Elemental analysis of organic samples

Abstract

The invention relates to an imaging elemental analyzer for imaging one or more analyte elements in an organic sample, wherein the analyzer comprises: a chamber for receiving an organic sample containing one or more analyte elements to be imaged, wherein the internal chamber pressure surrounding the sample is in the range of 10 -5 up to 10 -2mbar; at least one irradiant selected from: (i) an ion gun for irradiating the sample with a high-intensity primary ion beam, wherein the primary ions are generated in the ion gun at a pressure below 1 mbar, the ion gun serving to focus the primary ion beam onto a localized spot on the sample surface and to move the spot over time to a plurality of locations on the sample surface; (ii) a laser for irradiating a localized spot on the sample surface and to move the spot over time to a plurality of locations on the sample surface; a gas-filled RF ion guide for receiving the generated ions, comprising the elements released from the sample in response to irradiation by the primary ions or the laser, wherein the RF ion guide prevents the propagation of any ions whose m / z values ​​are less than the mass or mass range of the analyte elements;and a time-of-flight (TOF) mass analyzer for recording the ions or reaction products of the secondary ions formed from the RF ion guidance, wherein the TOF mass analyzer is configured for a repetition rate of at least 5 kHz. The invention also relates to a corresponding method and a corresponding device for imaging one or more inorganic, non-volatile elements.

Description

Field of invention

[0001] The invention relates to the field of imaging mass spectrometry, in particular imaging elemental mass spectrometry. In certain aspects, the invention relates to the analysis of the distribution of specific elements in a biological sample, which may have been introduced into the sample as element tags or may occur naturally in the sample. background

[0002] Determining the distribution of so-called inorganic elements in biological samples is important for several reasons. The term "inorganic elements" usually refers to elements other than those that typically form organic material, such as C, H, N, and O. The inorganic elements of interest are usually heavier than oxygen and are typically metallic or semimetallic. The natural distribution of inorganic elements in biological samples provides important information about biological processes at the level of genes, proteins, and metabolites, as demonstrated by the emerging field of metallomics. Furthermore, in an approach called element tagging, a number of such element tags (which can also be called markers) can be artificially added to the target objects in the sample, typically using specific binders (e.g., cyanide, cyanide, or cyanide).Antibodies, aptamers, metabolic tags, etc.) are used to focus on specific targets or processes in biological systems. Numerous different detection techniques can be used to measure the abundance of such tags, such as radioactivity, light (e.g., fluorescence or absorption), including X-ray fluorescence (XRF), secondary electron spectrometry (SES), X-ray photoelectron spectroscopy (XPS), electron microprobe analysis (EMPA), secondary ion mass spectrometry (SIMS), laser plasma ionization mass spectrometry (LPI MS), and inductively coupled plasma mass spectrometry (ICP MS), etc.

[0003] While fluorescence-based assays may be fast, they are characterized by low sensitivity and are limited to one or a few target objects per assay – in contrast to mass spectrometry techniques such as SIMS or ICP MS.

[0004] Mass spectrometry techniques enable a high degree of parallel multiplexed measurement of elements, e.g., using multicollector magnetic sector, time-of-flight, Orbitrap, or Fourier transform ion cyclotron resonance analyzers. However, when spatially resolved analysis is required, e.g., in tissue imaging, low elemental abundance poses a challenge for all these analyzers, as the spectra are dominated by intense matrix peaks from the tissues. These matrix peaks could originate from polyatomic species that constitute the bulk of the tissues, with important elements including not only C, H, N, O, but also S, P, alkali metals (Na, K), etc. Although polyatomic species can, in principle, be analyzed exclusively in RF-mode gas-filled reaction cells (e.g.,While reactions involving ions such as US5,767,512 A and US7,230,232 B2) could be eliminated, such reactions are highly analyte-dependent, could affect the metals of interest, and generally lead to losses of these ions. This is particularly noticeable in imaging applications where the initial amount of analyte is limited from the outset.

[0005] ICP-MS with laser ablation (LA / ICPMS) is known to exhibit a negligible contribution of multiatomic species; therefore, it has become one of the preferred methods for elementary tissue imaging, as described, for example, in WO2010 / 133196A1, DE10354787A1, WO0151907A1, WO02054057A1, US8274735B2, WO 2014 / 063246A1, WO2015128490A1, and others. Acquisition rates of up to several tens of pixels per second with a spatial resolution in the micrometer range (µm) have been demonstrated. Even at such high rates, acquiring a single image still takes several hours. However, a further increase in the acquisition speed is limited by the temporal propagation of the signal due to the propagation of the sample cloud as it travels from the surface to the ICP torch, since the transport process occurs mostly at atmospheric pressure and low transport speeds.Atmospheric pressure is essential for the ICP process. During this process, transmission lines can become coated with aerosol containing sample material, leading to carryover and contamination of the sample delivery unit. At higher throughputs, as required in any clinical application, excessive contamination results in increased analysis costs and longer turnaround times.

[0006] The relocation of the ionization process to a vacuum, as is known according to the state of the art in SIMS or laser plasma ionization approaches, leads to an extraordinarily long scanning process due to a relatively low current of the ions of interest generated, which necessitates long exposure times.

[0007] Such a weak current of generated ions is often caused not so much by ionizing agents or a low efficiency of secondary ion generation, but instead by a relatively low concentration of natural elements or tags in the cell / tissue matrix. This also precludes the use of other multichannel elemental imaging techniques such as SES, micro-XRF, etc. Another problem is the rapid contamination of the vacuum chamber and components of the analyzer by the organic matrix material. For example, the analysis of just a typical 5 µm thick tissue section with an area of ​​100 mm² could be problematic. 2A measuring instrument would become completely contaminated during analysis, given the required sensitivity. SIMS also suffers from the problem of a relatively low sample removal rate, which slows down the analysis of typical tissue samples, often at least 3-5 micrometers thick.

[0008] In the field of isotope ratio mass spectrometry (IRMS), particularly when the isotope ratio analyzer is connected to a gas chromatography (GC) or liquid chromatography (LC) separation stage, the samples are oxidized to remove gases such as CO2, NO. xto form H₂O, which is then analyzed to determine isotopic ratios of elements such as C, N, and / or O. Oxidation can be carried out in a combustion furnace (e.g., in GC-IRMS), as described in Z. Muccio and G.P. Jackson, Isotope ratio mass spectrometry, Analyst 134 (2009) 213–222, or it can involve a wet-chemical oxidation process (e.g., in LC-IRMS), as described in C. Osburn and G. St-Jean, Limnology and Oceanography: Methods 5 (2007) 296–308. “Dry” oxidation, e.g., by UV ozone, is also routinely used for the removal of contaminants from the surfaces of semiconductors, glass, etc.

[0009] DE 198 46 039 A1 discloses a method for sample pretreatment for elemental trace analysis measurements, characterized in that the oxidizable sample components are ashed at temperatures below 230 Kelvin by condensates of reactive gases. The condensation of the oxidizing agent can take place primarily at the location of the sample.

[0010] Against this background, the present invention was made. Summary

[0011] This invention relates to an approach that can be used to process tissue samples prior to their analysis by one of the aforementioned methods. Since the thickness of tissues is typically comparable to the required spatial resolution of the analysis, carefully controlled oxidation conditions can lead to a gradual removal of the organic matrix while simultaneously limiting the diffusion of heavier elements or tags from their initial position. Consequently, a significantly smaller amount of material is desorbed or removed during sample collection, while at the same time providing the necessary analytes of interest and imaging information.

[0012] This invention offers an improved approach to the elemental analysis of organic samples by pre-concentrating the The elements to be analyzed (which in this document may be referred to by different terms as elements of interest or analyte elements) are typically the inorganic elements present in the sample, either naturally occurring or introduced as tags. This allows for the application of a variety of elemental imaging techniques that have proven ineffective in previous approaches.

[0013] According to one aspect of the invention, a specific imaging elemental analyzer is used to image one or more Analytical elements provided in an organic sample, wherein the analyzer comprises: a chamber for receiving an organic sample containing one or more analyte elements to be imaged, wherein the internal chamber pressure surrounding the sample is in the range of 10 -5 up to 10 -2 mbar is; at least one primary irradiant selected from: (i) an ion gun for irradiating the sample with a high-intensity primary ion beam, wherein the primary ions are formed in the ion gun at a pressure below 1 mbar, the ion gun serving to focus the primary ion beam onto a localized spot on the sample surface and to move the spot over time to a plurality of locations on the sample surface; (ii) a laser, preferably a high-power laser, for irradiating a localized spot on the sample surface to form ions and to move the spot over time to a plurality of locations on the sample surface; a gas-filled RF ion guide for receiving formed ions comprising the analyte elements released from the sample in reaction to primary irradiation, wherein the RF ion guide prevents the further conduction of all ions whose m / z value is less than the mass or mass range of the analyte elements; wherein preferably at least some of the formed ions undergo an ion molecular reaction in said ion guide; and a time-of-flight (TOF) mass analyzer for recording the ions formed or reaction products of the ions formed from the RF ion guidance, wherein the TOF mass analyzer is configured to have a repetition rate of at least 5 kHz, preferably 50-100 kHz.

