanning, of sample material on a sample carrier that is to be subjected to ion spectrometric analysis. Of particular interest is the improvement and optimization of conditions for material removal during ionization for ion spectrometric analysis, which may include mass analysis. 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

[0015] According to a first aspect, the present disclosure relates to a method for analyzing sample material arranged on a sample carrier, comprising: - providing the sample carrier with sample material, - exciting fluorescent light emission from the sample material, - detecting emitted fluorescent light, - determining location data of the sample material along a z-axis substantially perpendicular to a plane of the sample carrier using the detected fluorescent light, - substantially registering a focus, object and / or image plane of an imaging optics assembly used in an (analytical) scan of the sample material with the sample material using the determined location data and - analyzing the sample material using ion spectrometry.

[0016] The sample material on the sample carrier may comprise a tissue section, which is provided, for example, from a frozen block or as an FFPE-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 individual or spot preparations on the sample carrier, e.g. a field or a plurality 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).

[0017] The sample carrier can be plate-shaped. The sample carrier can have the dimensions of a conventional 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-containing substrate that has a conductive coating on a surface supporting the sample material. Examples include indium tin oxide-coated glass slides. Principles of the present disclosure are particularly capable of optimizing and improving the removal conditions during the ionization of sample material for ion spectrometric analysis, since commercially available sample carriers can certainly be designed with a "thickness range" varying across the surface on the order of ±50 micrometers. Accordingly, the position of the sample material across the xy plane along the z axis can also vary.Even an analysis of sample material on a sample carrier that ideally has a completely uniform thickness can benefit from the principles of the present disclosure, particularly if the sample carrier is geometrically imperfectly mounted in its holder, e.g., if it is tilted. A variation in the thickness of the sample material itself (e.g., ±5 micrometers across a tissue section of a few square centimeters) can also be compensated for using the principles of the present disclosure.

[0018] In various embodiments, electromagnetic waves can be irradiated onto the sample material to excite fluorescent light emission. The wavelength of the irradiated electromagnetic waves can cover the ultraviolet spectral range, e.g., between 300 and 600 nanometers, in particular between 300 and 400 nanometers, preferably between 350 and 380 nanometers. In various embodiments, the electromagnetic waves can be conditioned to excite fluorescent light emission by filtering white light or by using a light source with chromatically limited Emission characteristics are used. In particular, a light-emitting diode (LED) can be used as the source of the radiated electromagnetic waves.

[0019] The area of ​​the sample material exposed to the irradiated electromagnetic waves to elicit a fluorescence response can comprise a largely circular or elliptical surface with a diameter or major axis of 100 micrometers or less, 80 micrometers or less, 50 micrometers or less, or 25 micrometers or less. It is also possible to irradiate the electromagnetic waves onto surfaces with other contours, such as rectangular or oval, and of a corresponding size, instead of circular or elliptical surfaces.

[0020] In various embodiments, the sample material can be treated with an exogenous fluorescent marker before exciting fluorescent light emission, or autofluorescence of the sample material can be stimulated. The exogenous fluorescent markers are preferably optically stable, can be applied over a large area, and serve to generate a clearly detectable fluorescence response from the sample material. Fluorescence markers can in particular comprise fluorophores in the form of small molecules, e.g. Hoechst 33342 (preferred excitation wavelength: 352 nanometers; emission maximum: 454 nanometers), which binds to DNA in the sample material, Hoechst 34580 (preferred excitation wavelength: 371 nanometers; emission maximum: 438 nanometers) or CellMask™ Green Actin (preferred excitation wavelength: 505 nanometers; emission maximum: 512 nanometers), which binds to actin in the sample material.To form an exogenous fluorescent marker, a fluorophore can be conjugated to a fluorophore carrier such as wheat germ agglutinin, which ultimately binds to the sample material. An example is a molecular complex of wheat germ agglutinin and FITC or Hoechst 33342.

[0021] Additionally or alternatively, staining using antibodies as a component of exogenous fluorescent markers is also conceivable. A primary antibody binds to the sample material, for example, to a ubiquitous protein, while a secondary antibody bound to the primary antibody is conjugated to a fluorophore. One example is a molecular complex consisting of a primary antibody, a secondary antibody, and Alexa 594 (preferred excitation wavelength: 590 nanometers; emission maximum: 618 nanometers). Such antibody complexes typically have molecular weights well beyond 100,000 atomic mass units.

[0022] A protocol for treating sample material, e.g. a tissue, with a fluorophore-antibody complex can be as follows: (a) Fixing the Sample material on a slide, (b) washing the sample material with a suitable wash buffer, (c) incubating the fixed and washed sample material with a primary antibody, (d) washing the treated sample material with a suitable wash buffer, (e) incubating the treated sample material with a secondary antibody, (f) washing the treated sample material with a suitable wash buffer, (g) applying a suitable fluorophore to the treated sample material, (h) washing the treated sample material with a suitable wash buffer and (i) applying a matrix substance suitable for matrix-assisted ionization, e.g. MALDI, to the treated sample material.

