Sample analysis method using an electron microscope

Abstract

This invention provides a novel method that enables electron microscopy observation and measurement of substances present in a single sample. [Solution] A sample analysis method using an electron microscope according to one embodiment of the present invention is characterized by comprising the steps of: applying a protective agent for electron microscope observation containing a survival environment-providing component, sugars, and electrolytes as the main components, or an auxiliary solution containing the protective agent for electron microscope observation and water, an organic solvent, or a mixed solvent of water and an organic solvent to a sample to be observed, irradiating the sample with an electron beam or plasma to form a thin film on the surface of the sample, and preparing a sample with a thin film; subjecting the sample with the thin film to electron microscope observation; and using the sample with the thin film after electron microscope observation, measuring the substances present in the sample without removing the thin film.

Description

Technical Field

[0001] The present invention relates to a sample analysis method using an electron microscope.

Background Art

[0002] In recent years, in the observation of pathological tissue specimens, cytological specimens, etc. using an electron microscope, in addition to two-dimensional data obtained by an optical microscope or a transmission electron microscope (TEM), attempts have been made to acquire three-dimensional data by a scanning electron microscope (SEM) and analyze information on the target pathological tissue, cells, etc.

[0003] Patent Document 1 discloses a protective agent for electron microscope observation under vacuum that can protect the living state of mammalian, plant tissues, cultured cells, single cells, etc. in a water-containing state without deforming them even under vacuum, and a method for observing and diagnosing samples using an electron microscope using the same. According to Patent Document 1, by applying a protective agent for electron microscope observation to a water-containing cancer cell or a tissue containing the same excised from a living body and irradiating with an electron beam or plasma to form and cover a thin film on the sample surface, image diagnosis by an electron microscope becomes possible. Specifically, for example, from the observation results of pathological specimens of human gastric cancer, it is possible to confirm the morphological differences between cancer cells and normal cells, and by observing a sample obtained by culturing cancer cells collected from a skin cancer patient, it is shown that it is possible to diagnose whether the cancer cells are metastatic or non-metastatic strains.

[0004] Patent Document 2 discloses a method for observing pathological tissue specimens or cytological specimens using SEM. According to Patent Document 2, a stained pathological tissue specimen or cytological specimen is observed with an optical microscope, the target area is marked, a protective solution for specimens containing a protective agent for electron microscopy observation mainly composed of survival environment-providing components, sugars, and electrolytes is applied to the surface of the specimen, and a thin film is formed on the surface of the specimen by irradiating it with an electron beam or plasma, making it possible to observe the marked area using SEM. With this technique, the same field of view can be observed with both an optical microscope and an SEM using a single specimen. In addition, after observation with SEM, the thin film (protective film for specimens) can be partially removed by treating the specimen with a predetermined solvent, and since the original staining state is maintained to a certain extent in the specimen from which the protective film has been removed, the same specimen can be used for optical microscope observation, etc., or stained again as needed. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 2015 / 115502 [Patent Document 2] Japanese Patent Publication No. 2020-34410 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] In conventional observation methods, when paraffin-embedded sections stained with hematoxylin-eosin (HE staining), special stains, immunostaining, etc., are subjected to SEM observation, it is necessary to apply metal or carbon deposition to the sample. Furthermore, due to concerns that the sample may become irreparable due to drying that inevitably occurs during the observation process, stained samples are not used directly for SEM observation; instead, serial sections must be newly prepared from the paraffin block. Therefore, the invention described in Patent Document 2 above was proposed to solve the problem that it is difficult to observe the area where an abnormality has been confirmed using conventional methods. Patent Document 2 discloses that the specimen after undergoing the series of observation steps disclosed therein (even if the protective film for the specimen is not completely removed, because the protective film is transparent to visible light) can be subjected to observation again using an optical microscope or SEM, or observation using other observation means. However, it cannot be said that sufficient consideration has been given to the application of observation means other than optical microscopes and SEM.

[0007] For example, in recent years, attention has been focused on a technique that uses mass spectrometry imaging (MSI) to analyze samples such as biological tissues, obtain imaging images from the resulting mass spectra, and analyze information about substances present in the sample. However, in the MSI method, stained samples cannot be used for analysis because peaks originating from the substances used in the staining process may appear. Furthermore, since the aforementioned protective film for specimens is transparent to visible light, it is thought that even if the protective film is not completely removed, there will be no problem with further observation using an optical microscope or SEM. However, the possibility of unintended damage to the original specimen (section) during the removal process cannot be ruled out. On the other hand, it is not entirely clear whether observation methods other than optical microscopes and SEMs can be applied without removing the protective film, and further concrete investigations have been desired.

[0008] This invention has been made in view of the above circumstances, and aims to provide a novel method that enables electron microscopy observation and measurement of substances present in a single sample. [Means for solving the problem]

[0009] The present invention provides a sample analysis method using an electron microscope, comprising the steps of: applying a protective agent for electron microscopy observation containing a survival environment-providing component, sugars, and electrolytes as the main components, or an auxiliary solution containing the said protective agent for electron microscopy observation and water, an organic solvent, or a mixed solvent of water and an organic solvent to a sample to be observed, irradiating the sample with an electron beam or plasma to form a thin film on the surface of the sample, and preparing a sample with a thin film; subjecting the sample with the thin film to electron microscopy observation; and using the sample with the thin film after electron microscopy observation, performing measurements on substances present in the sample without removing the thin film.