[0014] According to a further aspect of the invention, a method for imaging one or more analyte elements in an organic sample is provided, comprising: Providing the sample as a layer on a substrate; Oxidizing the sample on the substrate to produce one or more volatile products that escape from the sample and enter the gas phase, while one or more analyte elements remain in the sample, wherein a larger part by weight of the sample layer is removed from the substrate by the reaction (preferably oxidation) and the one or more analyte elements are enriched or concentrated in the remaining sample layer; and Detection of one or more analyte elements in the enriched or concentrated sample layer using an imaging elemental analyzer.

[0015] Preferably, the analyte elements are not spatially disturbed by the reaction to a greater extent than by the spatial resolution of the imaging analysis. While some individual analytes may be disturbed by a greater distance than this, on average the analyte elements are preferably not spatially disturbed by the reaction to a greater extent than by the spatial resolution of the imaging analysis.

[0016] According to another aspect of the invention, a device for imaging one or more analyte elements in an organic sample is provided, comprising: the imaging elemental analyzer; a reaction chamber (preferably an oxidation chamber) for receiving the sample, wherein the sample is provided in the form of a layer on a substrate; wherein the reaction (preferably oxidation) chamber comprises an electromagnetic radiation source and / or an inlet for introducing one or more chemical or ionic oxidizing agents into the chamber for oxidizing the sample, for the production of one or more volatile products that escape from the sample and enter the gas phase, while the one or more analyte elements remain in the sample, wherein a heavier part of the sample layer is removed from the substrate by oxidation and the remaining sample layer is enriched with the one or more analyte elements; and wherein the imaging elemental analyzer is located in a detection chamber to detect the spatial distribution of the aforementioned one or more analyte elements in the enriched sample layer.

[0017] According to a further aspect of the invention, an elemental analyzer is provided for mass analysis and preferably for imaging one or more analyte elements in a sample, wherein the analyzer comprises: a chamber for receiving a sample containing one or more analyte elements, wherein preferably the internal chamber pressure surrounding the sample is in the range of 10 -5 up to 10 -2 mbar is; a laser for irradiating a localized spot on the sample surface and for triggering the laser plasma ionization of at least one or more analyte elements in the sample, wherein preferably the laser serves to move the spot to a plurality of locations on the sample surface over time; a reaction cell for receiving ions of one or more analyte elements generated by laser plasma ionization, wherein the composition and emittance of the ions are modified – preferably reduced – as the ions pass through the reaction cell; and a mass analyzer, preferably a time-of-flight (TOF) mass analyzer, for collecting ions of one or more analyte elements and / or ions of reaction products of one or more analyte elements from the reaction cell, wherein the TOF mass analyzer is preferably configured to have a repetition rate of at least 5 kHz. Preferred embodiments

[0018] Preconcentration of analyte elements, which may be tags, on the substrate is achieved by enabling oxidation reactions that convert the organic matrix or sample material into volatile gases, which are then discarded, while the inorganic elements of interest remain on the substrate. Accordingly, the volatile products preferably contain substantially no analyte elements. Furthermore, the inorganic elemental species can end up on the substrate in oxidized form (i.e., in the oxidized sample).

[0019] The analyte elements enriched in the remaining sample can then be detected using an imaging elemental analyzer. Detection can occur at a different time and location (e.g., in a different chamber) than the oxidation. For example, detection typically takes place after oxidation. An image of the elements detected in the sample can then be generated from the detection. The imaging elemental analyzer can thus include a data acquisition system that receives input data from the detection of one or more elements and generates an image of that one or more elements in the sample. Ideally, the imaging elemental analyzer is a device capable of imaging multiple elements simultaneously in a short time, such as one that includes a mass analyzer or polychromator.

[0020] The starting sample is an organic sample, meaning it consists mostly of organic matter and contains a small amount or trace amount of inorganic matter, including the analyte elements to be measured. It can be any sample comprising an organic matrix containing one or more of the analyte elements to be measured. The mass or weight of the sample is predominantly comprised of the organic matrix. The organic matrix can account for at least 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 99.99% of the sample layer by weight.

[0021] The sample can be a biological sample, i.e., of biological origin. The biological sample can originate from an organism. The organism can be a plant, animal, or bacteria. In a preferred application of the invention, the biological sample is tissue and / or individual cells.

[0022] The analyte elements are generally different from those that typically form the organic matrix (C, H, N, and O). Typically, the analyte elements are heavier than oxygen. Typically, the elements are metallic or metalloid elements. The elements may preferably be metals with a mass greater than 16. The elements may be heavy metals. The elements may be selected from rare-earth elements (latethanides), transition metals, post-transition metals, alkali metals, alkaline earth metals, or metalloids. The elements may be radioisotopes. For a wide variety of analytes, the elements may represent any combination of the aforementioned classes.

[0023] The one or more analyte elements can comprise two or more different isotopes of the same element.

[0024] One or more analyte elements may occur naturally in the sample, such as trace elements in a sample like a biological sample. Such elements could also be used as internal standards for improved quantitative determination. One or more analyte elements may be introduced into the sample as Element tags could have been introduced, for example, using methods known in the art for element tagging. A class of preferred element tags are rare-earth elements (especially lanthanides). The tags can be radioisotopes that can be detected by a radioactivity analyzer.

[0025] The one or more element tags can exist as nanoparticles, nanorods, mass dots or quantum dots, e.g. as described in US 2014 / 0221241 A1.

[0026] The one or more element tags can be purified isotopes of rare earth or other elements, or combinations of these in a predetermined ratio.

[0027] One or more element tags can be attached to a binding element that binds to a target object in the sample. The binding element can be specific, binding to a particular target object in the sample. Each element tag (each mass), if more than one is present, can be attached to a different binding element specific to a particular target object in the sample. Thus, a multitude of different target objects can be present. Preferably, each element tag is attached to a different specific binding element. The one or more element tags can be attached directly or indirectly (e.g., via a linker) to the binding element. The binding element can be selected from a stain (e.g., a fluorescent stain), polypeptide, polynucleotide, antibody, affiliation body, aptamer, or SOMAmer™. The target object can be any organic molecule in the sample.In biological samples, the target object can be a biomolecule, e.g., a macromolecule such as those selected from proteins, polysaccharides, lipids, and nucleic acids, as well as small molecules such as metabolites and natural products.

[0028] The target can be an antigen. For example, the element tag can be attached to an antibody, so that it binds to an antibody-antigen complex after the antibody binds to an antigen. The target—or any target—is preferably a biomarker.

[0029] In some embodiments, one or more element tags may have been introduced into the sample metabolically, e.g., via food or carrier media. The element tag can therefore be part of a metabolic label.

[0030] Tagging can also involve using multiple elements in the form of a barcode, e.g. as described in US 2014 / 106976 A1 and in B. Bodenmiller et al., Nature Biotechnology 30 (2012) 858-867.

[0031] The one or more element tags can include two or more different isotope tags of the same element.

[0032] The sample is prepared as a layer on a substrate. The sample is preferably provided as a thin layer, particularly preferably with a thickness of at most (i) 20 µm or (ii) 10 µm or (iii) 5 µm or (iv) 3 µm.

[0033] The substrate is typically a microscope slide, e.g., a planar slide. The substrate or slide can be a flat plate made of metal, glass, or ceramic. In some embodiments, the substrate can have a titanium dioxide surface. For example, any of the aforementioned microscope slides or flat plates can have a titanium dioxide surface. For this purpose, the substrate can be coated with a layer of titanium dioxide, preferably in the form of a titanium dioxide film or immobilized titanium dioxide particles. One of the preferred embodiments of the substrate is a standard glass microscope slide with an indium tin oxide coating, as known from the prior art.

[0034] In some embodiments, the sample may contain a fixed and embedded tissue sample, e.g. formalin-fixed, paraffin-embedded (FFPE) tissue, preferably cut with a microtome, preferably to a thickness of 3-5 µm.

[0035] In some embodiments, the sample may comprise individual cells deposited on a substrate, e.g., by a flow cytometer or a high-content screening device. The cells may be deposited in a grid pattern (e.g., at 50 µm or 30 µm intervals in the X and Y directions). In this case, a typical cell may be up to 5 µm, up to 10 µm, or 5–10 µm in size. An example of grid-based sample preparation is shown in WO2014 / 063246A1.

[0036] In some embodiments, the sample may comprise a cell culture on a growth medium, e.g., a microbial or bacterial culture on a thin layer of a growth medium such as agarose. The culture is preferably up to 10 µm or up to 20 µm thick. Typically, the growth medium is thicker than this culture. The culture samples could be oxidized in the present form (e.g., by plasma etching), but oxidation and subsequent sampling will be less effective with thick growth medium layers. To improve oxidation and sampling, such a sample should preferably be cut to the thickness of, or nearly to the thickness of, a thin culture layer.