[0023] The use of exogenous fluorescent markers has the advantage of a significantly enhanced and thus more easily detectable fluorescence response. Furthermore, the optical setup for detecting the excited and emitted fluorescent light can be pre-configured and optimized for the corresponding emission characteristics.

[0024] In various embodiments, the sample material can be treated with a matrix substance for matrix-assisted ionization before exciting fluorescent light emission, and optionally after treatment with an exogenous fluorescent marker. A method described in this disclosure can be carried out in an ionization device for an ion analyzer that operates according to the principle of matrix-assisted ionization, in particular MALDI. Depending on the requirements, matrix-assisted methods in reflected light (in reflection) or transmitted light (in transmission) can be used for ablation and / or desorption. The MALDI method, for example, requires a specific sample preparation with a light-absorbing matrix substance, for example, sinapic acid, 2,5-dihydroxybenzoic acid, α-cyano-4-hydroxycinnamic acid, 2-(4-hydroxyphenylazo)benzoic acid, or 2,5-dihydroxyacetophenone, all of which absorb strongly in the ultraviolet spectral range.Suitable beam sources include laser light from a nitrogen laser at a wavelength of approximately 337 nanometers or a frequency-tripled solid-state Nd:YAG laser at approximately 355 nanometers. The beam energy is preferably in the range of 0.01–50 microjoules per laser pulse, whose pulse duration can typically be between 500 picoseconds and 20 nanoseconds. The lower limit of the beam energy is particularly applicable to small laser foci on the sample material, such as those achieved with transmission MALDI or transmitted-light MALDI. Ablation and / or desorption can be supplemented by a post-ionization modality, such as the so-called MALDI-2 method (see Jens Soltwisch et al., Science, March 5, 2015, Volume 348, Issue 6231, pages 211–215).

[0025] It is possible to apply an exogenous fluorescent marker to the sample material on one side that is in contact with the sample carrier. Alternatively or additionally, it is possible to Apply the exogenous fluorescent marker to the side of the sample material facing away from the sample carrier-sample material interface. This top-side application may correspond to an application at the sample material-matrix substance interface, e.g., if the sample material is prepared for matrix-assisted ionization after application of the exogenous fluorescent marker.

[0026] In various embodiments, the sample material and the sample carrier can be kept in a negative pressure range. A typical pressure, applicable for example for vacuum MALDI, is substantially greater than a high vacuum (> 10' 3 hectopascals) and less than about 10 2Hectopascals (< atmospheric pressure), e.g., 0.1-10 hectopascals. In various embodiments, ionization of the sample material for ion spectrometric analysis can be directed along the z-axis using determined location data. A control unit or guidance system can use the determined location data to modify the optical properties set for the sample material's examination to ensure the best possible examination.

[0027] In various embodiments, a detector capable of chromatically selective detection of light can be used to detect emitted fluorescent light. The functionality of selective detection preferably serves to remove interfering background from the received measurement signal. In particular, the detector can be equipped with an upstream emission filter. It is possible to use conventional fluorescence channels for filtering, e.g., DAPI (4,6-diamidino-2-phenylindole, emission at approximately 461–500 nanometers, depending on the binding state), FITC (fluorescein-5-isothiocyanate, emission at approximately 519 nanometers), or TRITC (tetramethylrhodamine isothiocyanate, emission at approximately 570 nanometers), and the like.

[0028] In various embodiments, the sample material can be flat, and location data of the emitted fluorescent light along the z-axis can be determined at various locations on the sample material (in the xy plane) and used to derive a topography profile of the sample material. In particular, deriving the topography profile can involve interpolation and / or extrapolation and / or regression based on determined location data of the emitted fluorescent light along the z-axis. This procedure allows an assumed continuity of the positional distribution of the sample material on the sample carrier along the z-axis to be translated into a reduced number of actual measurements or analytical scans in a time-saving manner.

[0029] In various embodiments, the focus, object, and / or image plane can correspond to (i) an object plane of a microscopic image of the sample material or (ii) a focus and / or image plane of an ablation beam directed at the sample material, which is used during ionization of the sample material. A microscopic image, for example a brightfield and / or darkfield image, allows a spatially high-resolution representation of structural features of the sample material. The lateral resolution can, for example, be in the submicrometer range, e.g., in the range of a few hundred nanometers, and can visualize structures within a cell (e.g., eukaryotic, either individually or in a tissue), which is difficult to achieve with material-ablating ionization methods.Although very high-resolution, laterally ablated scans using a primary ion beam in secondary ion mass spectrometry (SIMS) are possible, these require a high vacuum in the ionization region and are therefore complex to perform. Also worth mentioning is inductively coupled plasma mass spectrometry (ICP-MS), which is only truly suitable for the detection of elements, such as metal atoms, in or on the sample material and is therefore severely limited in terms of the evaluable mass range.

