Method for observing biological sample

Abstract

This method is for observing the same measurement place of the same biological sample by means of an X-ray microscope and an optical microscope by using a wax block in which a biological sample is embedded and the minimum value of the optical path length of the X-ray is 2 mm or less and the maximum value thereof is more than 2 mm. The method comprises: a step for imaging the biological sample by means of an X-ray microscope using an X-ray having an energy of 4-12 keV; a step for identifying, in reference to an arbitrarily defined position of the biological sample in an image captured by the X-ray microscope, positional information of an observation target region for imaging the biological sample by means of an optical microscope; and a step for obtaining a section of the biological sample including the observation target region on the basis of the identified positional information, and imaging the section by means of the optical microscope.

Description

Observation methods for biological samples

[0001] The present invention relates to a method for observing the same measurement location of the same biological sample using an X-ray microscope and an optical microscope.

[0002] Pathological evaluation of biological tissues is primarily based on a two-dimensional (two-dimensional) pathological assessment of tissues excised as a pathological specimen. However, pathological changes progress three-dimensionally within tissues. Furthermore, when preparing a pathological specimen, a portion of the specimen is discarded, making evaluation impossible. Given this current situation, the development of a method for three-dimensionally evaluating a specimen, or a method for evaluating the entire specimen, has been an important challenge. Conventional methods for observing biological specimens include a method for imaging a biological specimen using a microscope (Patent Document 1), a method for imaging rat kidneys (renal tubules) (Patent Document 2), and a method for infiltrating a biological specimen with a contrast agent, solidifying it, and then imaging it (Patent Document 3).

[0003] Patent Publication No. 2020-528557 Patent Publication No. 2014-211448 International Publication No. 2022 / 234844 Pamphlet

[0004] However, currently, no practical observation method exists that can evaluate pathological changes in specimens in three dimensions with cellular-level spatial resolution. Optical microscopes offer advantages such as a relatively high two-dimensional resolution of approximately 0.2 μm and accurate tissue identification using a variety of staining techniques. However, they face limitations, including sample deformation (destructive deformation) due to physical forces applied during sample preparation and insufficient depth resolution due to a sample thickness of approximately 4 μm. X-ray microscopes, on the other hand, offer advantages such as isotropic submicron three-dimensional resolution and non-destructiveness, allowing for sample reuse. However, the shading in X-ray microscope images reflects the electron density of the relatively light elements that make up the biological sample, resulting in low-contrast grayscale images that make up the biological sample, making tissue identification difficult. Therefore, a practical method for three-dimensional observation of the same measurement location on the same biological sample with cellular-level spatial resolution by combining X-ray and optical microscopes to overcome these limitations and difficulties was needed.

[0005] As a result of intensive research into solving the above-mentioned problems, the inventors have succeeded in observing the same measurement location of the same biological sample using an X-ray microscope and an optical microscope by imaging a plate-shaped wax block formed by embedding a biological sample in wax so that the block has a thickness of 2 mm or less, thereby completing the present invention.

[0006] That is, the present invention is as follows: [1] A method for observing the same measurement location of the same biological sample with an X-ray microscope and an optical microscope using a wax block in which a biological sample is embedded and in which the X-ray path length is a minimum of 2 mm or less and a maximum of more than 2 mm, the method comprising the steps of: imaging the biological sample with an X-ray microscope using X-rays with an energy of 4 to 12 keV; specifying positional information of an observation target region for imaging the biological sample with an optical microscope, using an arbitrary position of the biological sample in the image captured with the X-ray microscope as a reference; and sectioning the biological sample including the observation target region based on the specified positional information, and imaging the section with an optical microscope. [2] The wax block is formed by embedding a biological sample prepared by steps including chemical fixation, dehydration, and wax infiltration into a wax block having a base area of ​​9 to 900 mm. ２The method according to [1], wherein the wax block is placed in an embedding dish with a depth of 2 mm or less, and wax is poured into the embedding dish to form a plate with a thickness of 2 mm or less. [3] The method according to [1], wherein the arbitrary position belongs to the surface (reference surface) that contacts the upper end of the biological sample. [4] The method according to [1], wherein the wax block is placed in a cassette with a slit. [5] The method according to [4], wherein the size of the slit is larger than the imaging site of the biological sample. [6] The method according to [5], wherein the wax block is placed so that the area to be irradiated with X-rays is located within the slit area of ​​the cassette. [7] The method according to [4], wherein the slit has a strip-like or rectangular shape. [8] The method according to [1], wherein the observation using the X-ray microscope is performed between wax infiltration and microtome sectioning in a biological sample observation process in which a collected biological sample is subjected to chemical fixation, dehydration, wax infiltration, microtome sectioning, section extension, dewaxing, and staining, and then observed under an optical microscope. [9] The method according to [1], wherein the wax block is placed in a cassette having a slit through which X-rays can pass, and the step of imaging with the X-ray microscope involves imaging the wax block and the cassette in a combined state.

[10] The method according to [1], comprising the steps of placing a wax block not containing a biological sample in a cassette without a slit to prepare a combined body of the two (a two-part combined body), and further placing a biological sample-embedded wax block after X-ray microscope observation, in which a biological sample is embedded, on the upper surface of the wax block in the two-part combined body by adhering it with heat-molten wax as an adhesive, to prepare a combined body of the two-part combined body and the biological sample-embedded wax block (a three-part combined body).