[0037] In some embodiments, the sample can be applied by an automatic sampler (e.g., including the following types of automatic samplers: flow focusing, acoustic droplet ejection, induction, etc.).

[0038] The sample can be applied to the substrate, e.g. by the automatic sampler, in the form of individual droplets in or on a grid-like pattern (e.g. every 30-50 µm in the X and Y directions), or in or on microarrays or in a multi-well plate.

[0039] In some embodiments, a large number of samples (which could be different samples) could be applied to a substrate at different locations on the substrate, e.g. in a grid-like pattern as described above.

[0040] Tagging the sample with one or more analyte elements can be performed before or after the sample is placed on the substrate, preferably after.

[0041] In certain embodiments of the invention, the analyte elements are not element tags, but rather naturally occurring elements present in the sample (so-called native elements). Thus, in certain embodiments, the sample remains unprocessed in the sense that it is not labeled. Such methods can be used in applications to determine the distribution of native inorganic elements, such as metallic elements, in the sample, particularly native heavier inorganic elements (e.g., metals, such as Fe, Zn, Sn, etc., e.g., for metallomics experiments).

[0042] After sample preparation on the substrate is complete, the sample can be transferred to a process chamber (e.g., an oxidation chamber) for the oxidation step. Optionally, the sample could be lyophilized prior to this transfer to reduce its water content. The sample can be transferred to a hermetic reaction chamber where one or more preferably strong oxidizing agents act upon it. In one embodiment, the oxidation could involve heating the sample in an oxygen stream or atmosphere to induce combustion. Numerous different combustion or oxidation processes can be employed, provided that the analyte elements or tags are not spatially disturbed by the process to a greater extent than required by the desired spatial resolution of the analysis. The finer the spatial resolution of the analysis (e.g.,The higher the spatial resolution (typically 1 micron or 3-5 microns), the gentler the oxidation process should be. Under no circumstances should gas bubbles or boiling occur, as this would drastically disrupt the original spatial distribution of the elements. In some embodiments, if the desired spatial resolution is on the order of tens of microns, stronger and faster oxidations can be used. Although gas-phase oxidation is preferred, wet-chemical oxidation and etching by RF discharge plasma could also be implemented, as long as the requirement of minimal disturbance to the spatial distribution of element tags is met. A combination of several oxidation processes could be used to accelerate the enrichment of the remaining sample.

[0043] The hermetically sealed chamber can be a reaction chamber separate from the detection chamber, where the detection or analysis takes place, or it can be the same chamber as the detection or analysis chamber. Similarly, in some embodiments, the oxidation chamber is the same chamber as the detection chamber; that is, in these cases, there is a single oxidation and detection chamber. Preferably, the oxidation takes place in a chamber different from the detection chamber, which contains the imaging elemental analyzer. Accordingly, the oxidized sample typically needs to be transferred from the reaction (i.e., oxidation) chamber to the detection / analysis chamber (containing the imaging elemental analyzer). Thus, in these cases, once the oxidation process is complete, the substrate (microscope slide) is transferred to a vacuum chamber for elemental imaging by one of numerous applicable methods.The process as a whole is a two-stage process: conversion / concentration, followed by vacuum-based imaging analysis.

[0044] Preferably, the oxidation step comprises the action of one or more oxidizing agents on the sample, with the sample optionally being heated during the oxidation step. The apparatus, i.e., the reaction chamber, may therefore further include a heating device for heating the sample during the oxidation step. The reaction chamber may be a vacuum chamber. The oxidation may take place in the chamber at elevated (above atmospheric) pressure, at atmospheric pressure, or at reduced (below atmospheric) pressure. The reduced pressure regime may be between 100 and 1000 mbar, or between 1 and 100 mbar. In the latter case, the oxidation may be facilitated by a DC or RF gas discharge.The one or more oxidizing agents can be selected from (i) electromagnetic radiation and / or (ii) one or more chemical oxidizing agents of the gaseous phase and / or (iii) ions or electrons and / or (iv) one or more chemical oxidizing agents of the liquid phase. Thus, a source of one or more chemical oxidizing agents can be connected to the inlet, preferably one or more of which is the source of the oxidizing agent. The oxidizing agents are selected from the following: ozone, hydrogen peroxide, and, as an example of a liquid-phase oxidizing agent, a persulfate (e.g., ammonium, sodium, or potassium persulfate). The latter is preferably used with a catalyst, e.g., phosphoric acid and silver nitrate. The agents can be introduced into the chamber together or sequentially at reduced (less than atmospheric) pressure.

[0045] Preferred gas-phase oxidizing agents include ozone and hydrogen peroxide. The sample can be oxidized by exposure to electromagnetic radiation—in particular light, specifically light with a wavelength of < 400 nm (preferably UV light, but in some embodiments also X-rays). In the example of ozone oxidation, a UV ozone oxidation chamber at atmospheric or elevated pressure, as known in the art, could be used with the additional supply of moist air or moist oxygen and additional activation with UV light of 254 nm and / or 185 nm. If the oxidation is carried out with light, the sample is optionally located on a photocatalytic surface of the substrate (preferably a titanium dioxide surface) that is exposed to the light. The oxidizing light preferably irradiates the surface at luminous intensities above 0.1, 1.0, or 10 milliwatts / cm². 2or in the range of 0.1-10 milliwatts / cm² 2 .

[0046] Accordingly, the one or more oxidizing agents may: (i) be electromagnetic radiation and the oxidation step includes irradiating the sample with light with a wavelength of less than 400 nm, with the substrate acting as a photocatalyst to oxidize the sample; or (ii) be one or more chemical oxidizing agents and the oxidation step may consist of the action of one or more chemical oxidizing agents on the sample, selected from ozone and hydrogen peroxide.

[0047] A similar effect is achieved by bringing the sample into contact with RF or DC gas discharge at low pressure, so that the sample surface is bombarded by charged plasma particles and the oxidation is complemented by sputtering. This process is typically faster and more “aggressive” than UV ozone treatment and therefore better suited for larger clumps or crystals of elements.

[0048] The described oxidation processes are suitable for causing rapid oxidation of the organic matrix atoms: e.g., one or more of the following oxidation reactions: C → CO, CO₂, N → NO, NO₂, H → H₂O, etc. Thus, the one or more volatile products preferably comprise one or more oxides of the elements C, H, and / or N. Optionally, S is converted to SO₂ or other sulfur oxides. The volatile products generated by the oxidation are preferably pumped off, so that a portion—preferably the majority—of the sample mass is removed. Simultaneously, the analyte elements, e.g., heavier elements (heavier than O) and especially the metallic elements, do not form volatile products and therefore remain on the continuously thinning sample layer, mainly in oxidized form. Thus, the sample is enriched with the one or more analyte elements before analysis by the imaging elemental analyzer.Preferably, the reaction does not change the position of the analyte elements in the sample on the substrate (at least not significantly or substantially). The reaction rate can be controlled to prevent bubble formation or boiling, thus ensuring that the position of the analyte elements remains unchanged. Diffusion is low for heavier elements or larger clumps or crystals of such elements, but the reaction rate should be chosen so that the diffusion length is kept below a) 1 × sample thickness D, b) 0.5 × D, c) 2 × D. The resulting distribution of the analyte elements in the sample then accurately reflects the distribution of the analyte elements in the original sample (before the reaction).

[0049] Preferably, the reaction step removes the largest part of the sample layer by weight from the substrate. Particularly preferably, the reaction removes at least 60%, 70%, 80%, 90%, 95%, 99%, 99.9%, or 99.99% of the sample layer by weight. In some embodiments, the reaction step removes between 90% and 99% or between 90% and 99.9% of the sample layer by weight.

[0050] The reaction step is preferably continued until the oxidation process is substantially or nearly saturated, so that most of the organic matrix is ​​removed (most preferably by weight > 90% or > 95% or > 99%) and the typically heavier analyte elements have sufficient concentration for subsequent analysis.

[0051] Preferably, the reaction step comprises controlling the rate of the oxidation process by regulating the supply of oxidizing agents (including light or ions, if used) and / or the temperature of the sample. In some embodiments, the formation of at least one of the volatile products and / or their concentration in the gas phase can be monitored for process control (e.g., by using one or more gas sensors), for example, to determine the time required to complete the oxidation reaction (to monitor the completeness of the oxidation so that the proportion of unwanted products is minimized), or the oxidation rate (so that it is not too high, which could interfere with the location of the elements or tags), as well as for diagnostic purposes, e.g.,to measure the relative content of specific elements in the volatile products or their isotopes, in order to obtain further categories of information about the sample, such as byproducts, impurities, etc. Another embodiment of the reaction chamber involves the use of sample slides made of porous inorganic material, through which the oxidizing agents (ozone, hydrogen peroxide, persulfate) are drawn from a feeder located below. This approach relies on the rapid diffusion of these agents through the thin tissue section and therefore leads to a higher probability of bubbling and boiling. In this case, however, another porous slide can be positioned only a few micrometers away from the tissue to "capture" non-volatile oxides of heavier elements that are carried away by the resulting gas stream. This flow-through approach allows for a faster Oxidation process without loss of the analytes of interest, even in the case of bubble formation.