[0030] Images from different modalities, e.g., a microscopic image and an ion distribution map, can be computationally merged (image fusion) and thus provide 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, 366-372). In addition to or as an alternative to determining location data or coordinates of the sample material along the z-axis using the detected fluorescence light, optical scanning in the form of a microscopic image of the sample material can be used to determine location data.With the aid of principles of the present disclosure using excited (auto-)fluorescence, in particular an optimal focus or image plane for material removal can be found even if the microscopic image does not have sufficient contrast, e.g. due to excessive penetration of the sample material with matrix substance, provided that the sample material is prepared for matrix-assisted ionization.

[0031] In various embodiments, at least one of the following steps can be carried out in transmission through the sample carrier: (i) irradiating the sample material with electromagnetic waves, (ii) detecting emitted fluorescent light from the sample material, (iii) directing an ablation beam onto the sample material. For this purpose, the sample carrier can be designed to be transparent to electromagnetic waves or translucent. Performing the above-mentioned steps in transmission allows, in particular, the ionization region, in which sample material is ablated and gas-phase ions are generated, to be kept free of optical components. This design lessens the obstacles to handling the ablated sample material.

[0032] In various embodiments, at least two of the following steps can be performed using a same imaging optics assembly: (i) detecting emitted fluorescent light from the sample material, (ii) irradiating the sample material with electromagnetic waves, (iii) directing an ablation beam onto the sample material. In particular, the imaging optics assembly can comprise a microscope objective. The optical components in the microscope objective can be configured and / or optimized for guiding and focusing a few wavelength ranges, e.g., the wavelength of the excitation light for fluorescence, the wavelength of the emitted fluorescent light, and the wavelength of the ablation beam. The microscope objective can be set to infinity focus or can be adjustable.

[0033] In various embodiments, substantial registration can be achieved by changing the position of at least one of the following along the z-axis: (i) the sample carrier, (ii) an object plane of a microscopic image, (iii) a focus and / or image plane of an ablation beam. Movement of the sample carrier in the z-axis direction can be achieved using a suitable actuator, e.g., one capable of operating under vacuum, that can implement step sizes from a few micrometers down to nanometers, e.g., 100 nanometers. Such an actuator can, for example, comprise a piezoelectrically operated translation stage from the manufacturer SmarAct (Oldenburg, Germany), in particular from the CLL, CLS, and SLC series with a positioning accuracy of 40 nanometers. In general, the step size can be in the range of three-digit nanometers or single-digit micrometers.The position of an object, focus and / or image plane can also be changed by adjusting the distance between a microscope objective, through which corresponding radiation is guided and conditioned, and the sample carrier / sample material, for example by moving the microscope objective along the z-axis.

[0034] In various embodiments, the determination of location data or coordinates may include a contrast maximum determination along the z-axis of the fluorescent light detected from the sample material. In particular, several (auto)fluorescence images may be acquired in succession, each with a different orientation of the position of the sample carrier or the focus and / or image plane of the imaging optical assembly along the z-axis, which are then processed by a corresponding image evaluation algorithm to determine the point sharpest image or greatest contrast along the z-axis for the corresponding surface element on the sample material, which can correspond to a pixel or image element.

[0035] In various embodiments, the ion spectrometric analysis may include ion mobility separation and / or mass analysis. Mass analysis may be the final analytical step to which the ionized sample material is subjected. In particular, the ion spectrometric analysis may be performed using a mass analyzer or a coupled mobility separator-mass analyzer. Ion-conducting intermediate stages may be arranged upstream of the actual mass analyzer or the multiple separators and / or analyzers connected in series, and also in various sections between such separators and / or analyzers connected in series. For example, high-frequency voltage ion conductors such as rod multipoles, ring-stack ion conductors, or even RF funnel arrangements may be used. Likewise, different separators and / or analyzers and intermediate stages may be operated at different vacuum levels.

[0036] A mass analyzer separates charged molecules or molecular ions according to their mass-to-charge ratio, commonly referred to as m / z. Time-of-flight analyzers can be used, which can be linear or reflector-type, and / or 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.

[0037] An ion mobility separator separates charged molecules or molecular ions according to their collision cross-section-to-charge ratio, sometimes referred to as Q / z or o / z. This is based on the interaction of the ion species with an electric field that couples to the ion's charge, while simultaneously exposing the ion to a buffer gas that acts on the average cross-sectional area of ​​the ion. Drift tube mobility analyzers with a static electric field gradient are particularly well known. These drive ions through an essentially static gas, with the drift velocity of an ion species resulting from the driving force of the electric field and the decelerating force of the collisions with the gas particles.Also common are trapping ion mobility separators (TIMS), which use a steady laminar gas flow that propels the ions, counteracted by a gradually changing electric field gradient with a correspondingly variable stopping force. Traveling wave mobility analyzers are also worth mentioning. It is understood that analyzers and separators of the aforementioned types can be coupled to separate ion species. to be able to separate multidimensionally, i.e. according to more than one physico-chemical property such as m / z and Q / z or o / z.