[0010] In a preferred embodiment of the above method, the sample to be observed is a sample that has not been stained. Furthermore, in a preferred embodiment of the above method, the electron microscope observation is performed using a scanning electron microscope. Furthermore, in a preferred embodiment of the above method, the measurement is performed using at least one selected from the group consisting of imaging mass microscopes, optical microscopes, fluorescence microscopes, super-resolution microscopes, multiphoton laser microscopes, light sheet microscopes, devices or instruments configured to be attached to the above electron microscopes, immunostaining, and in situ hybridization. Furthermore, in a preferred embodiment of the above method, the step of subjecting the sample to be observed to observation for an observation other than electron microscopy is further included before the step of preparing the thin-film-attached sample. Here, observations other than electron microscopy observations may be made using at least one microscope selected from the group consisting of optical microscopes, fluorescence microscopes, super-resolution microscopes, multiphoton laser microscopes, and light-sheet microscopes. Furthermore, in a preferred embodiment of the above method, the auxiliary solution comprises the electron microscope observation protective agent and an amphiphilic solvent, or a mixed solvent of water and an amphiphilic solvent. Here, the amphiphilic solvent may be ethanol. [Effects of the Invention]

[0011] According to the present invention, a method is provided that enables electron microscopy observation and measurement of substances present in a single sample. [Brief explanation of the drawing]

[0012] [Figure 1] Mass spectrum obtained for the sample of Example 1. [Figure 2] (a)~(d) FE-SEM images obtained for the sample of Example 2. [Figure 3] Mass spectrum obtained for the sample of Example 2. [Figure 4] Optical microscope image (left) and mass spectrometry imaging image (right) obtained for the sample of Example 2. [Figure 5] Imaging images obtained for the sample of Example 2. Top row: Sample 2, Middle row: Sample X, Bottom row: Sample Y. [Figure 6] FE-SEM images obtained for the sample of Example 3. Left column: comparison sample, right column: sample 3. [Figure 7] FE-SEM images obtained for the sample of Example 3. Top row: comparison sample, bottom row: sample 3. [Figure 8] Optical microscope images obtained for the sample of Example 3. Left: control sample, center: sample 3, right: comparison sample. [Figure 9] Enlarged images of the upper center of each image in Figure 8. Left: Control sample, Center: Sample 3, Right: Comparison sample. [Figure 10] Enlarged images of the areas enclosed by squares in each image in Figure 9. Left: Control sample, Center: Sample 3, Right: Comparison sample. [Figure 11]FE-SEM images obtained for the sample of Example 4. Left column: comparative sample, right column: sample 4. [Figure 12] FE-SEM images obtained for the sample of Example 4. Upper row: comparative sample, lower row: sample 4. [Figure 13] Optical microscope images obtained for the sample of Example 4. Upper row: control sample, middle row: sample 4, lower row: comparative sample.

Mode for Carrying Out the Invention

[0013] Hereinafter, embodiments of the present invention will be described. Note that the specific forms are not limited to the following embodiments, and design changes and the like within the scope not departing from the gist of the present invention are also included in the present invention.

[0014] In this specification, the term "protective agent for electron microscope observation" refers to a solution (Surface Shielding Enhancer (SSE) solution) containing a living environment-providing component, saccharides, and electrolytes as main components, which is disclosed in Patent Document 1 described above.

[0015] As a result of repeatedly performing SEM observation and pathological diagnosis using the above-described protective agent for electron microscope observation on various pathological tissue specimens and cytological specimens, the present inventors specifically confirmed that it is possible to perform electron microscope observation and measurement of substances present in the sample without performing a removal treatment of the thin film formed on the surface of the sample using a single sample, and thus completed the present invention.

[0016] A method for analyzing a sample using an electron microscope according to an embodiment of the present invention includes a step of applying a protective agent for electron microscope observation containing a living environment-providing component, saccharides, and electrolytes as main components, or an auxiliary liquid containing the protective agent for electron microscope observation and water, an organic solvent, or a mixed solvent of water and an organic solvent to a sample to be observed, irradiating an electron beam or plasma to form a thin film on the surface of the sample, and preparing a sample with a thin film. Note that hereinafter, for the sake of clarity, an aspect in which the sample to be observed is a pathological tissue specimen will be described, but it should be noted that the present invention is not limited to this aspect.

[0017] The method for preparing pathological tissue specimens is well known in the field. For example, pathological tissue specimens (hereinafter also simply referred to as "specimens") are typically prepared by placing paraffin-embedded sections on a glass slide, deparaffinizing them, staining them, and then covering them with a coverslip. On the other hand, in this embodiment, if staining may adversely affect measurements of substances present in the specimen, the specimen may be prepared without staining. Also, if optical microscopy observation or the like is not performed before observation with an electron microscope, a so-called "no-cover specimen" without a coverslip may be used.

[0018] In one exemplary embodiment, serial sections may be prepared from a paraffin block (tissue block) in which abnormalities were observed in stained paraffin-embedded sections, and one of these serial sections may be used to prepare the sample (specimen) to be observed in this embodiment. In other words, in the present invention, "single sample" means that the sample to which the above-mentioned electron microscope observation protective agent or auxiliary solution is applied and to which measurements regarding the substances present in the sample are performed after electron microscope observation are the same, and it is not intended to limit whether or not observations using another sample prepared from the same tissue block have been performed prior to the electron microscope observation.

[0019] Here, if necessary, the prepared specimen may be observed with an optical microscope before the electron microscope observation described later. Alternatively, images of the specimen may be obtained by taking photographs with an optical microscope or by scanning with a commercially available glass slide scanner. Furthermore, in addition to, or instead of, optical microscope observation, observation using a fluorescence microscope, super-resolution microscope, multiphoton laser microscope, light sheet microscope, or other microscope may be performed.

[0020] Next, remove the cover glass from the specimen using a solvent (e.g., xylene) and dry the solvent from the specimen. If the specimen is an uncovered specimen as described above, this step can be omitted. After that, if necessary, treat the specimen with ethanol and then water to make the surface of the specimen hydrophilic. At this point, a light drying treatment to remove excess moisture may be performed.

[0021] Next, the specimen is coated with a protective agent for electron microscopy observation (or an auxiliary solution described later). While there are no particular limitations on the means of applying the protective agent, it is desirable to apply it without directly touching the specimen in order to avoid damaging it. For example, the protective agent can be applied to the specimen using a commonly used pipette. Alternatively, a device that applies minute droplets may be used.