[0052] Accordingly, in some embodiments, the process of the sample reacting on the substrate involves passing one or more oxidizing agents from a side of the substrate opposite the sample through pores in the substrate to reach the sample, wherein a second substrate is located near the sample but at a certain distance from it and directed towards it, the one or more oxidizing agents diffusing through the sample and thereby generating volatile products from the sample and causing non-volatile (heavier) analyte elements and / or their oxides to reach the surface of the second substrate and remain there to be detected by the imaging analyzer. If there is a sufficiently small gap between the two substrates, e.g.,The space between the two substrates is 5-10 micrometers in diameter and is designed such that the spatial distribution of the heavier analyte elements in the sample is essentially preserved after the elements are transferred to the second substrate. The analyte elements transferred to the second substrate are, at least preferably on average, not spatially disturbed more by oxidation than by the spatial resolution of the imaging analysis to be performed.

[0053] The imaging elemental analyzer can be selected from a group consisting of: a secondary electron spectrometer (SES), an X-ray photoelectron spectrometer (XPS), an X-ray fluorescence spectrometer (XRF), an energy-dispersive X-ray microanalyzer, a radioactivity analyzer, an ion mobility analyzer and a mass spectrometer (MS), preferably a mass spectrometer.

[0054] Preferably, the detection of one or more elements comprises irradiating the concentrated sample with a beam of primary particles, such as ions or photons, focused on a localized spot on the sample surface to emit secondary particles from the spot and to analyze the secondary particles in order to determine the presence and optionally the amount of one or more elements at the spot, wherein the spot transforms into a plurality of elements over time.

[0055] The sample is moved to different spots on the sample surface to obtain an image of the one or more elements in the sample, with each spot on the sample surface corresponding to a pixel of the image. At least some of the secondary ions are ions comprising the one or more analyte elements. The secondary particles can be analyzed directly upon release from the sample surface (e.g., if the secondary particles are already elemental ions or their oxide ions and the analyzer is a mass spectrometer, or if the secondary particles are photons or electrons emitted by the one or more analyte elements that are characteristic of that element), or they can be converted into another form for analytical purposes, e.g., from released neutral or ionic polyatomic particles into monatomic elemental ions for mass analysis (e.g.,in an ICP ion source of a mass analyzer), or are converted into reaction products (by ion molecule or ion-ion reactions) in a reaction or collision cell upstream of the mass analyzer.

[0056] Thus, the imaging elemental analyzer can include a primary particle source (e.g., an ion gun) or a photon source (e.g., a laser) for generating the primary particle beam and focusing the beam onto a localized spot on the sample surface to emit secondary particles from the spot, and it includes a secondary particle analyzer to analyze the secondary particles to determine the presence and optionally the amount of the one or more elements at the spot, wherein the primary particle source is configured to move the spot over time to a plurality of locations on the sample surface to obtain an image of the one or more elements in the sample, with each location of the spot on the sample surface corresponding to a pixel of the image.The imaging element analyzer can capture the image at a speed of at least 100 pixels per second, or at least 1000 pixels per second, or in the range of 1000-10000 pixels per second.

[0057] The primary particles are preferably selected from IR photons or photons in the visible wavelength range, or UV or X-ray photons, electrons, and ions. Likewise, the secondary particles are preferably selected photons (especially X-ray photons), electrons, and ions.

[0058] Preferably, the primary particles have an energy above 1 keV.

[0059] Preferably, the primary particles are ions, and thus the primary particle source particularly preferably comprises an ion gun. Preferably, the primary ion beam is continuous. In some embodiments, however, the primary ion beam is pulsed. Preferably, the primary particles are ions generated at a pressure below 1 mbar, and the secondary ions are ions for analysis by the mass analyzer. Thus, the imaging elemental analyzer can be an imaging secondary ion mass spectrometer (SIMS) configured to create a vacuum by pumping out the vacuum, wherein the primary particles are ions generated in the source at a pressure below 1 mbar, and wherein the secondary particles are ions for analysis by a mass analyzer of the SIMS.

[0060] The sample can be scanned by guide plates of the ion gun and / or by moving a stage on which the sample or substrate is located. Higher spatial resolution for subcellular and suborganic resolution could be achieved, for example, by using a thinner sample (e.g., a tissue section with a thickness of 3 µm or less) and / or stronger spatial focusing of the primary beam and / or a weaker primary beam current to reduce defocusing of the space charge.

[0061] The primary ion beam preferably has an intensity of up to 100 nA per 1 µm spot size (i.e., spot diameter 1 µm). Preferably, the primary ion beam has an intensity of at least: a) 1 pA or b) 100 pA or c) 1 nA or d) 10 nA at a spot size of 1 µm.

[0062] When photons are used as primary particles, the fluence in the ionization pulse is preferably above a) 5, b) 10, c) 20, d) 50 J / cm². 2This enables the formation of high-density plasma and the achievement of a high degree of ionization, as known from the prior art. A laser device is preferably used as the photon source. Such a laser is preferably a pulsed laser, wherein the individual pulses preferably have the aforementioned fluence. Lasers of any wavelength could be used, but preferably not longer than the required spatial resolution and preferably with an extinction length (i.e., the length to be attenuated for the laser intensity by a factor e (Euler's number)) of at most a) 100 nm, b) 200 nm, or c) 500 nm. For example, the fluence in the irradiation pulse exceeds a) 5 or b) 10 J / cm². 2(or the other preferred fluence values) and has an extinction length of at most 500 nm. Most preferably, solid-state lasers such as Nd:YAG are used for ionization with or without frequency multiplication (the latter might be necessary to achieve sufficiently short extinction lengths).

[0063] The laser radiation could strike the sample from either the front or the back. In the latter case, focusing the laser beam, ensuring the orthogonality of the laser light on the sample, and thus creating a round spot is simplified—however, glass with a high radiation limit should be used to avoid radiation damage to the slide. When irradiated from the front, the laser light typically strikes at an angle (to simplify more demanding extraction optics requirements), while optical observation of the focal spot from the back through the glass slide is possible.

[0064] In embodiments, the mass analyzer may further comprise a distance-of-flight mass analyzer, a quadrupole ion-trap mass analyzer, an electrostatic trap (EST) mass analyzer (such as an orbital EST, e.g., Orbitrap), an ion cyclotron resonance mass analyzer (FT-ICR), in particular an EST or ICR operating with imaging current detection, a magnetic sector mass analyzer, and an array or any combination thereof. The time-of-flight (TOF) analyzer may be an orthogonal acceleration (OA-TOF) analyzer, in particular a high-repetition-rate OA-TOF as known in the art, preferably with gridless orthogonal acceleration, optionally with one or more ion mirrors providing single or multiple reflection of ions in the analyzer (single reflection is particularly preferred).The mass analyzer is preferably capable of simultaneously analyzing a continuous secondary ion beam over a large mass range of ions, such as a TOF or Orbitrap or an electrostatic orbital trap. Preferably, a large number of analyte elements are to be analyzed (imaging), e.g., from a large number of element tags. Particularly preferably, at least 5 or at least 10 analyte elements or element tags are analyzed by the elemental analyzer. A mass spectrometer, especially a TOF mass spectrometer, is easily capable of performing such multichannel analysis in short timeframes. A combination of analyzers could be used for multimodal analysis, e.g., a TOF mass analyzer for nominal mass element analysis and an electrostatic trap with image current detection, such as an Orbitrap, for organic analysis or high-resolution interference analysis.

[0065] The secondary ions of the analyte elements (tags) can be reduced to a mean vacuum pressure of 10 -5 -10 -2 mbar instead of a higher vacuum are released, which is why lower requirements apply to sample dehumidification and transfer times compared to high-vacuum (e.g., SIMS) instruments. Thus, the pressure in the chamber for holding the primary ion-irradiated sample is in the range of 10 mbar when the internal chamber pressure surrounding the sample is around 10 mbar. -5 up to 10 -2 mbar.

[0066] Preferably, after emission from the surface, the secondary particles are transferred to an RF ion guide for analysis by the mass analyzer and then transferred from the ion guide to the mass analyzer. Thus, the imaging elemental analyzer preferably comprises an RF ion guide for capturing the secondary ions after their emission from the surface and for transporting the secondary ions to the mass analyzer. The ion guide used is preferably designed to withstand a pressure of at least 10 -2 The chamber is filled with gas to a density of mbar, whereby all m / z values ​​below the mass range of the analyte elements or their oxides – if oxides of the elemental ions are detected – are removed. The ion guide can thus advantageously function as a reaction or collision cell.