[0038] According to a further aspect, the present disclosure relates to an apparatus for carrying out a method as described above. Short description of the figures

[0039] 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.

[0040] Figure 1 schematically shows an apparatus for ion spectrometric analysis in which principles of the present disclosure can be implemented.

[0041] Figure 2A schematically shows an arrangement with three optical assemblies that can be used to carry out principles of the present disclosure.

[0042] Figure 2B shows a schematic diagram of the operation of the first optical assembly shown in Figure 2A.

[0043] Figure 2C shows a schematic diagram of the operation of the second optical assembly from Figure 2A.

[0044] Figure 2D shows a schematic diagram of the operation of the third optical assembly shown in Figure 2A.

[0045] Figure 3 schematically shows an apparatus for combined ion mobility separation and mass analysis with which principles of the present disclosure can be implemented. Detailed description

[0046] 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.

[0047] Figure 1 schematically shows an apparatus for ion spectrometric analysis (10) in which principles of the present disclosure can be implemented and serves to contextualize the disclosed technical teaching.

[0048] The illustration in Figure 1 shows a simplified diagram. Typical normal operation with intermediate storage and possible collisional fragmentation of ions in the ion storage (19) can be as follows: In an ion source with a first laser system (11), a first laser light pulse beam (12) is generated, which passes through a lens (not shown) into the ionization region and impinges on the sample material in transmission through the sample carrier (15). This ionized sample material (16) is pushed into a conventional radio-frequency (RF) ion funnel (17) by a potential at the electrode (14).Optionally, the ion generation can be supported by a second laser light pulse beam (12*) from a second laser system (11*), which is focused laterally into the desorption cloud of the ablated sample material spreading over the sample carrier (15) before a beam catcher (13) absorbs the unused energy away from the sample carrier (15).

[0049] The ions then enter the RF quadrupole rod system (18), which can be operated either as a simple ion guide or ion conductor or as a mass filter for selecting a type of precursor ion to be fragmented. The unselected or selected ions are then fed into the RF quadrupole ion storage (19), where they can be fragmented by energetic collisions depending on their acceleration. The ion storage (19) is enclosed in a gas-tight enclosure and is supplied with collision gas such as nitrogen or argon via the gas supply (20) to focus the ions through collisions and gather them on the axis.

[0050] At predetermined times, ions are extracted from the ion storage (19) through an extraction switching lens (21), formed into a fine primary ion beam (22), and sent to the ion pulser (23). The ion pulser (23) pulses a portion of the primary ion beam (22) orthogonally into the drift zone, which is at a high electrical potential, creating the elongated ion beam (24). The elongated ion beam (24) is reflected in the reflector (25) in a velocity-focused manner and received in the detector (26). The mass spectrometer is evacuated by connected pumps (27), (28), and (29).

[0051] Figure 1 shows how ionized sample material, which moves away from the sample carrier (15) on which the sample material is initially stored, basically along a surface normal of the sample carrier (15), is deflected by means of a deflection electrode (14) and - supported by the gas flow from the ion formation area in the fine vacuum into a subsequent section, which is kept at lower pressure, is led into a connected ion guide, here designed as an RF funnel (17). Those skilled in the art will understand that this design is not to be regarded as restrictive. Equally conceivable is an extraction direction of the ionized ablated sample material which is substantially parallel to a surface normal of the sample carrier (15), which in turn is aligned substantially parallel to a z-axis. In relation to Figure 1, this could mean that the sample carrier (15) is arranged substantially opposite the wide end of the RF funnel (17) (e.g. by displacing the sample carrier clockwise by 90°). The deflection electrode (14) could then be removed or retained and converted into an extraction electrode by utilizing the central opening. The beam paths of the first and second laser light pulse beams (12, 12*), if applicable.by removing and / or adding suitable beam deflectors, as well as the position of the beam catcher (13) for the second laser light pulse beam (12*) would have to be adapted accordingly in such a modified embodiment.

[0052] In a further variant of the structure shown in Figure 1, and also generally within the scope of the present disclosure, it is possible to guide a laser light pulse beam (12*) for the purpose of post-ionization in transmitted light (in transmission) and substantially along a surface normal of the sample carrier (15), as described in the patent specification DE 10 2021 105 327 B3 (corresponds to US 2022 / 0285142 A1 and GB 2 606 441 A), to which reference is expressly made here.

[0053] Figure 2A schematically shows several optical assemblies (30, 32, 34) arranged in an ionization area and having various functionalities that can interact in the analysis of sample material. The ionization area is schematically represented by a sample carrier (15), on the front of which a sample material (36) is placed, e.g. a tissue section. The sample material (36) can be treated with a matrix substance for matrix-assisted ionization and in particular for MALDI. Additionally or alternatively, the sample material (36) can be treated with an exogenous fluorescent marker in order to elicit a clearly detectable and known fluorescence response if an autofluorescence response does not exist or is insufficient. The sample carrier (15) is transparent and allows the transmission of electromagnetic waves, e.g. in the ultraviolet and visible spectral range of approx.300-800 nanometers wavelength. The sample carrier (15) can be designed, for example, as an indium tin oxide-coated glass slide. The sample carrier (15) extends essentially in an xy plane, to which a z axis is substantially perpendicular.