[0022] The composition of the protective agent for electron microscopy observation is not particularly limited, but the following compositions are typical examples and can be used as the stock solution. Specific example of the composition and preparation method of the stock solution for electron microscope observation protective agent: Dissolve 5g of sucrose, 5g of fructose, and 5g of sodium chloride in 500mL of water, add 1.25g of citric acid and 0.05g of sodium glutamate, adjust the pH to 7.4, and mix this aqueous solution with glycerin in a ratio of aqueous solution:glycerin = 1:2.

[0023] The protective agent for electron microscopy observation may be used as is from the stock solution described above, or it may be diluted with any solvent. Examples of solvents include water, organic solvents, and mixed solvents of water and organic solvents. In this specification, a solution prepared by diluting the stock solution of the protective agent for electron microscopy observation, represented by the above composition, with any solvent is referred to as an "auxiliary solution."

[0024] In one exemplary embodiment of this product, the stock solution is diluted with water to prepare an auxiliary solution. The dilution ratio can be, for example, stock solution to water in the range of 1:1 to 1:50, preferably in the range of 1:1 to 1:30, more preferably in the range of 1:2 to 1:20, and even more preferably in the range of 1:5 to 1:10.

[0025] In another embodiment of this model, the stock solution is diluted with an organic solvent to prepare an auxiliary solution. The organic solvent is not particularly limited, but for example, an amphiphilic solvent such as ethanol can be used. As for the dilution ratio of the stock solution, for example, the ratio of stock solution to organic solvent can be in the range of 1:1 to 1:50, preferably in the range of 1:1 to 1:30, more preferably in the range of 1:2 to 1:20, and even more preferably in the range of 1:5 to 1:10. By using the auxiliary solution of this model, the hydrophilization treatment step of the specimen surface can be omitted. That is, in this model, the cover glass of the specimen is removed, the solvent on the specimen is dried, and then the auxiliary solution is applied. This further reduces the observation time. Naturally, if the sample is the aforementioned uncovered specimen, the observation time can be further reduced.

[0026] In addition, thin films formed from auxiliary solutions prepared by dilution with organic solvents can exhibit superior charge suppression effects during SEM observation. Specifically, in the case of an auxiliary solution prepared with a stock solution:ethanol ratio of 1:10, sufficient charge suppression effects were confirmed during SEM observation under an accelerating voltage of 15kV. This indicates that the same sample can be subjected to EDX analysis, etc. On the other hand, for SEM observations under acceleration voltage conditions of 10kV or less (for example, acceleration voltage conditions in the range of 5kV to 10kV), an excellent anti-static effect can be achieved even when using an auxiliary solution prepared by diluting the above stock solution with water. When using a mixed solvent of water and an organic solvent, the water and organic solvent should be mixed in any proportion, and the resulting mixed solvent should be used to dilute the stock solution to satisfy the above-mentioned dilution ratio range.

[0027] It is preferable to allow specimens coated with an electron microscope protective agent or auxiliary solution to stand for a certain period of time as needed, and then spin-dry them using a spin-coating device, thereby making the layer of the electron microscope protective agent or auxiliary solution on the specimen more uniform.

[0028] Next, a thin film is formed on the surface of the specimen by irradiating it with an electron beam or plasma. In a typical embodiment of this specification, the specimen is placed on the sample stage of the SEM, and a thin film is formed on the surface of the specimen by irradiating it with an electron beam or plasma. This creates a specimen with a thin film. In other words, in this embodiment, a specimen with a thin film can be created using the principle of SEM. To put it another way, any configuration that allows for irradiation of the specimen with an electron beam or plasma can be used to create a specimen with a thin film, but when performing SEM observation as electron microscopy observation as described later, it is efficient to create a specimen with a thin film using SEM. In this specification, the thin film formed by irradiating the undiluted or auxiliary solution of the electron microscopy observation protective agent applied to the specimen with an electron beam or plasma is also referred to as the "protective film".

[0029] Next, the sample with the thin film attached is subjected to electron microscope observation. In a typical embodiment of this model, the specimen with the protective film attached is subjected to SEM observation to observe the target lesion, etc. In this embodiment, the protective film reduces the charging (charge-up) of the specimen, and a clear image can be obtained, so that lesions, etc., which are usually identified by optical microscopes, can be identified from the SEM image. Alternatively, if lesions, etc., have been previously identified using another sample (stained sample) prepared from the same tissue block, it is relatively easy to identify the corresponding area in the thin film attached specimen. Furthermore, if observation with an optical microscope or the like has been performed first, the specimen image obtained there and the SEM image can be combined.

[0030] In the invention described in Patent Document 2 mentioned above, after observation by SEM, the specimen can be treated in the order of water, ethanol, and xylene (a solvent selected depending on the type of mounting medium used) to partially remove the protective film formed on the surface of the specimen. Even after this removal treatment, the specimen remains stained to a degree that it can be used directly for optical microscopy observation, etc. Furthermore, the specimen after removal of the protective film can be stained again as needed, thereby returning it to the stained state before SEM observation.

[0031] In contrast, in this embodiment, the sample after electron microscopy observation is used as is, and measurements are performed on the substances present in the sample. That is, the sample analysis method using an electron microscope according to this embodiment includes a step of performing measurements on the substances present in the sample using a thin-film sample after electron microscopy observation, without removing the thin film. In the following, this step may be simplified and referred to as the "measurement step."

[0032] The "measurement" performed in the measurement process is intended to be different from the electron microscope observation described above. That is, the sample analysis method according to this embodiment makes it possible to correlate the information obtained from electron microscope observation with the information obtained from the measurement for a single sample. Therefore, any information obtained that is different from the information obtained from electron microscope observation can be included in the scope of the measurement. For example, in a typical embodiment of this embodiment, the electron microscope observation is SEM observation, so any measurement that obtains information different from the information obtained from SEM observation may be performed using an apparatus or instrument configured to be attached to the apparatus (scanning electron microscope) used for SEM observation. Alternatively, the sample can be removed from the scanning electron microscope, subjected to appropriate pretreatment, etc., and then subjected to SEM observation again, or it can be subjected to measurements other than SEM observation.