[0067] In some embodiments, the ion guide can contain a reactive gas for the formation of reaction products with the secondary particles (e.g., via ion-molecular reactions), wherein the secondary particles are ions comprising one or more of the elements. For example, the reactive gas can comprise a gas such as NO or O₂ to completely oxidize most analyte elements (e.g., native metals or rare earth elements) to oxides, as is known in the art (see, e.g., BG Koyanagi, D. Bohme. J. Phys. Chem. A, 105 (2001) 8964-8968), thereby reducing interferences (by mass shifting these element peaks by 16 amu while other peaks remain unshifted) and increasing the number of channels that can be analyzed in parallel. If a reactive gas is used, all oxides and other molecularly induced interferences could be eliminated.Thus, preferably at least some of the generated (secondary) ions undergo an ion-molecular reaction in the ion guide. The ion guide can cause the ions to cool during transport. This alters (preferably reduces) the energy distribution or emittance of the ions at the ion guide's outlet compared to the ions at the inlet. Similarly, in some embodiments, the composition and / or energy distribution of the ions at the ion guide's outlet can be altered compared to the ions at the inlet. The ion guide is therefore preferably configured as a reaction cell. It is known in the art that gases such as N₂O, NO₂, O₂, CO₂, and NO facilitate the oxidation of numerous metal ions, particularly those with m / z values ​​> 100, which in turn allows the number of analysis channels per isotope to be reduced to one.NH3 and NO could also be used to eliminate polyatomic species such as hydrides before this oxidation. The reaction cell can therefore be filled with one or more of these gases, which act as one or more reactive gases within the reaction cell.

[0068] In certain embodiments, heavier elements, when concentrated on the slide surface, can form a new matrix type that may begin to interfere with the analysis, leading to a lower ionization yield or effects on the topography. For example, phosphorus and phospholipids can concentrate around cell membranes (including the nuclear membrane) and form DNA within the nucleus. Alkali metals (e.g., Na, K) could be present in varying concentrations on opposite sides of the membranes, and so on. Therefore, in such embodiments, one or more of these elements (e.g., P, Na, K) should preferably also be monitored, and corrections made according to their abundance. For example, if, in some embodiments, the concentrations of such abundant elements (e.g., Na, K, etc.)If the analytes do not exhibit the expected concentrations in known sample ranges, then the observed deviation from their expected concentration can be used as a basis for correcting the detected frequencies of the (typically heavier) analyte elements.

[0069] Furthermore, the sample could undergo additional treatment, such as the application of an extra matrix to enhance ionization and compensate for matrix effects. For example, a uniform coating containing one or more elements to support ionization can be applied to the sample (e.g., oxygen is known to increase the yield of secondary ions from metals). Irradiation and / or ionization by a laser can also result in the sample being coated with a light-absorbing layer.

[0070] The mass analyzer is preferably of the OA-TOF type, and the repetition rate of the TOF mass analyzer is at least equal to or greater than (a) 5 kHz, (b) 20 kHz, (c) 50 kHz, or (d) 100 kHz. The repetition rate can be, for example, 50–100 kHz. Here, the repetition rate refers to the rate of pulsed ions entering the TOF analyzer for ion separation according to their mass-to-charge ratio (m / z). A detected ion signal (intensity versus m / z) is obtained from each ion pulse entering the TOF analyzer. Summing the detection signals from each ion pulse entering the TOF analyzer at a given spot on the sample surface can be used to generate the corresponding pixel of the elemental distribution image. Preferably, the elementary distribution image is acquired at a speed of at least 100 pixels per second or at least 1000 pixels per second or in the range of 100-10000 pixels per second.Preferably, the mass analyzer is configured to detect elements or oxides of the elements.

[0071] Preferably, no more than (a) 2, (b) 5, or (c) 10 pulses of the TOF analyzer are required for the analysis of each spot using the TOF mass analyzer. The analysis time for each spot is preferably the time required to acquire a mass spectrum with a sufficient or desirable signal-to-noise ratio or sensitivity for use in the pixels of the image.

[0072] This invention can be used for one of the following applications: i. Tissue imaging, e.g., as used for anatomical pathology, especially cancer, including fixed, paraffin-embedded tissues and in conjunction with hematoxylin and eosin (H&E) staining; ii. High-content cell analysis; iii. microarray-based targeted assays for clinically relevant biomarkers for diseases; iv. pharmaceutical, chemical and clinical high-throughput analysis; v. Identification and resistance determination of bacteria (using cultures on thin media layers). vi. Cytometry, including offline flow cytometry;

[0073] In cases where the sample is a biological sample, particularly clinically relevant samples, the procedure may further include the use of the image of one or more elements in the sample to determine a physiological condition or to diagnose a disease state in an organism from which the biological sample is taken.

[0074] The sample pre-concentration approach according to the invention can offer numerous advantages, such as increased pixel acquisition speed and thus the throughput of elemental imaging; reduced contamination of the instruments, applicability with a wider range of elemental imaging techniques, and increased ionization efficiency due to the presence of oxidized elemental ion species on the oxidized surface. Description of the drawings Fig. Figure 1 schematically shows an embodiment of a device for imaging one or more elements in an organic sample according to the invention. Fig. Figure 2 schematically shows another embodiment of a device for imaging one or more elements in an organic sample according to the invention. Fig. Figure 3 schematically shows a flow-through design with transfer of the analytes of interest to an opposite slide. Detailed description of embodiments

[0075] To enable a more detailed understanding of the invention, numerous embodiments are now described by way of example and with reference to the accompanying drawing.

[0076] With reference to Fig. Figure 1 schematically depicts a device for imaging one or more elements in an organic sample. A thin layer of the sample to be analyzed is applied to the slide (2). The sample can be arranged as a microarray on the slide. The slide is typically a flat glass slide with an ITO coating. Alternatively, it could be a metal plate.

[0077] The sample is, for example, a bio(organic) sample, such as a tissue sample or cell line. However, the sample is generally not limited to a specific type. The sample could include any of the following: - a biological or a chemical (organic but non-biological) sample - a fixed and embedded tissue, e.g. a formalin-fixed, paraffin-embedded (FFPE) tissue, preferably cut with a microtome, preferably to a thickness of 3-5 µm - Individual cells deposited on a slide, e.g., from a flow cytometer or a high-content screening device, e.g., in a grid-like pattern (e.g., deposited at 50 µm intervals in the X and Y directions, with a typical cell size of up to 5–10 µm). Typically, each grid cell or square is occupied by no more than a single biological cell. The cells in the grid may all be different (i.e., from different samples or experiments), or at least some, optionally all, of the cells in the grid may be from a single sample or population to detect variation within that sample or population. - a cell culture on a growth medium, e.g. a microbial or bacterial culture on a thin layer of a growth medium such as agarose (preferably the culture is up to 10 µm or up to 20 µm thick) - a sample, such as a non-cellular sample, applied to the slide, e.g. by an automatic sampler of any known type (including by flow focusing, acoustic droplet ejection, induction, etc.). The sample may be applied in the form of individual droplets on a grid-like pattern (e.g. every 50 µm in the X and Y directions), or in a microarray.

[0078] For the reasons described below, in some embodiments the microscope slide is coated with a layer of titanium dioxide, e.g. in the form of a film or immobilized particles.

[0079] In some embodiments, the sample on the slide could be analyzed in its present form (i.e., unprocessed, untagged) to determine a distribution of native heavier inorganic elements (e.g., Fe, Zn, Sn, etc., for example, for metallomics experiments). In other embodiments, the sample could be tagged, preferably with one or more elements that are non-native to the sample (hereinafter referred to as non-native elements). Rare-earth elements represent a class of element tags known from the prior art. The one or more tags are typically specific to one or more different corresponding target objects in the sample. The sample could be tagged before or after it is applied to the slide, but preferably afterward. The specificity of the tagging can be achieved using binding elements, such as…Antibodies, aptamers, somamers, metabolic markers, and other known methods. Tags could include polymer chains, nanoparticles (as shown in US 8,679,858 B2), quantum dots, etc. Tagging could also utilize multiple elements in a barcode-like manner, as shown in US 2014 / 106976 A1 and B. Bodenmiller et al., Nature Biotechnology 30 (2012) 858-867.

[0080] The next step in the procedure for analyzing the sample consists of placing the slide containing the (tagged) sample into a reaction or oxidation chamber (10), where an oxidation reaction takes place to reduce the mass of the sample by removing most of the organic matrix, leaving behind the sample containing the analyte elements of interest, typically in the form of oxides. One or both of two approaches to oxidation could be used. The first approach involves using light with a wavelength of < 400 nm, such as UV light (12), to irradiate the slide and the sample, preferably at intensities of or above 0.1–10 milliwatts / cm². 2and a wavelength of < 400 nm (e.g., from a gas discharge source as known in the prior art). These are embodiments in which the microscope slide exposed to UV light is preferably provided with a layer of titanium dioxide or another photocatalyst. Titanium dioxide exhibits very strong photocatalytic properties that lead to the rapid oxidation of the matrix atoms. Alternatively or additionally, a chemical oxidizing agent, preferably ozone or hydrogen peroxide vapor, is introduced into the oxidation chamber (10) through the inlet (14) and expelled from it through an outlet (16). The sample can be heated on the microscope slide to assist the oxidation process, as is known in the prior art.