[0054] Close to the rear of the sample carrier (15) is a microscope objective (38) designed to guide and image multiple optical beams. The focal length of the microscope objective (38) can be in the range of a few tens of millimeters, e.g., 15 millimeters. A gas-tight yet light-permeable partition (not shown) can be arranged between the microscope objective (38) and the sample carrier (15), shielding a low-pressure region in the ionization range from ambient conditions outside it. The microscope objective (38) can have a plurality of imaging lenses (not shown). On the side facing the sample carrier (15), the microscope objective (38) has a flange-like, peripheral ring structure (40) containing light sources designed and constructed to illuminate the sample material (36) or parts thereof for the purpose of microscopic bright-field or dark-field imaging.

[0055] In the rear area further away from the sample carrier (15), there are also three optical assemblies (30, 32, 34) which can, on the one hand, ensure the irradiation of energy onto the sample carrier (15) and the sample material (36) and, on the other hand, can detect and focus the response of the sample material (36) to the energy irradiation.

[0056] The first optical assembly (30) is a fluorescence assembly comprising a light-emitting diode (42), an optical waveguide (44) connected thereto in the form of a glass fiber for flexible light guidance, a collimating optical component (46), an excitation filter (48) for selecting a suitable wavelength range (provided the emission characteristic is broadband), a dichroic and / or dielectrically coated mirror (50) for deflection and coupling onto the central optical axis, and an emission filter (52) for selectively transmitting fluorescent light.

[0057] The second optical assembly (32) is an image receiving assembly comprising an imaging optical component (54) in the form of an imaging lens or a more complex lens system and an optical detector (56), e.g. in the form of a digital, CCD or CMOS camera, for detecting a light-optical response from the sample material (36).

[0058] The third optical assembly (34) is an ablation beam assembly and contains a laser system (58) for generating an energetic ablation beam, a lens system (60) for conditioning the ablation beam and two dichroic and / or dielectrically coated mirrors (50', 50") for deflecting and coupling the ablation beam onto the central optical axis.

[0059] A possible way of working with this arrangement is briefly explained below:

[0060] Figure 2B shows a schematic illustration of how the LED light source (42) generates radiation (62, dot-dash) and couples it into the optical waveguide (44), with the excitation radiation being collimated and filtered upon exiting the optical waveguide (44). The filtered light, which now covers a wavelength range suitable for exciting fluorescence in the sample material (36), is deflected by the first dichroic and / or dielectric mirror (50) onto the central optical axis and passes through the second dichroic and / or dielectric mirror (50") on the central optical axis as well as the microscope objective (38), in which the incident light is further conditioned, if necessary, for optimal incidence on the sample carrier (15) and the sample material (36). Finally, after passing through the translucent sample carrier (15), it reaches the sample material (36).There, the excitation light interacts with endogenous fluorophores in the sample material (36) or exogenous fluorescent markers on the sample material (36), the latter being able to have been added to the sample material (36) in a treatment step, and initiates the slightly delayed emission of fluorescent light.Although a not insignificant part of the fluorescent light radiating in all directions is lost in the space of the ionization area or in the environment, the portion that is radiated in the direction of the microscope objective (38) is collected and passes as a beam (64, dash-double-dotted line) bundled by the dichroic and / or dielectric mirrors (50, 50") on the central optical axis, through the emission filter (52), which largely removes light components that do not originate from the fluorescence process in the sample material (36) from the beam path, and the imaging lens (54) or the lens system into the detector (56) where it is received and recorded.

[0061] In order to determine whether the optical properties, in particular of the microscope objective (38), are adjusted with sufficient precision for imaging radiation onto the sample material (36) or for collecting and focusing radiation emanating from the sample material (36), the images of the recorded fluorescent light are evaluated. In particular, in order to be able to determine the position of the sample material (36) in the direction of the z-axis, which corresponds to the central optical axis, the imaging conditions are changed. In one variant, this can be achieved by adjusting the sample carrier (15) along the z-axis, as indicated by the double-headed arrow (66), e.g. using an appropriately adjusted translation stage (not shown), which can also be used to move to positions in the xy plane.The corresponding actuator can incrementally move to different positions on the sample carrier (15) over a specific distance along the z-axis. It is assumed that the fluorescent light is emitted from the sample material (36), which must be true in the case of intrinsic fluorescence (triggered by endogenous fluorophores of the sample material) and in the case of fluorescence using exogenous fluorophores. Fluorescence markers added to the sample material (36) represent a reliable assumption. The further the sample material (36) is located outside the object plane for recording the fluorescent light, the blurrier the fluorescence image becomes. In other words, in such a scenario, the contrast is not optimal. By gradually incrementally changing the position of the sample carrier (15) while simultaneously recording the fluorescent light under these changed conditions, the settings at which the fluorescence image is sharpest or the contrast is greatest can be determined. For example, an area of ​​100 micrometers along the z-axis can be recorded in 5 millimeter increments.The image sharpness or contrast information can be easily translated mathematically into the position of the sample material (36) along the z-axis in the corresponding surface element under observation via the general properties of the optical system, if necessary using interpolation.