[0033] The above measurements can be performed using microscopes that utilize various imaging technologies, such as imaging mass microscopes, fluorescence microscopes, super-resolution microscopes, multiphoton laser microscopes, and light-sheet microscopes. Furthermore, an energy-dispersive X-ray spectrometer (EDX) can be used as an attachment to a scanning electron microscope (SEM). Alternatively, the measurement method can be selected according to the type of substance of interest; for example, immunostaining can be used when focusing on proteins in the sample, while in situ hybridization can be used when focusing on DNA or mRNA.

[0034] In one exemplary embodiment, scanning electron microscopy (SEM) observation is performed to observe the target lesion, etc. After the specimen is removed from the SEM sample chamber and subjected to necessary pretreatment, observation is performed using an imaging mass microscope (IMS). This allows for the correlation and analysis of information obtained from SEM observation and imaging images obtained from the IMS. Furthermore, if the IMS has optical microscope functionality, it is possible to perform analysis combining SEM images, imaging images, and optical microscope images. Moreover, if observation using a microscope as exemplified above is performed before electron microscopy (SEM observation), it is possible to obtain more multifaceted and comprehensive information from these observation results.

[0035] In addition, it is expected that the present invention can be applied to spatial transcriptome analysis, which has been attracting increasing attention in recent years (for example, the Visium platform (10x Genomics) and the CosMx® RNA assay (nanoString)), enabling comprehensive analysis of gene expression profiles related to target pathological tissues and cells. Similarly, it is expected that the present invention can be applied to techniques that perform high-level multiplexing of multiple proteins from a single sample, such as spatial proteomics analysis (for example, the CosMx® protein assay (nanoString)), enabling comprehensive analysis of protein expression profiles related to target pathological tissues and cells, and that these can be combined and applied to spatial proteogenomics. [Examples]

[0036] The present invention will be described in more detail below using examples, but the present invention is not limited in any way to these examples.

[0037] In the following examples, SEM observations were typically performed using a field emission scanning electron microscope (FE-SEM; Hitachi S-4800) with an accelerating voltage of 1.0 kV (secondary electron mode). Photographs were also taken at magnifications of 30,000x, 20,000x, 10,000x, 5,000x, 2,500x, 1,000x, 400x, and / or 30x, as needed.

[0038] <Example 1> We prepared the sample for observation using bovine serum albumin (BSA), which is used as a protein standard substance in various research experiments on proteins. Specifically, BSA was immobilized on conductive glass (ITO-coated slide glass, resistance value 20Ω) using a commercially available glutaraldehyde fixative. The immobilized BSA was embedded in paraffin and thinly sectioned (10 μm) onto another conductive glass (same specifications as above) to prepare paraffin sections.

[0039] Next, the paraffin sections were deparaffinized by immersing them in xylene for 1 minute four times. Then, they were washed twice each in 99% ethanol, 70% ethanol, and 50% ethanol for 2 minutes each, followed by two washes in ultrapure water (DDW) for 1 minute each to perform hydrophilization. After that, they were dried overnight in an incubator set to 54°C. The prepared samples were not stained.

[0040] Next, an auxiliary solution (original solution:ethanol = 1:50) prepared by diluting the undiluted solution of the aforementioned electron microscope observation protective agent with ethanol was applied to the surface of the sample, left to stand for about 30 seconds, and then spin-dried using a spin-coating apparatus.

[0041] Next, the sample was placed on the sample stage of the SEM, and an electron beam emitted from the SEM's electron source was irradiated onto the surface of the sample to form a thin film, thereby creating a sample with a thin film. Subsequently, the sample with the thin film was subjected to SEM observation and then removed from the SEM's sample chamber.

[0042] The removed thin-film-attached samples were subjected to antigen retrieval by heating in a citrate buffer (10 mM, pH 6.0) at 100°C for 30 minutes using an autoclave. Afterward, they were cooled to room temperature and washed with distilled water. They were then cooled in a water bath set to 10°C for 15 minutes, rinsed 10 times with DDW, and dried in a desiccator.

[0043] Next, a trypsin solution for protein digestion (0.05 μg / μL, 10% acetonitrile, 50 mM ammonium bicarbonate) was applied to the dried, thin-film-attached sample. Specifically, approximately 200 μL of the trypsin solution was applied 10 to 12 times using a hand brush, with each application being approximately 200 μL. The sample was then left to stand for 2.5 hours in an incubator set to 37°C.

[0044] Next, a matrix solution for mass spectrometry (5 mg / mL α-cyano-4-hydroxycinnamic acid (CHCA), 0.1% trifluoroacetic acid, 50% acetonitrile) was applied to the surface of the sample using a spray device (imaging mass spectrometry sample preparation device; Techno Alpha TM-sprayer). The main conditions (parameters) during application were as follows. Nozzle temperature: 90℃ Flow rate: 0.1mL / min Password number: 8 Path type: Criss-cross (CC)

[0045] Furthermore, none of the above-mentioned antigen retrieval treatment, trypsin treatment, or matrix coating treatment constitutes a treatment for removing the thin film (protective film) formed on the sample surface.

[0046] Next, the sample was subjected to mass spectrometry using an imaging mass microscope (FT-ICR MS; Bruker Solarix XR). The main conditions (parameters) used during the measurement were as follows. Calibration: NaF + TFA Mass range m / z: 150~3000Da Polarity: Positive Ion Mode Data Magnitude: 1M ToF time: 1.2ms Accumulation time: 0.05 Raster: 200 μm Laser power: 30% Laser focus: medium Laser Shot: 100 Laser frequency: 1000Hz

[0047] Figure 1 shows the obtained mass spectrum. For comparison, the same measurements were performed on paraffin sections (control samples) prepared in the same manner as above, except that the trypsin solution described above was not applied. As a result, clear peaks were observed in the sample of this example at m / z values ​​where no peaks were observed in the mass spectrum of the control sample (for example, m / z = 834.43, 842.45, 1350.59, etc.). From this, it was found that the mass spectrum of the sample of this example shown in Figure 1 shows multiple peaks attributable to BSA peptides produced by trypsin digestion.