[0081] Under the influence of UV light and / or oxidizing agents, rapid oxidation of matrix atoms occurs: e.g., C→CO2, N → NO, NO2, H→H2O, etc. The volatile products are pumped away by a vacuum pump connected to the chamber (not shown); thus, most of the sample mass is removed. Meanwhile, heavier atoms, including the analyte elements of the tags or the native heavier elements, do not form volatile products and therefore remain on the thinning sample layer, mainly in oxidized form. After the process reaches saturation and the organic matrix has been largely removed (preferably > 90% or > 99% by weight, e.g., 90-99%), the heavier atoms are sufficiently removed. concentrated for subsequent analysis. The rate of the oxidation process can be regulated by controlling the supply of oxidizing agents, the incident light intensity, and / or the sample temperature. Oxidation that is undesirably too rapid could lead to gas bubbles that carry away the heavier atoms of interest. Therefore, the oxidation rate should be carefully adjusted. Sample production in the reaction chamber can be adjusted. The volatile products can be used for process control (e.g., to determine the time required to complete oxidation) and / or for diagnostics (e.g., to measure the relative content of elements or their isotopes to obtain additional information). For example, the elements in the volatile products could be monitored. If, for instance, the monitored ratio of C to O is 1:1, this indicates incomplete oxidation; however, if the ratio of C to O is 1:2, this indicates complete oxidation to CO2. In another example, the isotopic ratios, e.g., C 12 / C 13 They can serve as process indicators. For example, this ratio could be used to distinguish when the process has finished oxidizing a bacterial culture and begins oxidizing the medium (which has a different C ratio). 12 / C 13(might have an effect on bacteria).

[0082] An alternative approach is in Fig. Figure 3 shows that this allows for higher reaction rates, which in principle even permits boiling and bubbling of the sample. In this case, one or more oxidizing agents (ozone, hydrogen peroxide, persulfate) are driven (101) from a supply (not shown) beneath the slide (102), which carries a sample (104), e.g., a thin tissue sample. In this embodiment, two sample slides (102, 106) are arranged at a distance but close together, each made of porous inorganic material (e.g., glass, ceramic, ITO). The one or more oxidizing agents are driven through the two closely spaced sample slides. These agents diffuse rapidly through the thin tissue section, generating light gases (e.g., volatile products) along the way. This mixture then flows through a gap of 5–10 mm. Micrometers from the sample slide (102) to the opposite slide (106) and through the latter. If the sample is sufficiently lyophilized, it could actually be brought into direct contact with the opposite slide (106). The size of the pores in the opposite slide is chosen (preferably in the range of 1–10 nm) such that non-volatile heavier elements and their oxides cannot enter the pores but remain on the surface of the opposite slide for subsequent analysis. Thus, the analysis, as described in this document, can be performed on the opposite slide. If the gap between the slides is small enough, the spatial distribution of the heavier elements in the sample is essentially preserved. This flow-through approach allows for a faster oxidation process without loss of analytes of interest, even in the event of bubble formation.Preferably, the opposing slide is transparent in the UV range to allow ozone formation and titanium dioxide reactions (e.g., if the sample substrate has a titanium dioxide surface), supported by UV radiation from a UV source (108). This can promote the oxidation process.

[0083] The reaction could also be facilitated by a focused laser scanned across the surface. In one embodiment, the laser power can be high enough to cause local heating, which accelerates the breakdown of organic substances and the oxidation rate.

[0084] It is understood that several sample slides could be processed simultaneously in the oxidation chamber.

[0085] For subsequent analysis, the sample is removed from the reaction chamber and transferred to a device suitable for the rapid parallel imaging of multiple elements, preferably with an acquisition rate of > 100 or > 1000 pixels / second (e.g., 100-1000 pixels / second, or 2000 pixels / second). The rate can be increased to up to 10 in some cases. 5 pixels / second can be achieved. The pixel size for such an acquisition rate can be 10 µm or less, or 5 µm or less, or 2 µm or less, or ideally achieve a subcellular resolution of 1 µm or less (e.g., 0.5–1.0 µm). It should be noted that the reaction chamber could alternatively be integrated into the imaging device, which would be preferable if, for example, a means within the latter (e.g., ion or electron gun or X-ray gun of the imaging analyzer) could be used to accelerate the oxidation.

[0086] A preferred imaging device in the form of a secondary ion mass spectrometer (SIMS) or LPI (laser plasma ionization) mass spectrometer is described in Fig. Figure 1 shows. It comprises a high-power laser or ion gun (20) as the irradiant for generating ions from the sample. With reference to Fig. 2, which share many characteristics with Fig. Figure 1 shows an ion gun (20) as a primary ion source for irradiating the sample. The ion gun comprises the ionization chamber (22), the lens system (24), the optional collimator system (26), the focusing optics (28) for focusing the ion beam onto a small spot on the sample surface, and scanning electrode plates (30) for scanning the ion spot across the sample. The primary ion beam ionizes the element tags of interest or native heavier elements, causing their release from the surface. Alternatively, laser ablation (LA) or laser plasma ionization (LPI) could be used, preferably at high fluences > 1–10 joules / cm², to facilitate the dissociation of molecular bonds and the release of ions from elements. Scanning the continuous primary beam (ion or laser beam) allows the sample to be imaged more quickly than would be possible by mechanically moving the sample holder.Typically, a combination of rasterization with deflection plates over an area of ​​0.5 × 0.5 or 1 × 1 mm could be combined with mechanical rasterization over larger distances, e.g. 50 × 100 or 100 × 200 mm.

[0087] Generally, a continuous primary ion beam with high intensity (36) of up to 100 nA is generated in a 1 µm spot. For example, a suitable ion source is an oxygen ion beam that is equipped with an RF- A gas-phase ion source, as described in NS Smith, Appl. Surf. Science, 255 (2008) 1606-1609, is generated. This generates secondary ions (38) from the sample surface, encompassing the element tags, at a mean vacuum pressure of 10⁻⁵ 10⁻² mbar in the chamber containing the sample, thus reducing the required sample dehumidification and transfer time compared to typical SIMS instruments. In general, the ions generated from the samples by SIMS or LPI are produced at a mean vacuum pressure of 10⁻⁵ 10⁻² mbar in the chamber containing the sample.

[0088] The generated ions (38) (secondary ions from irradiation of the samples by primary ions) are accelerated by a short, gas-filled, radio-frequency (RF)-driven ion guide or collision cell (40) at elevated pressure (typically > 10⁻² mbar), removing all ions with m / z values ​​below the mass range of the tags (or their oxides). The emittance of the ion beam is also reduced as the ions pass through the guide. The RF ion guide typically comprises a multipole, such as a quadrupole (42), located within a gas-filled housing (44). This increased pressure in the RF ion guide or collision cell makes it possible – unlike in US 7,910,882 B2 – to use an optional reactive gas in the ion guide (in particular an oxidation gas, such as NO, O2) to oxidize essentially most of the metal ions to oxides, as is known according to the prior art (see e.g. BG Koyanagi, D.Bohme. J. Phys. Chem. A, 105 (2001) 8964-8968, S. Tanner, V. Baranov, D. Bandura, Spectrochimica Acta B, 57 (2002) 1361-1452), and thus reduce interferences by monitoring the oxides of the elements or tags and increase the number of channels to be analyzed in parallel. In certain embodiments, the ion guide is configured as a reaction cell. Preferably, the RF ion guide features a DC gradient to accelerate and control ion transport.

[0089] After passing through the RF ion guide (40), the produced ions (38) are separated from element tags or native elements using a high-speed (OA) TOF mass spectrometer with orthogonal Acceleration (50), as known from the prior art, subjected to mass analysis, but preferably in operation at a repetition rate of 50-100 kHz (i.e., multiple MS scans are available per spot (pixel) of the ionized sample). Preferably, as in Fig. As shown in Figure 1, this TOF-MS has a gridless orthogonal accelerator (52), as described in WO 01 / 11660 A1, and a single-stage ion mirror (54). and a detector for the high dynamic range (56), comprising an electron multiplier, e.g., as described in one of the following documents: US 6,940,066 B2, US 6,864,479 B1, US 2013 / 264474 A1, or others. The produced ions (38) are thus separated according to their m / z value and detected as shown. The TOF analyzer enables the simultaneous analysis of a large mass range of ions. Preferably, a large number of analyte elements are to be analyzed (imaging).

[0090] The sample surface is scanned using deflection (raster) plates of the ion gun or, in the case of a laser, by using a movable mirror and / or by moving the sample stage (e.g., in the x and / or y direction). Higher spatial resolution for subcellular and suborganic resolution could be achieved by using a thinner sample (e.g., a tissue section 3 µm thick or less) and / or by increasing the spatial focus on the primary ion beam and using a weaker primary beam current to reduce space charge defocus.