[0062] An alternative embodiment of the change in the optical properties consists in changing the imaging optical properties, e.g. by shifting an object plane for the imaging in the detector (56), indicated here by the double-headed arrow (68). This can be implemented using an optical assembly assigned to the microscope objective (38), such as a lens or a lens system (not shown) with variable imaging properties. The lens or at least one lens of the lens system can be adjustable along the optical axis to change the imaging properties. By gradually incrementally changing the imaging optical properties of this overall optical assembly comprising the microscope objective (38) and simultaneously detecting the fluorescent light under these changed conditions, the settings at which the fluorescent image is sharpest or brightest can be determined.the contrast is greatest, similar to what was previously described for changing the position of the sample carrier (15).

[0063] Figure 2C schematically shows an optional step according to which a microscopic image of the sample material (36) is recorded. For this purpose, the ring of light sources (40), which is arranged directly at the microscope objective (38), is activated, and the generated light (70, dot-dashed line), for example white light, is projected in transmission through the sample carrier (15) onto the sample material (36). The light (72, double-dash line), which is also reflected in transmission through the sample carrier (15), passes through the microscope objective (38) itself, the dichroic and / or dielectric mirrors (50, 50") located on the central optical axis, the lens system (54), and is finally received and recorded by the detector (56). The emission filter (52), which was used in the previous step for the selective transmission of the fluorescent light, can be used for the microscopic Recording can be swung out of the beam path, e.g. by turning a filter wheel (not shown) in which the emission filter (52) is mounted.

[0064] If the settings of the microscopic image allow for sufficient contrast, e.g. because the sample material (36) has been prepared accordingly, a method for determining the position of the sample material (36) along the z-axis can also be carried out without fluorescent light excitation, as explained as an embodiment in the international patent application PCT / DE2022 / 100627. In any case, however, the position determination of the sample material (36) along the z-axis using the emitted fluorescent light can also be used to improve the image quality of the microscopic image. For this purpose, the object plane of the microscope objective (38) can be brought into alignment with the position of the sample material (36), e.g. by changing and adjusting the imaging optical properties of the microscope objective (38) or the position of the sample carrier (15) in the ionization region accordingly.This particularly benefits a light-optical microscopic scanning of the sample material, which is carried out for the purpose of structural visualization of the sample material (36).

[0065] Figure 2D shows schematically how an ablation beam (74, dot-dashed line), whose focus and / or image plane is aligned with the position of the sample material (36) along the z-axis determined using fluorescence, e.g., by adjusting the imaging properties of the lens system (60) or adjusting the sample carrier (15) along the z-axis, is directed in transmission through the sample carrier (15) onto the sample material (36). The laser system (58) generates laser pulses that can have a typical duration between 500 picoseconds and 20 nanoseconds and are deflected and coupled to the central optical axis via the lens system (60) and the dichroic and / or dielectric mirrors (50', 50"), where they pass through the microscope objective (38), where they are finally conditioned for incidence on the sample material (36).If the sample material (36) is prepared using MALDI, ions are generated in the ablation cloud (76) from the molecules of the sample material (36) at the point of impact. These ions can be transferred to a connected analyzer, as shown in Figure 1. To increase the degree of ionization or improve the ionization yield, a post-ionization modality can optionally be used, which introduces energy into the ablated sample material. In Figure 2D, the post-ionization modality is exemplified by a second laser pulse (78, dotted line) irradiated laterally into the ablation cloud and timed to match the transmission laser pulse.

[0066] The sequence of steps from Figures 2B, 2C, and 2D can be repeated for additional image elements, pixels, or point preparations of the sample material (36), e.g., using an actuator for the sample carrier (15) that changes its position in the xy plane. In this way, a topography profile map of the z-position of the sample material (36) located on the sample carrier (15) can be created, for example, across the surface of a tissue section or a field of point preparations.

[0067] Figure 3 schematically illustrates a device for combined ion mobility separation and mass analysis (10') with which the principles of the present disclosure can be implemented. Like Figure 1, it serves to contextualize the present disclosure. Figure 3 schematically depicts the setup of a timsTOF fleX (MALDI-TIMS-Qq-OTOF) from Bruker.

[0068] Sample material or molecules from sample material can be ionized in an ionization region (92), e.g., using matrix-assisted laser desorption / ionization (MALDI), and form precursor ions. Ionization can be improved and optimized using position determination by detecting fluorescent light emitted by the sample material, as previously explained with reference to Figures 2A-2D. The precursor ions can be introduced into an ion mobility separator (IMS, 94), which releases the precursor ions sequentially according to their ion mobility. In the embodiment shown, the IMS (94) may be a so-called trapped ion mobility separator (TIMS), in which the ions are preferably (temporarily) stored by the opposing forces of a gas flow and a DC electric field along an axial direction.The ions can typically be radially confined by RF electric fields. The design and operation of a TIMS are explained in detail in US Pat. No. 7,838,826 B1, for example, which is hereby incorporated by reference.