[0048] Furthermore, when the BSA protein used in this example was subjected to mass spectrometry according to a standard procedure without SEM observation, a mass spectrum similar to that in Figure 1 was obtained for the trypsin-treated sample, thus confirming the validity of the mass spectrometry performed using the sample in this example.

[0049] These results suggest the following, for example: The thin film formed on the sample surface protects the substances present in the sample (proteins in this example) even after SEM observation. This is thought to be due to the water / gas barrier properties (Surface Shielding Effect, SS effect) of the thin film (protective film). On the other hand, the thin film does not produce any negative effects in the pretreatment required for the measurement process after SEM observation. In this embodiment, trypsin treatment of the sample with the thin film allowed for the digestion of BSA protein, and mass spectrometry could be performed using the sample with the matrix-treated thin film. This suggests that the thin film not only possesses the aforementioned water / gas barrier properties, but also has the potential to broaden the range of options for the measurement process performed after SEM observation.

[0050] <Example 2> Pathological tissue specimens for observation were prepared using serial sections (FFPE serial sections) made from paraffin blocks that had been previously diagnosed with AA amyloidosis by observation of HE specimens. In this example, some samples were stained to confirm the effectiveness of the sample analysis method according to the present invention, but it should be noted that this does not mean that staining is essential for carrying out the method.

[0051] The paraffin sections were deparaffinized by immersing them in xylene for 1 minute four times. Next, they were washed twice each in 99% ethanol, 70% ethanol, and 50% ethanol for 2 minutes each, followed by two washes in DDW for 1 minute each to induce hydrophilization. Finally, they were left to stand overnight in an incubator set at 54°C and dried. The prepared samples were not stained.

[0052] Next, an auxiliary solution (undiluted solution:ethanol = 1:50) prepared in the same manner as in Example 1 was applied to the surface of the sample, left to stand for about 30 seconds, and then spin-dried using a spin-coating apparatus. Then, the sample was placed on the sample stage of the SEM, and the electron beam emitted from the electron source of the SEM was irradiated onto the surface of the sample to form a thin film, thereby creating a sample with a thin film. Subsequently, the sample with the thin film was subjected to SEM observation and then removed from the sample chamber of the SEM.

[0053] Figures 2(a) to 2(d) show examples of obtained FE-SEM images. All images in Figures 2(a) to 2(d) are of the same sample, with the magnification increasing from (a) to (d). The scale bars in the figures are 1 mm for Figure 2(a), 100 μm for Figure 2(b), 5 μm for Figure 2(c), and 2 μm for Figure 2(d). In the images in Figures 2(c) and 2(d), amyloid fibrils can be seen in the areas indicated by the arrows.

[0054] After SEM observation, the thin-film-attached sample was subjected to antigen retrieval by heating in a citrate buffer (10 mM, pH 6.0) at 100°C for 30 minutes using an autoclave. The sample was then cooled to room temperature and washed with distilled water. Next, it was cooled in a water bath set to 10°C for 15 minutes, rinsed 10 times with DDW, and dried in a desiccator.

[0055] Next, a trypsin solution for protein digestion (0.05 μg / μL, 10% acetonitrile, 50 mM ammonium bicarbonate) was applied to the dried, thin-film-attached sample. Specifically, approximately 400 μL of the trypsin solution was applied 10 to 12 times using a hand brush, with each application being approximately 400 μL. The sample was then left to stand for 2.5 hours in an incubator set to 37°C.

[0056] Next, a matrix solution for mass spectrometry (5 mg / mL CHCA, 0.1% trifluoroacetic acid, 50% acetonitrile) was applied to the surface of the sample using a sprayer (TM-sprayer). The main conditions (parameters) during application were as follows. Nozzle temperature: 90℃ Flow rate: 0.1mL / min Password number: 8 Path type: Criss-cross (CC)

[0057] Furthermore, none of the above-mentioned antigen retrieval treatment, trypsin treatment, or matrix coating treatment constitutes a treatment for removing the thin film (protective film) formed on the sample surface.

[0058] Next, the samples were subjected to mass spectrometry using imaging mass microscopes (TOF-MS; Shimadzu iMScope QT, and FT-ICR MS; Bruker Solarix XR). The main conditions (parameters) used during the measurement were as follows. (iMScope QT) Regarding the m / z range of 900 to 1800 Pitch size: 50 μm Laser power: 70 Laser diameter: 2 Laser Shot: 100 Laser frequency: 1000Hz Detector voltage: 2.2kV Ion mode: +Ve Heat block: 450℃ DL temperature: 250℃ Regarding the m / z range of 500-900 Pitch size: 50 μm Laser power: 60 Laser diameter: 2 Laser Shot: 50 Laser frequency: 1000Hz Detector voltage: 2.2kV Ion mode: +Ve Heat block: 450℃ DL temperature: 250℃ (Solarix XR) Pitch size: 150 μm Data size: 512K Laser power: 24% m / z: 900~1800 Accumulation time: 0.05 ToF time: 1.2ms Laser focus: minimum Polarity: +Ve Calibration: NaF + TFA

[0059] Figure 3 shows an example of a mass spectrum obtained with iMScope QT. Figure 3 reveals that multiple peaks attributable to different peptides appear within the m / z range of 500 to 900. Although not shown, multiple peaks attributable to different peptides were also observed in the mass spectrum within the m / z range of 900 to 1800.

[0060] Figure 4 shows an image (left) acquired using the optical microscope mounted on the iMScope QT and an imaging image (right) of the same sample. The scale bar in both images is 500 μm. The imaging image on the right visualizes the signal intensity at an m / z value of 887.51, which was identified as a peptide derived from vitronectin (VTN). The rectangular frame in the image indicates a region of high brightness, meaning that the above peptide is present in large quantities in that region. In fact, this region includes the area shown in the SEM images in Figures 2(a) to 2(d), which suggests that VTN is expressed at the amyloid fibril deposition site.

