Immunostaining method, sample exchange chamber and charged particle beam device

Abstract

In order to enable the new immunostaining to be used for pathological diagnosis and research, a sample exchange chamber is provided, which comprises: a container 2 capable of introducing a substrate for carrying a sample, a specific solution inlet 3 as a staining mechanism for staining the sample, a cleaning liquid inlet 4 as a cleaning mechanism for cleaning the sample, a vacuum exhaust port 5 as an exhaust mechanism for vacuum exhausting the container, a waste liquid port 6, and a sterilization mechanism 7 for sterilizing the interior of the container and the sample.

Description

Technical Field

[0001] The present invention relates to an immunostaining method, a sample exchange chamber with a staining mechanism, and a charged particle beam device. Background Art

[0002] In pathological diagnosis, biological tissues are made into formalin fixed paraffin embedded (FFPE) specimens, and various staining methods are used to stain the target cell components with different colors, and the tissues are identified and diagnosed based on the differences in morphology and color tones. Immunostaining, one of the staining methods, is an indispensable technology in pathological diagnosis and research. It mainly uses the antigen-antibody reaction, which is a specific reaction between antigenic substances such as proteins and antibodies, to visualize the expression of specific genes and various marker proteins, thereby identifying the functions of tissues and small organs.

[0003] There are high expectations for the immunoelectron microscopy method that applies this technology. Since it depends on the resolution of the microscope, the transmission electron microscope (TEM) is mainly used. TEM has a high resolution and is suitable for observing the structure of fine cells. It is used for pathological diagnosis and research, but it requires spacious facilities and a high budget, so the institutions that can own it are limited. In addition, since the complicated preprocessing such as the preparation of ultra-thin sections takes time and effort, and the electron beam is used in the line source and the observation image is displayed in black and white, it is impossible to identify cell components by color like an optical microscope. Therefore, understanding the observed image also requires professional knowledge and proficiency.

[0004] As mentioned above, there are many problems when using TEM in pathological diagnosis. In order to solve this problem, in recent years, it is expected that the method of pathological diagnosis is performed by observing FFPE specimens for optical microscope by scanning electron microscope (SEM: Scanning Electron Microscope), which is cheaper than TEM and can save space. Along with this, the expectation of immunoelectron microscope using SEM is also improved, requiring a simpler method that can support pathological diagnosis. Related prior art exists, for example, patent document 1, which discloses a SEM that generates an enlarged image of the sample surface configured in the atmosphere.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application Publication No. 2018-92952 Summary of the invention

[0008] Problems to be solved by the invention

[0009] Immunoelectron microscopy has high expectations but is not widespread for two reasons.

[0010] (1) Fixatives, permeabilizing agents, activation methods, etc. used in optical microscopic specimen preparation and immunostaining are limited to agents that reduce the morphology of the microstructure of tissues that are important for electron microscopic observation.

[0011] (2) Pre- and post-fixative solutions and epoxy resins used to prepare electron microscope specimens to preserve fine structures reduce the antigenicity of the specimens and inhibit immune responses.

[0012] In the immunoelectron microscopy method to improve the above problems, there are two methods. One method uses horseradish peroxidase (HRP: Horseradish peroxidase) as a labeled antibody for the target antigen, and makes the positive part DAB color, and then makes osmium react, and the osmium black generated is observed by TEM. The other method uses a second antibody labeled with gold particles of about 5 to 15nm, and the gold particles in the positive part are observed by TEM or SEM, which is not commonly used. However, both methods are more complicated than conventional immunostaining techniques, and the stability of staining is also weak. In addition, it is difficult to observe osmium black or tiny gold particles by SEM.

[0013] The present invention aims to solve the above-mentioned problems and to use a simpler and more stable new immunostaining for pathological diagnosis and research using a SEM which is cheaper and more space-saving than a TEM.

[0014] Means for solving problems

[0015] In order to achieve the above-mentioned purpose, in the present invention, based on the development of a new immunostaining method, a sample exchange chamber for a charged particle beam device is provided, which is equipped with a container capable of introducing a substrate for carrying samples, a staining mechanism for staining the samples, a cleaning mechanism for cleaning the samples, an exhaust mechanism for vacuum exhausting the container, and a sterilization mechanism for sterilizing the interior of the container and the samples.