[0069] The TIMS device (94) shown may include an RF ion entrance funnel (96) for collimating and compressing the ion beam, a first TIMS (94-1), a second TIMS (94-2), and an RF ion exit funnel (98). The first TIMS (94-1) may be configured to continuously receive precursor ions and to forward the received precursor ions to the second TIMS (94-2) in a time-controlled manner, enabling the implementation of a parallel accumulation and serial fragmentation method (PASEF®, see, for example, Florian Meier et al., J Proteome Res. 2015 Dec. 4; 14(12): 5378-5387). The second TIMS (94-2) can be configured to perform ion mobility runs in which the precursor ions are released sequentially as ion packets from the second TIMS (94-2) according to their ion mobility. In the illustrated embodiment, precursor ions with lower ion mobility can be released before precursor ions with higher ion mobility in each ion mobility run. In other embodiments, the ion mobility separator can be a traveling-wave IMS (TWIMS), a drift-tube IMS (dt-IMS), or a field-asymmetric IMS (FAIMS). These ion mobility separators can generally release precursor ions with higher ion mobility before precursor ions with lower ion mobility due to different separation mechanisms.

[0070] The ion mobility separator (94) can be operated with ion mobility separation enabled to output ion packets sorted by their mobility, and it can also be operated with ion mobility separation disabled to pass incoming ions through the ion mobility separator (94) without ion mobility separation. In this case, the ion mobility separator (94) can be temporarily considered an ion conductor operating at slightly increased pressure.

[0071] A mass filter (100) is provided downstream of the ion mobility separator (94). The mass filter (100) can be switched to selectively pass precursor ions with m / z values ​​that fall within an adjustable mass selection window. In the embodiment shown, the mass filter can be a quadrupole RF device (100). The mass filter (100) can be operated with a narrow bandpass mode "on" to allow only ions with a comparatively narrow mass range m / z to pass through the mass filter (100), for example within a range extending between 1 AMU and 25 AMU from the lower limit to the upper limit, e.g. between 1000 AMU and 1001 AMU or between 1000 AMU and 1025 AMU, and can also be operated with the narrow bandpass mode "off" or, in other words, in a pure RF mode to allow essentially all incoming ions to pass through the mass filter (100) without narrow bandpass selection.

[0072] Precursor ions that fall within the current mass window of the quadrupole RF device (100) can be forwarded to a fragmentation device (102), in which the precursor ions can be split into fragment ions. In the embodiment shown, the fragmentation device can be a collision cell (102) in which a neutral gas atmosphere is maintained at slightly elevated pressure and into which ions can be shot after acceleration in order to fragment them by collisions with the gas molecules, such as molecular nitrogen or sulfur hexafluoride or mixtures thereof. However, other types of fragmentation devices are also conceivable, such as surface-induced dissociation fragmentation devices, electron transfer dissociation devices (electron transfer dissociation (ETD), electron capture dissociation (ECD) devices, ultraviolet photodissociation (UVPD) devices, and the like.

[0073] The collision cell (102) can be operated in a fragmentation mode "on" to dissociate incoming ions and generate fragment ions therefrom, and can also be operated in a fragmentation mode "off" to only receive and temporarily store incoming ions without breaking them into fragments and passing them on to the next component in the device (10').

[0074] The device (10') may further comprise a mass analyzer (104) that performs a mass, or more precisely, m / z, measurement on the fragment ions and, optionally, on the precursor ions. In the embodiment shown, the mass analyzer is a time-of-light (TOF) analyzer that uses orthogonal injection and has a reflector to extend the trajectory and improve mass resolution.

[0075] Finally, the device (10') comprises a control system (106) configured to coordinate and control each of the components of the device (10') and also to perform data analysis of the mass analysis data or mass spectra obtained from the TOF analyzer (104) that form part of a spectral data set. The control system (106) may comprise one or more microprocessors and a memory for storing suitable computer code to perform system control and data analysis functions. The control system (106) may consist of a single unit or be a distributed system comprising various control units with individual processors and / or special control circuits, ASICs, and the like that communicate with each other. Contrary to the illustration in Figure 3, the control system (106) may also be decentralized, i.e.different sub-areas can be arranged distributed on different components of the overall analytical device (10').

[0076] The device (10') shown in Figure 3 can operate in different modes, two of which stand out, which can be referred to as MSI and MS2 mode (the latter also referred to as tandem MS or MS / MS). When operating in MSI mode, the mass filter (100) and the collision cell (102) can be deactivated, so that all precursor ions released successively from the second TIMS (94-2) are passed to the TOF analyzer (104) without mass selection and fragmentation, thus obtaining high-resolution m / z spectral data of the precursor ions at high speed. This precursor ion spectral data can be used to select samples of interest and therefore to be used in a subsequent measurement run. fragmenting ion species, e.g. using a representation of the precursor ions in a mass mobility map.