[0061] Figure 5 shows several imaging images obtained with iMScope QT. For comparison, two thin-film samples were prepared using paraffin sections made from the same paraffin block as the sample described above. One sample was not subjected to SEM observation, but mass analysis was performed using an imaging mass microscope in the same procedure as described above. The other sample was not subjected to SEM observation, nor was protein digestion with trypsin solution performed, but mass analysis was performed using an imaging mass microscope in the same procedure as described above. For convenience, the sample from this example will be referred to as "Sample 2," and the two comparative samples will be referred to as "Sample X" and "Sample Y."

[0062] In Figure 5, the top row shows images of sample 2, the middle row shows images of sample X, and the bottom row shows images of sample Y. For clarity, the leftmost section of each row shows an optical microscope image of a section of the sample before (or after) staining with DFS (Direct Fast Scarlet) staining solution for amyloid staining. These are intended to objectively demonstrate the presence of amyloid-derived proteins in each sample and do not imply that staining is an essential step in the sample analysis method according to the present invention.

[0063] Each row in Figure 5 shows six imaging images. These images, from left to right, represent the signal intensity for m / z values ​​of 515.33, 523.29, 659.34, 764.44, 887.51, and 1314.68. The areas with higher brightness indicate a greater concentration of peptides corresponding to each m / z value.

[0064] Looking at sample Y, shown in the lower part of Figure 5, we can see that the brightness of the image is generally low for all m / z values. Since sample Y has not undergone protein digestion with trypsin solution, these results are reasonable.

[0065] Looking at sample X shown in the middle of Figure 5, for example, a region of high brightness is observed in the image with an m / z value of 515.33, and the shape of this region is similar to the outer shape of the sample in the optical microscope image to its left. In addition, areas of high brightness are also seen in the images with m / z values ​​of 523.29 and 887.51. Compared with the results for sample Y described above, it was suggested that in sample X, protein digestion with trypsin solution digested amyloid-derived proteins, resulting in the production of multiple peptides. However, the images with m / z values ​​of 659.34 and 764.44 appear to be brighter overall than those of sample Y, making it difficult to identify regions where the target peptide is abundant.

[0066] In contrast, looking at sample 2 shown in the upper part of Figure 5, it can be seen that the contrast between light and dark is improved in all images with m / z values ​​compared to sample X. For example, in the images with m / z values ​​of 523.29 and 887.51, it is clear that the target peptide is abundant in the upper part of the image. Also, in the images with m / z values ​​of 659.34 and 764.44, areas of high brightness can be seen in the upper part of the image, and the clarity of the image is clearly higher compared to the results for sample X mentioned above. In addition, in the image with m / z value of 1314.68, the brightness in the upper part of the image is also high, suggesting the presence of peptides that could not be confirmed in the images of sample Y and sample X.

[0067] Furthermore, analysis of the mass spectrum peaks revealed that the m / z value of 659.34 was derived from serum amyloid A (SAA) peptide, 764.44 from serum amyloid P (SAP) peptide, and 887.51 and 1314.68 from vitronectin (VTN) peptide.

[0068] These results suggest that, according to the sample analysis method of the present invention, subjecting a sample with a thin film to electron microscopy observation (SEM observation in this embodiment) can improve the accuracy of measurement results for the target substance compared to simply using a sample with a thin film to perform measurements on the substance present in the sample. In the case of mass spectrometry imaging performed in this embodiment, the "improvement in the accuracy of measurement results" refers, for example, to an improvement in the contrast of the brightness and darkness of the imaging image. Thus, according to the present invention, not only is it possible to perform electron microscopy observation and measurement of the substance present in the sample using the same (single) sample, but the electron microscopy observation process may also have a favorable effect and / or benefit on the latter measurement.

[0069] <Example 3> Pathological tissue specimens were prepared as samples for observation using serial sections (FFPE serial sections) made from paraffin blocks diagnosed with lymphoproliferative disorders.

[0070] The paraffin sections were deparaffinized by immersing them in xylene for 1 minute four times. Next, they were washed twice each in 99% ethanol, 70% ethanol, and 50% ethanol for 2 minutes each, followed by two washes in DDW for 1 minute each to perform hydrophilization. Finally, excess moisture was removed using a spin coating apparatus, and the samples were lightly dried. The prepared samples were not stained.

[0071] Next, an auxiliary solution (undiluted solution:ethanol = 1:50) prepared in the same manner as in Example 1 was applied to the surface of the sample, left to stand for about 30 seconds, and then spin-dried using a spin-coating apparatus. Then, the sample was placed on the sample stage of the SEM, and the electron beam emitted from the electron source of the SEM was irradiated onto the surface of the sample to form a thin film, thereby creating a sample with a thin film. Subsequently, the sample with the thin film was subjected to SEM observation and then removed from the sample chamber of the SEM.

[0072] Figure 6 shows an example of the obtained FE-SEM image. For comparison, FE-SEM images of a sample (hereinafter also referred to as the "comparative sample") prepared from the same paraffin block as the above sample, but without the application of the auxiliary solution, are also shown.

[0073] The left column of Figure 6 shows FE-SEM images of the comparison sample, and the right column shows FE-SEM images of sample 3. The top images are at 5,000x magnification (scale bar 10.0 μm), the middle images are at 2,500x magnification (scale bar 20.0 μm), and the bottom images are at 1,000x magnification (scale bar 50.0 μm).

[0074] As shown in Figure 6, there appear to be no particularly significant differences between the FE-SEM images of the comparison sample and sample 3. However, in the image taken at 30x magnification (scale bar 1.00 mm) shown in Figure 7, damage that appears to be caused by electron beam irradiation was clearly observed in the upper comparison sample in the area enclosed by the dotted line near the center of the image, whereas no such damage (electron beam irradiation marks) was observed in sample 3 in the lower panel.