[0016] In addition, in order to achieve the above-mentioned purpose, the present invention provides a charged particle beam device, which is equipped with a sample chamber, a sample exchange chamber, and an automatic loading mechanism. The sample exchange chamber has a container that can introduce a substrate for carrying samples, a dyeing mechanism for dyeing the samples, a cleaning mechanism for cleaning the samples, an exhaust mechanism for vacuum exhausting the container, and a sterilization mechanism for sterilizing the interior of the container and the samples. The automatic loading mechanism can transfer the sample between the sample exchange chamber and the sample chamber without exposing it to the atmosphere.

[0017] Effects of the Invention

[0018] According to the present invention, in the sample room, all operations from staining-related work to observation using SEM, characteristic X-ray analysis, and analysis of the results can be automated.

[0019] Brief description of the attached figure

[0020] [ Figure 1 ] is a diagram showing a structural example of the sample exchange chamber involved in Example 1.

[0021] [ Figure 2 ] is a schematic diagram showing a structural example of a charged particle beam device involved in Example 1.

[0022] [ Figure 3 ] is a flowchart showing the operation of the charged particle beam device involved in Example 1.

[0023] [ Figure 4 ] is a flowchart showing the procedure of the new dyeing method involved in Example 1.

[0024] [ Figure 5 ] is a diagram showing an example of staining of a pathological section involved in Example 1.

[0025] [ Figure 6 ] is a diagram showing the results of characteristic X-ray analysis of pathological sections stained using the staining method involved in Example 1.

[0026] Specific embodiments of the invention

[0027] Hereinafter, the embodiments are described in detail based on the accompanying drawings. In addition, in all the figures used to illustrate the embodiments, the same symbols are marked on the components with the same functions, and their repeated descriptions are omitted. In addition, in the following embodiments, the description of the same or identical parts is not repeated in principle except when it is particularly necessary.

[0028] Example 1

[0029] Embodiment 1 is an embodiment of a sample exchange chamber and a charged particle beam device using the same, wherein the sample exchange chamber comprises a container capable of introducing a substrate for carrying a sample, a staining mechanism for staining the sample, a cleaning mechanism for cleaning the sample, an exhaust mechanism for vacuum exhausting the container, and a sterilization mechanism for sterilizing the interior of the container and the sample.

[0030] Figure 1This is a structural diagram of a sample exchange chamber used in the charged particle beam device involved in Example 1. The sample exchange chamber 1 has a container 2, a specific solution inlet 3 with a tube, a cleaning liquid inlet 4, a vacuum exhaust port 5, a waste liquid port 6, and a sterilization mechanism 7. The sample exchange chamber 1 also includes a control circuit for controlling each component, but its illustration is omitted here. The specific solution inlet 3 constitutes a dyeing mechanism for dyeing the sample. The cleaning liquid inlet 4 constitutes a cleaning mechanism for cleaning the sample. The vacuum exhaust port 5 constitutes an exhaust mechanism for vacuum exhausting the container. The sterilization mechanism 7 is a sterilization mechanism for sterilizing the inside of the container and the sample.

[0031] The container 2 has a structure capable of introducing a plurality of substrates with samples placed therein, such as a box-type container. In addition, when the substrates are introduced, sufficient space is maintained in the container 2 so that the samples placed on the substrates do not contact the container 2 or other substrates.

[0032] The specific solution introduction port 3 with a tube can be opened and closed, and is connected to the sample exchange chamber 1 or the container 2. The specific solution is, for example, a heavy metal such as silver or gold used in the sensitization reaction of the sample. As shown in the figure, a tube is connected to the specific solution introduction port 3, and the other end of the tube is installed in a specific solution container 8 provided outside the sample exchange chamber 1. Through the tube, a sufficient amount of specific solution for dyeing the sample in the container 2 can be introduced into the sample exchange chamber 1 or the container 2.

[0033] Similar to the specific solution introduction port 3, the cleaning solution introduction port 4 with a tube can be opened and closed, and is connected to the sample exchange chamber 1 or the container 2. The cleaning solution is, for example, water. A tube is connected to the cleaning solution introduction port 4, and the other end of the tube is installed in a cleaning solution container 9 provided outside the sample exchange chamber 1. The cleaning solution is introduced into the sample exchange chamber 1 or the container 2 through the tube to rinse the specific solution on the sample.

[0034] The vacuum exhaust port 5 is connected to a pump 10 outside the sample exchange chamber 1 in order to vacuum exhaust the sample exchange chamber 1. The waste liquid port 6 with a tube is connected to a waste liquid container 11 outside the sample exchange chamber 1. The waste liquid port 6 can discharge a specific solution or a cleaning solution from the sample exchange chamber 1 or the container 2 to the waste liquid container 11.