[0077] When operating in MS2 mode, the mass filter (100) and the fragmentation cell (102) can be activated so that the incoming precursor ions are mobility-separated, selected according to the set mass selection window of the mass filter (100), and then fragmented in the collision cell (102). This allows high-resolution m / z spectra of the fragment ions resulting from the precursor ions in the collision cell (102). This allows the features in a resulting spectral data set to be sorted according to a greater number of physicochemical properties than in MSI mode. It goes without saying that the fragment ions can be assigned a corresponding mobility or a mobility-related value, such as the collision cross section (CCS) or the voltage setting of the second TIMS (94-2) upon release.

[0078] In the above description, reference was regularly made to (auto-)fluorescence. This means that upon excitation, characteristic light is emitted and detected by the sample material, whether treated with a fluorescent marker or untreated. For the purposes of this disclosure, phosphorescence of the sample material is considered synonymous with fluorescence, since, according to scientific convention, the difference is limited solely to the duration of light emission after excitation.

[0079] 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 not to 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, comprising: - Providing the sample carrier with sample material, - Excitation of fluorescent light emission from the sample material, - Detection of emitted fluorescent light, - Determining location data of the sample material along a z-axis substantially perpendicular to a plane of the sample carrier using the detected fluorescent light, - substantially aligning a focus, object and / or image plane of an imaging optical assembly used in scanning the sample material with the sample material using the determined location data and - ion spectrometric analysis of the sample material.

2. The method according to claim 1, wherein electromagnetic waves are irradiated onto the sample material to stimulate fluorescent light emission.

3. A method according to claim 2, wherein the electromagnetic waves are conditioned to excite fluorescent light emission by filtering white light or using a light source with chromatically limited emission characteristics.

4. A method according to claim 2 or claim 3, wherein a light-emitting diode (LED) is used as the source of the radiated electromagnetic waves.

5. Method according to one of claims 1 to 4, wherein the sample material is treated with an exogenous fluorescent marker before exciting fluorescent light emission or wherein autofluorescence of the sample material is excited.

6. Method according to one of claims 1 to 5, wherein the sample material is treated with a matrix substance for matrix-assisted ionization before exciting fluorescent light emission, and optionally after treatment with an exogenous fluorescent marker.

7. A method according to any one of claims 1 to 6, which is carried out in an ionization device for an ion analyzer which operates according to the principle of matrix-assisted ionization.

8. Method according to one of claims 1 to 7, wherein the sample material and the sample carrier are kept in a negative pressure area.

9. Method according to one of claims 1 to 8, wherein ionization of the sample material for the ion spectrometric analysis of determined location data is conducted along the z-axis.

10. A method according to any one of claims 1 to 9, wherein a detector capable of detecting light chromatically selectively is used to detect emitted fluorescent light.

11. The method according to claim 10, wherein the detector is equipped with an emission filter.

12. Method according to one of claims 1 to 11, in which the sample material is flat, location data of the emitted fluorescent light along the z-axis are determined at different locations on the sample material and are used to derive a topography profile of the sample material.

13. The method according to claim 12, wherein deriving the topography profile comprises interpolation and / or extrapolation and / or regression based on determined location data of the emitted fluorescent light along the z-axis.

14. Method according to one of claims 1 to 13, wherein the focus, object and / or image plane corresponds to (i) an object plane of a microscopic image of the sample material or (ii) a focus and / or image plane of an ablation beam directed onto the sample material, which is used in the course of ionization of the sample material.

15. Method according to one of claims 1 to 14, in which at least one of the following steps is carried out in transmission through the sample carrier: (i) irradiating electromagnetic waves onto the sample material, (ii) detecting emitted fluorescent light from the sample material, (iii) directing an ablation beam onto the sample material.

16. A method according to any one of claims 1 to 15, wherein at least two of the following steps are performed using a same imaging optics assembly: (i) detecting emitted fluorescent light from the sample material, (ii) irradiating electromagnetic waves onto the sample material, and (iii) directing an ablation beam onto the sample material.

17. The method according to any one of claims 1 to 16, wherein the imaging optical assembly comprises a microscope objective.

18. The method according to any one of claims 1 to 17, wherein the substantial registration is carried out by changing a position of at least one of the following along the z-axis: (i) the sample carrier, (ii) an object plane of a micrograph, (iii) a focus and / or image plane of an ablation beam.

19. The method according to any one of claims 1 to 18, wherein determining location data comprises determining the contrast maximum along the z-axis of the fluorescent light detected by the sample material.

20. The method according to any one of claims 1 to 19, wherein the ion spectrometric analysis comprises ion mobility separation and / or mass analysis.

21. Apparatus for carrying out a method according to one of claims 1 to 20.

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