[0075] These results suggest that samples without the aforementioned protective agent for electron microscopy observation may experience various problems during electron microscopy observation (SEM observation in this example), including the degradation of nucleic acids such as RNA. This makes it difficult to perform other measurements (e.g., in situ hybridization using multiple probes) using the same sample. In contrast, samples with a thin film formed using the protective agent for electron microscopy observation are expected to suppress the occurrence of the aforementioned problems. Therefore, in situ hybridization (ISH) targeting EBER (EBV encoded small RNA) was performed using sample 3, which was removed from the SEM sample chamber. Note that none of the following processes involve the removal of the thin film (protective film) formed on the sample surface.

[0076] First, sample 3 underwent antigen retrieval. Specifically, a citrate buffer (weakly acidic) for antigen retrieval was applied to the slide of sample 3, incubated at 90°C for 12 minutes, then the slide was cooled to room temperature and washed with PBS.

[0077] Next, sample 3, which had been washed with DEPC-treated water, was treated with proteinase K. Specifically, the slide of sample 3 was washed with RNase-free PBS for 5 minutes, then the tissue of sample 3 was treated with proteinase K solution (20 μg / mL) at 37°C for 10 minutes, and then the slide was washed again with RNase-free PBS.

[0078] Next, as a pre-treatment for hybridization, the slide of sample 3 was incubated with pre-hybridization buffer at 37°C for 30 minutes.

[0079] Next, as a hybridization treatment, the hybridization buffer was applied to the slide of sample 3 and covered with a coverslip. The hybridization buffer contained an EBER probe. After that, the slide of sample 3 was incubated for 1 to 2 hours in a humidified chamber set at 37°C.

[0080] The slide of sample 3 was removed from the chamber and washed with wash buffer to remove unbound EBER probes. This washing process was performed at room temperature, with a total of three 5-minute washes.

[0081] Next, the detection process was carried out using the following procedure. After blocking the slide of sample 3 with the blocking buffer for 30 minutes, the biotinylated antibody was applied to the slide and incubated at room temperature for 1 hour. After washing the slide with PBS, HRP-labeled streptavidin was applied and incubated at room temperature for 30 minutes. The slide was then washed again with PBS.

[0082] Next, the DAB substrate (DAB solution + H2O2) for DAB color development was applied to the slide of sample 3, and the color development was observed under a light microscope. Generally, DAB color development is completed within a few minutes. After observation under the light microscope, the slide of sample 3 was washed with water to stop the reaction.

[0083] Next, counterstaining was performed. For example, hematoxylin can be used for nuclear staining. After that, the slide of sample 3 was washed with water, dehydrated with an ethanol series solution, and immersed in xylene to clear the tissue.

[0084] Next, the coverslip was attached to the slide of sample 3 using mounting media, and observation with an optical microscope was performed.

[0085] Note that the ISH protocol is not limited to those described above. For example, it is possible to use commercially available fully automated staining systems (e.g., Leica Biosystems' BOND RX, Roche Diagnostics' VENTANA BenchMark ULTRA, etc.) and customize the reagents and processing conditions as needed.

[0086] Figure 8 shows the obtained optical microscope images. For comparison, optical microscope images of the above comparison sample (after SEM observation) and paraffin sections prepared from the same paraffin block as the above sample, but treated only for ISH (without SEM observation; hereinafter also referred to as the "control sample") are also shown.

[0087] As shown in Figure 8, sample 3 exhibits a similar level of color development (hue) as the control sample. Furthermore, images confirming the presence or absence of color development were obtained for the comparison sample. This is likely due to the short recognition region of the EBER probe contained in the hybridization buffer, which is approximately several tens of bp. Therefore, even if RNA disruption or cleavage occurred during electron microscopy observation, a detectable level of color development was achieved.

[0088] Figure 9 shows a magnified view of the upper center of each image in Figure 8. Furthermore, Figure 10 shows a magnified view of the area enclosed by a rectangle in each image in Figure 9.

[0089] As shown in Figures 9 and 10, it was found that even after being subjected to SEM observation, sample 3 maintained (protected) the substances present in the sample (RNA in this example) to the extent that it exhibited a similar color development (hue) to that of the control sample that had not been subjected to SEM observation. Furthermore, in the comparative sample, EBER (or short RNA derived from it) could be partially detected, and the same inference as described above, referring to Figure 8, applies.

[0090] <Example 4> Pathological tissue specimens were prepared as observational samples using serial sections (FFPE serial sections) made from paraffin blocks of vascular wall specimens.

[0091] The paraffin sections were deparaffinized by immersing them in xylene for 1 minute four times. Next, they were washed twice each in 99% ethanol, 70% ethanol, and 50% ethanol for 2 minutes each, followed by two washes in DDW for 1 minute each to perform hydrophilization. Finally, excess moisture was removed using a spin coating apparatus, and the samples were lightly dried. The prepared samples were not stained.

[0092] Next, an auxiliary solution (undiluted solution:ethanol = 1:50) prepared in the same manner as in Example 1 was applied to the surface of the sample, left to stand for about 30 seconds, and then spin-dried using a spin-coating apparatus. Then, the sample was placed on the sample stage of the SEM, and the electron beam emitted from the electron source of the SEM was irradiated onto the surface of the sample to form a thin film, thereby creating a sample with a thin film. Subsequently, the sample with the thin film was subjected to SEM observation and then removed from the sample chamber of the SEM.

[0093] Figure 11 shows an example of the obtained FE-SEM image. For comparison, FE-SEM images of a sample (hereinafter also referred to as the "comparative sample") prepared from the same paraffin block as the above sample but without the application of the auxiliary solution, are also shown.

[0094] The left column of Figure 11 shows FE-SEM images of the comparison sample, and the right column shows FE-SEM images of sample 4. The upper images are at a magnification of 2,500x (scale bar 20.0 μm), and the lower images are at a magnification of 1,000x (scale bar 50.0 μm).

[0095] As shown in Figure 11, the irregular structure of the blood vessel wall was more clearly observed in the image of sample 4 compared to the comparison sample. Although not all images taken in this example are shown, a similar trend was observed in images at other magnifications.