[0091] Due to the aforementioned high primary ion current or the use of powerful lasers, the secondary ion current from the generated ions could reach hundreds of picoamperes (pA) (e.g., up to 100, 200, 500, or 1000 picoamperes or more), with a significant fraction of the current consisting of ions from the analyte elements (or tags). This means that a complete pixel could be analyzed in just one TOF MS pulse, reducing the acquisition rate in some applications (e.g., analyzing iron in brain sections) to nearly 10⁻⁶. 5The method can achieve pixels per second. Thus, a complete slide could be analyzed within one minute, significantly reducing the cost per analysis. Images of 500×500 pixels or more, suitable for histological imaging, can be produced. The image can represent a 500×500 µm field of the sample (e.g., with a pixel size of 1 µm). Compared to conventional fluorescence detection of tagged samples, the method according to the invention provides an output equivalent to up to ten to one hundred colors or channels, at a speed comparable to that of single-color measurement.

[0092] From the above description and Fig. As shown in Figure 1, various preferred imaging arrangements can be used to visualize the elements in the sample that has undergone reaction or oxidation. In one arrangement, laser plasma ionization can be used to produce ions from the sample, which are introduced into a downstream reaction cell (where the composition and emittance of the produced ions are modified, preferably towards smaller variations) and subsequently into the TOF mass analyzer. The laser plasma ionization can be used to scan the sample to obtain an elemental image of the sample.In another arrangement, a SIMS system can be used in which a primary ion beam is employed to generate secondary ions from the sample. These secondary ions are then introduced into a downstream reaction cell (where the composition and emittance of the produced ions are modified, preferably towards smaller variations) and subsequently into the TOF mass analyzer. The primary ion beam can be scanned across the sample to obtain an elemental image of the sample.

[0093] This described method can also yield higher absolute sensitivity in approaches for the detection of analyte elements due to increased ion yield, which is attributable to the presence of oxidized analyte element atoms on the oxidized surface, as is known in the prior art. With an ionization efficiency of 0.1–1% and low-loss transport under vacuum conditions, the SIMS or LPI method can offer an order-of-magnitude advantage over, for example, LA / ICP-MS due to high losses (which are typically associated with LA / ICP-MS). (leading to losses of 1 × 10⁴ to 1 × 10⁵ times) during transport using the latter method. On the other hand, the proposed method for sample preparation is also fully compatible with LA / ICP-MS. compatible, and it could also reduce contamination and carryover of samples.

[0094] The results of the analysis could be presented in an analog or quantitative mode (e.g., by determining the concentration of an element or tag, preferably taking matrix effects into account), or in a digital or qualitative mode (e.g., by determining whether an element or tag is present or not). The results can be compiled by software into an image, such as an elementary distribution image (and thus a target distribution, insofar as the element has been tagged to a target object in the sample).

[0095] The following steps are given as an example of a workflow using the invention: 1) Preparing a sample, for example a tissue sample, 2) Oxidizing the sample with light by photocatalysis and / or with chemical oxidizing agents to remove almost all of the organic matrix; 3) Irradiation of the oxidized sample surface with an ion gun using continuous current or focused laser pulses with a spatial resolution of 0.5-10 µm, with an intensity such that the sample is broken down into its constituent elements, which are released from the surface as ions; the irradiation spot is scanned over the area of ​​the sample surface to be analyzed; 4) Parallel detection of signals from the ions of the elements (multi-channel detection) for each individual irradiation spot using a mass spectrometer; 5) Determining the presence or absence of the spatial distribution of elements in the sample, based on the mass spectrometric information.

[0096] Such a workflow can be varied according to the above description; for example, element distributions could be used as a substitute for corresponding antigens when used as element tags.

[0097] As an alternative to the primary ion method described above for the ionization of the imaging SIMS analyzer from Fig. 2. Alternatively, other ionization methods in a vacuum suitable for mass spectrometric analysis could be used: e.g., laser plasma ionization or laser ablation with laser positioning. It should be noted that laser radiation could alternatively be emitted from the back of the slide, thereby utilizing its transparency and simplifying the optical system.

[0098] As alternatives to mass analysis of ions generated from the sample surface, other elemental imaging methods, e.g., non-destructive ones, could also be used to image elemental tags, for example: - Micro-XRF (µXRF) enabling analysis under atmospheric pressure conditions, preferably using a multi-element detector to enable parallel detection - X-ray photoelectron spectroscopy (XPS) - Electron microprobe analyzer (EMPA), especially when integrated into an electron microscope - Secondary electron spectrometry (SES) - Energy-dispersive X-ray microanalysis, preferably using a silicon drift detector

[0099] Each of the elementary imaging techniques could be combined with other types of sample imaging (e.g., optical imaging). Such optical imaging could serve as an internal standard for improved quantitative determination of the sample.

[0100] It is evident from the foregoing description that the invention comprises providing a substrate or surface with a thin layer of a (bio-)organic sample which is subjected to an oxidation process that converts the organic matrix, e.g. C, H, N, O, S, into volatile Species are converted that enter the gas phase, leaving mainly heavier inorganic elements in the sample, particularly in oxidized form. The resulting sample is then subjected to high-speed imaging analysis of the remaining heavier elements by mass spectrometry or other techniques in a vacuum to measure the spatial distribution of the elements within the sample.

[0101] In embodiments, the invention enables an elemental imaging mass spectrometer that can offer subcellular lateral resolution in conjunction with highly multiplexed sample output at a significantly higher throughput and low cost per analysis. The invention is preferably based on secondary ion or laser plasma mass spectrometry in a vacuum with time-of-flight mass analysis ( Fig. 1).

[0102] The invention is used in many of today's growth markets, such as: - Tissue imaging, as used, for example, in anatomical pathology, especially in cancer; - Microarray-based targeted assays for clinically relevant biomarkers for diseases or biomarker panels and for use in research and development in life sciences; - High-content cell analysis; - Pharmaceutical and clinical high-throughput analysis; - Identification and resistance determination of bacteria.

[0103] It is understood that modifications may be made to the foregoing embodiments of the invention, which, however, still fall within the scope of the invention. Unless otherwise stated, each feature disclosed in the specification may be replaced by alternative features serving the same, equivalent, or similar purpose. Thus, unless otherwise stated, each disclosed feature represents an example of a generic set of equivalent or similar features.

[0104] The use of one or more of the examples provided herein, or of exemplary language (“for example,” “such as,” “for instance,” and the like), is intended solely to better illustrate the invention and does not constitute a limitation with respect to the scope of the invention, unless otherwise claimed. No linguistic formulation in the specification shall be construed as indicating any unclaimed element as essential to the practice of the invention.

[0105] For the purposes of their use in this document, including the claims, singular forms of terms in this document shall be interpreted as including the plural form and vice versa, unless the context suggests otherwise. For example, unless the context suggests otherwise, a singular reference in this document, including in the claims, such as "a" or "an", means "one or more".

[0106] Throughout the description and claims of this specification, the words "comprise", "include", "include" and "contain" and variants of the words, for example "comprehensive" and "encompasses", etc., mean "including, but not limited to", and are not intended to exclude (and do not exclude) other components.

[0107] All steps described in this specification can be performed in any order or simultaneously, unless otherwise specified or the context requires otherwise.

[0108] All features disclosed in this specification may be combined in any combination, except for combinations in which at least some of these features and / or steps are mutually exclusive. In particular, the preferred features of the invention apply to all aspects of the invention and may be used in any combination. Likewise, features described in non-essential combinations may be used separately (not combined with each other).