[0035] The sterilization mechanism 7 is installed in the sample exchange chamber 1 and has the function of sterilizing the sample exchange chamber 1, such as an ultraviolet (UV) irradiator. When a toxic virus is contained in the sample, there are cases where the activity does not decrease even after fixation, but by being able to sterilize in the sample exchange chamber 1, the virus will not be exposed to the atmosphere and the charged particle beam device. That is, the possibility of the virus that cannot be inactivated by fixation affecting the operator can be reduced.

[0036] Figure 2 It shows that Figure 1The sample exchange chamber 1 of the present embodiment shown in the figure is applied to the charged particle beam device 21. The charged particle beam device 21 can be configured as, for example, a scanning electron microscope (SEM) for photographing an observed image of the sample. As for the sliced ​​sample in the scanning electron microscope, a thickness of several μm or so for an optical microscope is sufficient, so the slice preparation becomes easy. Therefore, pretreatment can be performed more simply and quickly.

[0037] like Figure 2 As shown, the charged particle beam device 21 includes a device body 22 , a sample exchange chamber 1 , an automatic loading mechanism 23 , and a control device 24 .

[0038] The apparatus body 22 is formed by integrating a lens barrel 25 and a sample chamber 26. The lens barrel 25 has an electron gun 27, a focusing lens 28, a deflection coil 29, and an objective lens 30. The focusing lens 28 and the objective lens 30 are electromagnets with coils, and the electromagnetic fields generated by them respectively exert a focusing effect on the electron beam irradiated from the electron gun 27, and function as lenses for the electron beam EB. In addition, the lens barrel 25 also includes a control circuit for controlling each structure, but its illustration is omitted here.

[0039] When the sample on the sample stage 33 is irradiated with the electron beam EB, for example, secondary electrons, reflected electrons, characteristic X-rays, etc. are generated. The detector 31 is disposed at an appropriate position in the column 25 or the sample chamber 26 to detect various signals.

[0040] The sample chamber 26 includes an openable and closable introduction and outlet port 32 and has a structure for accommodating a sample stage 33. The sample is placed on the sample stage 33.

[0041] The automatic loading mechanism 23 is connected to the import / export port 32 of the sample chamber 26. The automatic loading mechanism 23 is also connected to the sample exchange chamber 1. The automatic loading mechanism 23 has a mechanism that can transfer the sample from the sample exchange chamber 1 to the sample chamber 26 and from the sample chamber 26 to the sample exchange chamber 1 without exposing the sample to the atmosphere. Since the automatic loading mechanism 23 is also vacuum-exhausted, the vacuum exhaust and the time of opening to the atmosphere when transferring the sample to the sample chamber 26 and the sample exchange chamber 1 can be shortened.

[0042] The control device 24 is composed of, for example, a personal computer (PC) and has a function of controlling all operations in the device body 22, the sample exchange chamber 1, and the automatic loading mechanism 23. For example, through the user's pre-setting, the adjustment of brightness and focus during imaging, imaging, and characteristic X-ray analysis can be automatically performed in multiple samples, and positive sites can be identified. In the sample exchange chamber 1, the presence or absence and frequency of staining and washing, and the presence or absence and timing of sterilization can be set and automatically performed.

[0043] Figure 3This is a flowchart showing the operation of the device from the time when the user introduces the sample to the time when the charged particle beam device 21 images the sample. Figure 3 The following describes each step.

[0044] ( Figure 3 :Step S1)

[0045] The user loads the substrate carrying the sample into the container 2 in the sample exchange chamber 1. After the substrate is loaded, automation can be achieved by inputting instructions to the sample exchange chamber 1, the automatic loading mechanism 23, and the charged particle beam device 21 to the PC as the control device 24. The input instructions include, for example, the presence and sequence of dyeing, washing, and sterilization in the sample exchange chamber 1, time, imaging settings, and identification of positive sites by characteristic X-ray analysis.

[0046] ( Figure 3 :Steps S2~S3)

[0047] The sample exchange chamber 1 injects a specific solution into the sample exchange chamber 1 or the container 2 according to the instruction input by the user to sensitize and dye the sample (step S2). The specific solution is discharged from the waste liquid port 6 after the dyeing is completed (step S3).