[0096] The upper and lower panels of Figure 12 show images of the comparison sample and sample 4 taken at 30x magnification (scale bar is 1.00 mm), respectively. As indicated by the arrows in each image, immunostaining was performed using the following procedure to stain the actin filaments present in the smooth muscle of the blood vessel wall. Note that none of the following processes constitute removal of the thin film (protective film) formed on the sample surface.

[0097] First, sample 4, which had been washed with running water, underwent antigen retrieval treatment. Specifically, the slide of sample 4 was immersed in antigen retrieval citrate buffer (pH 6.0), heated in a boiling water bath for 20 minutes, then the slide was cooled to room temperature and washed with PBS.

[0098] Next, the slides of sample 4 were incubated in 3% H2O2 in PBS for 10 minutes to block endogenous peroxidase, and then the slides were washed with PBS.

[0099] Next, the slide of sample 4 was incubated in PBS with 1% BSA for 30 minutes to block nonspecific binding.

[0100] Next, anti-α-smooth muscle actin antibody was applied to the slide of sample 4 as the primary antibody, incubated at room temperature for 30 minutes, and then washed with PBS. The PBS washing was performed three times, each time for 5 minutes.

[0101] Next, the HRP-labeled secondary antibody was applied to the slide of sample 4, incubated at room temperature for 30 minutes, and then the slide was washed with PBS. The PBS washing was performed three times, each time for 5 minutes.

[0102] Next, the DAB substrate (DAB solution + H2O2) for DAB color development was applied to the slide of sample 4, and the color development was observed under a light microscope. Generally, DAB color development is completed within a few minutes. After observation under the light microscope, the slide of sample 4 was washed with water to stop the reaction.

[0103] Next, counterstaining was performed. For example, hematoxylin can be used for nuclear staining. After that, the slide of sample 4 was washed with water, dehydrated with an ethanol series solution, and immersed in xylene to clear the tissue.

[0104] Next, the coverslip was attached to the slide of sample 4 using mounting media, and observation with an optical microscope was performed. The comparison sample was also immunostained using the same procedure and observed with an optical microscope.

[0105] Figure 13 shows the obtained optical microscope images. For comparison, optical microscope images of paraffin sections prepared from the same paraffin block as the above sample, which were treated only for immunostaining (without SEM observation; hereinafter referred to as the "control sample"), are also shown.

[0106] As shown in Figure 13, the contours of actin filaments were confirmed in sample 4 to the same extent as in the control sample, whereas in the comparative sample, reduced staining was observed in the areas indicated by arrows in the image. The aforementioned reduction in staining in the comparative sample is evident from the comparison with the areas indicated by arrows in the image of sample 4, and is thought to be due to damage to the vascular wall tissue caused by electron beam irradiation of the sample surface during SEM observation. In other words, it was found that in sample 4, the formation of the thin film described above reduces undesirable effects (damage, etc.) on the tissue due to electron beam irradiation during SEM observation, and that measurements regarding substances (epitopes in this example) present in the sample can be performed well using the same sample after SEM observation. [Industrial applicability]

[0107] As described above, the inventors have specifically confirmed that, after electron microscopy observation using the above-mentioned protective agent for electron microscopy observation, it is possible to perform electron microscopy observation and measurement of substances present in the sample using the same sample without removing the thin film formed on the surface of the sample. In the above-described examples, the experimenter (i.e., a human) actually observes each image, but by applying AI / ML (Artificial Intelligence / Machine Learning) technology, which has advanced remarkably in recent years, it is expected that it will be possible to detect lesions that have not been noticed by experts such as pathologists and clinicians, and to further expedite intraoperative pathological diagnosis, in which some cells or tissues are collected during surgery and pathologists make pathological diagnoses in a short time. Furthermore, in recent years, research on multi-omics analysis integrating gene analysis (genomics), RNA analysis (transcriptomics), protein analysis (proteomics), metabolite analysis (metabolomics), etc. has been actively conducted, and the present invention may also contribute to obtaining multiple omics data using a single sample. This will allow for the full utilization of valuable cases, leading to the discovery of new pathological findings that had not been previously identified, and is expected to inspire the development of new diagnostic criteria and diagnostic methods.

Claims

1. The process involves applying a protective agent for electron microscopy observation, mainly containing a survival environment-providing component, sugars, and electrolytes, or an auxiliary solution containing the said protective agent for electron microscopy observation and water, an organic solvent, or a mixed solvent of water and an organic solvent, to the sample to be observed, irradiating it with an electron beam or plasma to form a thin film on the surface of the sample, and preparing a sample with a thin film. The process involves subjecting the thin film-attached sample to electron microscope observation, A process of measuring substances present in a sample with a thin film attached after electron microscope observation, without removing the thin film. A sample analysis method using an electron microscope, including [details omitted].

2. The sample analysis method using an electron microscope according to claim 1, wherein the sample to be observed is a sample that has not been stained.

3. The electron microscope observation is performed using a scanning electron microscope, as described in claim 1, for sample analysis using an electron microscope.

4. The method for analyzing a sample using an electron microscope according to claim 1, wherein the measurement is performed using at least one selected from the group consisting of an imaging mass microscope, an optical microscope, a fluorescence microscope, a super-resolution microscope, a multiphoton laser microscope, a light sheet microscope, an apparatus or instrument configured to be attached to the electron microscope, immunostaining, and in situ hybridization.

5. The method for analyzing a sample using an electron microscope according to claim 1, further comprising the step of subjecting the sample to be observed to observation for an observation other than electron microscopy observation, prior to the step of preparing the thin film-attached sample.

6. The electron microscope method for analyzing a sample according to claim 5, wherein the observation other than the electron microscope observation is an observation using at least one microscope selected from the group consisting of an optical microscope, a fluorescence microscope, a super-resolution microscope, a multiphoton laser microscope, and a light sheet microscope.

7. The method for analyzing a sample using an electron microscope according to any one of claims 1 to 6, wherein the auxiliary solution comprises the protective agent for electron microscope observation and an amphiphilic solvent, or a mixed solvent of water and an amphiphilic solvent.

8. The method for analyzing a sample using an electron microscope according to claim 7, wherein the amphiphilic solvent is ethanol.

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