Claims

[1] Imaging elemental analyzer for imaging one or more analyte elements in an organic sample, the elemental analyzer comprising: a chamber for receiving an organic sample containing one or more analyte elements to be imaged, wherein the internal chamber pressure surrounding the sample is in the range of 10 -5 up to 10 -2 mbar is; at least one irradiating agent selected from: (i) an ion gun (20) for irradiating the sample with a primary ion beam (36) of high intensity, wherein the primary ions are generated in the ion gun at a pressure below 1 mbar, the ion gun (20) serving to focus the primary ion beam (36) onto a localized spot on the sample surface and to move the spot over time to a plurality of locations on the sample surface; (ii) a laser for irradiating a localized spot on the sample surface and to move the spot over time to a plurality of locations on the sample surface; a gas-filled RF ion guide (40) for receiving formed ions (38), comprising the analyte elements released from the sample in response to irradiation by the primary ions or the laser, wherein the RF ion guide (40) prevents the passage of all ions whose m / z values ​​are less than the mass or mass range of the analyte elements; and a time-of-flight (TOF) mass analyzer (50) for recording the ions (38) formed or reaction products of the ions (38) formed from the RF ion guide (40), wherein the TOF mass analyzer (50) is configured for a repetition rate of at least 5 kHz. [2] Device for imaging one or more inorganic, non-volatile elements in an organic sample, comprising the imaging elemental analyzer according to claim 1, further comprising: a reaction chamber (10) for receiving the sample, wherein the sample is provided in the form of a layer on a substrate; wherein the reaction chamber (10) comprises an electromagnetic radiation source and / or an inlet (14) for introducing one or more chemical or ionic oxidizing agents into the chamber for oxidizing the sample to produce one or more volatile products that escape from the sample and enter the gas phase, while the one or more inorganic, non-volatile elements remain in the sample and are, on average, not spatially disturbed by the oxidation to a greater extent than the spatial resolution of the imaging analysis, wherein a larger proportion by weight of the sample layer is removed from the substrate by the oxidation and the remaining sample layer is enriched in the one or more inorganic, non-volatile elements; and wherein the imaging elemental analyzer is provided in a detection chamber for detecting the spatial distribution of one or more inorganic, non-volatile elements in the concentrated sample layer. [3] Device according to claim 2, wherein the imaging elemental analyzer comprises a data acquisition system that receives input from the detection of the one or more elements and generates an image of the one or more elements in the enriched sample. [4] Device according to claim 2 or 3, wherein the sample is a biological sample selected from tissue, bacteria and cells, and wherein one or more elements occur naturally in the sample and / or have been introduced into the sample as element tags. [5] Device according to one of claims 2 to 4, wherein by oxidation at least 60% or at least 70% or at least 80% or at least 90% or at least 95% or at least 99% or at least 99.9% or at least 99.99% or between 90% and 99% or between 90% and 99.9% of the sample layer is removed by weight. [6] Device according to any one of claims 2 to 5, wherein the radiation source comprises an ultraviolet or X-ray light source. [7] Device according to any one of claims 2 to 6, wherein a source of one or more chemical oxidizing agents is connected to the inlet, wherein the one or more oxidizing agents are selected from: ozone, persulfate and hydrogen peroxide. [8] Device according to one of claims 2 to 7, wherein the formed ions (38) or reaction products of the formed ions (38) are analyzed to determine the presence and optionally the amount of one or more inorganic, non-volatile elements at the spot, wherein the spot is moved over time to a plurality of locations on the sample surface in order to obtain an image of the one or more elements in the sample, wherein each location of the spot on the sample surface corresponds to a pixel of the image. [9] Device according to any one of claims 2 to 8, wherein the imaging elemental analyzer comprises a data acquisition system which receives input from the detection of one or more inorganic non-volatile elements and generates an image of one or more inorganic non-volatile elements in the sample, wherein the imaging elemental analyzer can acquire the image at a rate of at least 100 pixels per second or at least 1000 pixels per second or in the range of 1000-10000 pixels per second. [10] Device according to claim 8 or 9, wherein the primary ions or photons provided by the at least one irradiating means have an energy of more than 1 keV. [11] Device according to one of claims 8 to 10, wherein the imaging elemental analyzer is an imaging secondary ion mass spectrometer (SIMS) configured to produce a vacuum by pumping, wherein the at least one irradiant is an ion gun (20) and the primary ions are generated in the ion gun (20) at a pressure of less than 1 mbar and wherein the ions (38) formed are secondary ions for analysis by a mass analyzer of the SIMS. [12] Device according to any one of claims 8 to 11, wherein the imaging elemental analyzer comprises a further mass analyzer, wherein the further mass analyzer is selected from: a distance-of-flight mass analyzer, a quadrupolon trap mass analyzer, an electrostatic trap mass analyzer, an ion cyclotron resonance mass analyzer, and a magnetic sector mass analyzer and / or an array or combination thereof. [13] Device according to any one of claims 8 to 12, wherein the ion guide is configured to receive a reactive gas which fills the ion guide to form reaction products with the secondary ions, wherein the secondary ions are ions comprising one or more of the elements. [14] Device according to any one of claims 12 to 13, wherein the repetition rate of the TOF mass analyzer is at least equal to or greater than (a) 20 kHz or (b) 50 kHz or (c) 100 kHz. [15] Method for imaging one or more inorganic, non-volatile elements in an organic sample, comprising: Provision of the sample as a layer on a substrate; Oxidizing the sample on the substrate to produce one or more volatile products that escape from the sample and enter the gas phase, while the one or more inorganic, non-volatile elements remain in the sample and are, on average, not spatially disturbed by the oxidation more than the spatial resolution of the imaging analysis, wherein a larger proportion of the sample layer by weight is removed from the substrate by the oxidation and the remaining sample layer is enriched with the one or more inorganic, non-volatile elements; and subsequently Detecting one or more inorganic, non-volatile elements in the enriched sample layer using the imaging elemental analyzer according to claim 1. [16] Method according to claim 15, wherein the sample is a biological sample selected from tissue, cells, bacterial culture and bioorganic solution. [17] Method according to claim 15 or 16, wherein one or more elements are elements naturally occurring in the sample and / or have been introduced into the sample as element tags. [18] Method according to claim 17, wherein the one or more element tags comprise one or more rare earth elements, heavy metal elements and / or radioisotopes. [19] Method according to one of claims 17 or 18, wherein the one or more element tags are attached to a binding element selected from a polypeptide, polynucleotide, antibody, affiliator and an aptamer, the binding element binding to a target object in the sample. [20] Method according to any one of claims 15 to 19, wherein one or more elements are metals or semimetals. [21] Method according to any one of claims 15 to 20, wherein the layer is a thin layer, in particular having a thickness of at most (i) 20 µm or (ii) 10 µm or (iii) 5 µm. [22] Method according to any one of claims 15 to 21, wherein at least 60% or at least 70% or at least 80% or at least 90% or at least 95% or at least 99% or at least 99.9% or at least 99.99% of the sample layer is removed by weight by oxidation. [23] Method according to any one of claims 15 to 22, wherein the oxidation comprises the action of one or more oxidizing agents on the sample, wherein optionally the sample is heated during the oxidation step. [24] Method according to claim 23, wherein the one or more oxidizing agents are selected from (i) electromagnetic radiation and / or (ii) ions and excited species generated by gas discharge, and / or (iii) one or more chemical oxidizing agents. [25] Method according to claim 24, wherein the one or more oxidizing agents: (i) are electromagnetic radiation and the oxidation step comprises irradiating the sample with light having a wavelength of less than 400 nm, wherein the substrate acts as a photocatalyst to oxidize the sample, or (ii) are one or more chemical oxidizing agents and the oxidation step comprises the action of one or more chemical oxidizing agents on the sample, selected from: ozone, persulfate and hydrogen peroxide. [26] Method according to any one of claims 15 to 25, wherein the substrate is a planar slide (102, 106), in particular a metal, a coated or uncoated glass or a coated or uncoated flat ceramic plate or a substrate with a titanium dioxide surface. [27] Method according to any one of claims 15 to 26, wherein the step of oxidizing the sample on the substrate comprises passing one or more oxidizing agents from a side of the substrate opposite the sample through pores in the substrate to reach the sample. [28] Method according to claim 27, wherein a second substrate is located near the sample, but at a certain distance from and directed towards the sample, wherein one or more oxidizing agents diffuse through the sample, thereby generating volatile products from the sample and causing non-volatile elements and / or their oxides to reach the surface of the second substrate and remain there to be detected by the imaging elemental analyzer, wherein a gap between the substrates is such that the spatial distribution of the non-volatile elements transferred to the second substrate is on average no more disturbed by the oxidation than the spatial resolution of an imaging analysis performed by the imaging elemental analyzer. [29] Method according to any one of claims 15 to 28, further comprising generating an image of the elements detected in the sample. [30] Method according to claim 29, wherein the detection of the one or more elements comprises irradiating the concentrated sample with a beam of primary ions or photons focused on a localized spot on the sample surface to emit formed ions (38) or reaction products of the formed ions (38) from the spot and analyzing the formed ions (38) or reaction products of the formed ions (38) to determine the presence and optionally the amount of the one or more elements at the spot, wherein the spot is moved over time to a plurality of locations on the sample surface to obtain an image of the one or more elements in the sample, each location of the spot on the sample surface corresponding to a pixel of the image. [31] Method according to one of claims 29 or 30, wherein the image is captured at a speed of at least 100 pixels per second or at least 1000 pixels per second or in the range of 1000 - 10000 pixels per second. [32] Method according to one of claims 30 or 31, wherein the primary ions or photons have an energy of over 1 keV. [33] Method according to any one of claims 30 to 32, wherein the beam comprises a primary ion beam and has an intensity of 1 to 100 nA per 1 µm spot diameter. [34] Method according to any one of claims 30 to 33, wherein the imaging elemental analyzer comprises a further mass analyzer, wherein the further mass analyzer is selected from: a distance-of-flight mass analyzer, a quadrupole ion trap mass analyzer, an electrostatic trap mass analyzer, an ion cyclotron resonance mass analyzer and a magnetic sector mass analyzer or an array or a combination thereof. [35] Method according to any one of claims 30 to 34, wherein the ion guide comprises a reactive gas to form reaction products with the formed ions (38), wherein the formed ions (38) are ions comprising one or more of the elements, wherein a composition and / or energy distribution of the ions at the outlet of the ion guide is changed compared to the ions at the inlet of the ion guide. [36] Method according to any one of claims 34 to 35, wherein the repetition rate of the TOF mass analyzer is at least equal to or greater than (a) 20 kHz or (b) 50 kHz or (c) 100 kHz, wherein optionally no more than (a) 2 or (b) 5 or (c) 10 pulses of the TOF mass analyzer are required for the analysis of each spot with the TOF mass analyzer.

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