[0048] ( Figure 3 :Steps S4~S5)

[0049] The sample exchange chamber 1 injects the cleaning liquid into the sample exchange chamber 1 or the container 2 according to the instruction input by the user to clean the sample (step S4). After the cleaning is completed, the cleaning liquid is discharged from the waste liquid port 6 (step S5).

[0050] ( Figure 3 : Step S6)

[0051] The sample exchange chamber 1 is evacuated.

[0052] ( Figure 3 :Steps S7~S10)

[0053] The multiple samples in the sample exchange chamber 1 are introduced into the sample chamber 26 one by one by the automatic loading mechanism 23 and placed on the sample stage 33 (step S7). The charged particle beam device 21 images the sample according to the instructions such as the imaging range and imaging conditions input in advance by the user, and performs characteristic X-ray analysis (step S8).

[0054] After the imaging and analysis are completed, the sample is transferred from the sample chamber 26 to the sample exchange chamber 1 by the automatic loading mechanism 23 and returned to the original position in the container 2 (step S9). The sterilization mechanism 7 sterilizes the sample (step S10). In the sample exchange chamber 1, the sample is sterilized for a sufficient time (set in step S1) to inactivate toxic or infectious viruses. In addition, the sterilization in step S10 can also be implemented in the sample exchange chamber 1 before the sample is introduced into the sample chamber 26. Thereafter, steps S7 to S10 are repeated until the imaging of the number of samples indicated by the user is completed.

[0055] In this embodiment, the sample exchange chamber 1, the automatic loading mechanism 23 and the charged particle beam device body 22 are integrated, so that the operations from dyeing to imaging can be fully automated. That is, dyeing, washing, sample introduction, imaging, and sample extraction that have been performed by the user so far can be automated. Therefore, the number of processes for the user can be greatly reduced.

[0056] exist Figure 4 The flowchart of the new staining method of this embodiment is shown. In addition, the inventors of the present application sometimes refer to the new staining method as the Takaki method. That is, the sample is immunostained, and the immunostained sample is sensitized to heavy metals. In the new staining method 41, from tissue collection to immunostaining, it is the same method as the conventional immunostaining method using a labeled antibody such as HRP as a target antigen and performing DAB color development on the positive part.

[0057] Next, osmium staining S42 using 1% osmium tetroxide is performed as necessary, but it may be omitted. This step is to make the immune reaction stronger.

[0058] In heavy metal sensitization staining S43, heavy metals such as thiosemicarbazide, gold, silver, and sodium are combined for staining. For example, after being treated with 1% sodium bisulfite (room temperature) for 1 minute, the sample is placed in hexamethylenetetramine silver for 15 minutes (60°C) for sensitization. The concentration of hexamethylenetetramine silver used here is changed to the optimal concentration according to the observation site. Then, it is treated with gold chloride (room temperature) for 3 minutes and fixed with 5% sodium thiosulfate (1 minute). Finally, it is washed with water, dried, and photographed. By adding heavy metal sensitization staining S43, the positive site is sensitized. Furthermore, SEM can also obtain a clear contrast that can fully distinguish the positive site.

[0059] For example, if the area of ​​the sensitized dyed part does not exceed the threshold value in the sample that has been imaged, and if the setting of indicating the alarm of dyeing failure is set, the operation of re-dyeing can be returned to before imaging all the samples loaded in the sample exchange chamber 1, and time loss can be prevented. In addition, the area of ​​the sensitized part can be automatically derived based on the contrast difference of the image or the result of characteristic X-ray analysis.

[0060] In addition, the captured image is binarized, for example, by using a PC constituting the control device 24, and the area ratio of the positive site is calculated. The calculated area ratio can be used to determine the disease, identify specific cells, and determine the presence or absence of viruses. In addition, for example, based on the results of the characteristic X-ray analysis of the sample, by displaying only the elements used in the sensitizing staining, the site sensitized by the staining can be identified. In this way, it is possible to identify tiny positive sites that cannot be perceived with an optical microscope. Therefore, in Figure 4 In the new staining method 41, since image analysis can be performed, judgment can be made regardless of the proficiency level.

[0061] An example of a pathological section stained by the new staining method 41 is shown in comparison with the case of the conventional method. Figure 5 The lower part of the figure is a magnified part of the upper part. As shown in (a) of the figure, it is difficult to dye only the target part in the existing method of osmium black method. Furthermore, even if dyeing is possible, the frequency of over-staining is high, and pretreatment requires time and proficiency. In addition, in SEM, it becomes difficult to distinguish the stained part from other parts.

[0062] However, as shown in (b) of the figure, the heavy metal sensitization positive site is expressed with a bright color in the new staining method 41. As shown in (c) of the figure, since a clear contrast can be obtained in the case of this embodiment, the microscopic intracellular tissue that has undergone an immune reaction can be easily visualized.

[0063] Figure 6 The figure shows the result of characteristic X-ray analysis of a pathological section stained by heavy metal sensitization staining S43 of the new staining method 41. (a) of the figure shows an image based on a signal of reflected electrons, and (b) of the figure shows an image in which the positive part is visualized by sensing gold used in the sensitization staining through characteristic X-ray analysis. In this way, in the new staining method 41, even tiny positive parts become clear, enabling more rapid pathological diagnosis.

[0064] According to the present invention described in detail above, since the operations related to dyeing, observation by SEM, characteristic X-ray analysis, and analysis of the results can be automated in the sample chamber, the time and number of processes required for sample pretreatment can be reduced. In addition, since a mechanism capable of sterilizing the sample is provided in the sample exchange chamber, toxic or infectious viruses can be observed without being exposed to the atmosphere or the inside of the device.

[0065] In addition, according to the present invention, the thickness of each slice can be made into several μm, and the time and skill required for making the slice are not required. By sensitizing the positive part with heavy metal after immunostaining, high contrast can be obtained. Even if the immunoelectron microscopy of SEM, which has been difficult so far, can be used, the immunopositive reaction can be simply visualized. Furthermore, by performing characteristic X-ray analysis on the part, more accurate part identification and quantification based on area ratio of the positive part can be achieved.

[0066] Explanation of symbols

[0067] 1: Sample exchange room

[0068] 2: Container

[0069] 3: Specific solution inlet

[0070] 4: Cleaning fluid inlet

[0071] 5: Vacuum exhaust port

[0072] 6: Waste liquid outlet

[0073] 7: Sterilization mechanism

[0074] 8: Containers for specific solutions

[0075] 9: Container for cleaning fluid

[0076] 10: Pump

[0077] 11: Waste liquid container

[0078] 21: Charged particle beam device

[0079] 22: Device body

[0080] 23: Automatic loading mechanism

[0081] 24: Control Device

[0082] 25: Lens tube

[0083] 26: Sample room

[0084] 27: Electron Gun

[0085] 28: Focusing lens

[0086] 29: Deflection Coil

[0087] 30: Objective lens

[0088] 31: Detector

[0089] 32: Import / Export port

[0090] 33: Sample table

[0091] 41: New dyeing method

Claims

1. A sample exchange chamber for a charged particle beam device, characterized in that have: a container capable of introducing a substrate on which a sample is placed; a staining mechanism having a specific solution introduction port for introducing a specific solution capable of staining the sample into the container, wherein the specific solution is a heavy metal solution and stains the immunostained sample with the heavy metal; A cleaning mechanism for cleaning the sample; An exhaust mechanism having a vacuum exhaust port for vacuum exhausting the container; as well as A sterilization mechanism is installed in the sample exchange chamber to sterilize the interior of the container and the sample.

2. The sample exchange chamber according to claim 1, characterized in that have: A control device controls the dyeing mechanism, the cleaning mechanism, and the sterilization mechanism.

3. The sample exchange chamber according to claim 1, characterized in that: The cleaning mechanism includes a cleaning liquid introduction port for introducing a cleaning liquid capable of cleaning the sample.

4. A charged particle beam device, characterized in that have: Sample room; A sample exchange chamber, comprising: a container capable of introducing a substrate for placing a sample, a specific solution introduction port for introducing a specific solution capable of dyeing the sample into the container, the specific solution being a heavy metal solution, a dyeing mechanism capable of dyeing the immunostained sample with a heavy metal, a washing mechanism for washing the sample, an exhaust mechanism having a vacuum exhaust port for vacuum exhausting the container, and a sterilization mechanism installed in the sample exchange chamber for sterilizing the interior of the container and the sample; and An automatic loading mechanism is capable of transferring the sample between the sample exchange chamber and the sample chamber without exposing the sample to the atmosphere.

5. The charged particle beam device according to claim 4, characterized in that: The charged particle beam device is a scanning electron microscope.

6. The charged particle beam device according to claim 4, characterized in that have: A control device, which controls the sample chamber, the sample exchange chamber, and the automatic loading mechanism; The control device can automatically transfer and image the sample.

Images