[11] An embedding dish for embedding a biological sample in wax, the dish having an area of ​​9 to 900 mm ２, and an embedding dish having a depth of 2 mm or less.

[12] A method for producing a plate-shaped biological sample embedded block, comprising the steps of placing a biological sample prepared by a process including chemical fixation and dehydration in the embedding dish described in

[11] so that the embedding dish satisfies the imaging angle range of 150° or more of an X-ray microscope, and pouring wax into the embedding dish.

[13] A cassette for placing a wax block in which a biological sample is embedded and whose X-ray path length is 2 mm or less at a minimum and more than 2 mm at a maximum, the cassette having a notch for X-ray irradiation of the biological sample, for realizing the method described in any of [1] to [9].

[14] A holding device for holding the cassette described in

[13] for X-ray microscope imaging, the device comprising: a stage on which the cassette is placed; and a fixing member holder for fixing the placed cassette.

[15] The device described in

[14] , further comprising a position adjustment mechanism for moving the stage up and down or left and right.

[16] A microscope image processing device comprising: means for acquiring positional information of a biological sample for observation with an optical microscope from an X-ray microscope image acquired by irradiating the biological sample with X-rays and taking an image of the biological sample, using an arbitrary position of the biological sample as a reference; and means for outputting the positional information.

[17] The device according to

[16] , wherein the arbitrary position belongs to a plane (reference plane) that is in contact with the upper end of the biological sample.

[0007] The present invention makes it possible to easily obtain images of the same measurement location of the same biological sample taken with an X-ray microscope and an optical microscope, with the orientations of the two images approximately aligned.The present invention provides images necessary for a practical method of three-dimensionally observing the same measurement location of the same biological sample with cellular-level spatial resolution by using an X-ray microscope and an optical microscope complementary to each other.

[0008] 1 shows a typical sample preparation process for biological sample observation using an X-ray microscope and an optical microscope.

[0023] FIG. 1 shows an embedding dish for embedding a biological sample in wax.

[0024] FIG. 2 shows a process for embedding a biological sample in wax to produce an embedded block.

[0025] FIG. 3 shows an embedded block placed in an embedding cassette.

[0026] FIG. 4 shows the shape of a wax imaging cassette.

[0027] FIG. 5 shows a sample placement method for X-ray microscope measurement.

[0028] FIG. 6 shows a cassette with a block placed therein fixed to a stage.

[0029] FIG. 7 shows the relationship between the biological sample and the X-ray irradiation direction.

[0029] FIG. 8 shows a position adjustment mechanism for adjusting the position of the stage.

[0029] FIG. 9 shows the process for optical microscope observation performed after X-ray microscope observation using the method of the present invention.

[0029] FIG. 10 shows an overview of obtaining position information from a three-dimensional image.

[0029] FIG. 11 shows an overview of obtaining position information from an actual three-dimensional X-ray microscope image using a biological sample.

[0029] A: CT slice edited to have the same perspective as optical microscope observation. The position of the renal corpuscle is indicated by an arrow.

[0029] B: Partial enlargement of a renal corpuscle. The internal structure necessary for identifying kidney lesions can be confirmed.

[0029] C: Partial enlargement of a renal corpuscle obtained by reducing the imaging data to 150°. Essentially the same results were obtained as with the full data. 1 is a diagram showing an overview of a microscope image processing device; FIG. 2 is a block diagram showing the operation of an information processing device; FIG. 3 is a diagram showing the process of preparing a wax block for optical microscope observation from a plate-shaped wax block sample after X-ray microscope observation without being bound to an embedding cassette; FIG. 4 is a diagram showing an embodiment in which an X-ray microscope observation process is incorporated into a biological sample observation process using an optical microscope; and FIG. 5 is a diagram showing an example of observation of the same measurement location of the same biological sample using an X-ray microscope (top) and an optical microscope (bottom).

[0009] The present invention relates to a method for observing the same measurement location of the same biological sample with an X-ray microscope and an optical microscope, using a wax block in which the biological sample is embedded and in which the X-ray path length is a minimum of 2 mm or less and a maximum of more than 2 mm. The method of the present invention comprises the following steps: imaging the biological sample with an X-ray microscope using X-rays with an energy of 4 to 12 keV; specifying positional information of an observation target region for imaging the biological sample with an optical microscope, using an arbitrary position of the biological sample in the image captured with the X-ray microscope as a reference; and sectioning the biological sample including the observation target region based on the specified positional information, and imaging the section with the optical microscope.

[0010] In the present invention, a block containing a biological sample embedded in wax is placed so that the sample plate surface is aligned with the rotation axis of the X-ray microscope, and imaging is performed over a rotation angle range of 150° or more, thereby successfully achieving spatial resolution useful for pathological diagnosis. Furthermore, the present invention makes it possible to section the sample after X-ray microscopy observation and observe it under an optical microscope using position information based on the X-ray imaging data. Furthermore, the present invention provides an embedding dish for embedding a biological sample in wax, and a cassette for placing a block containing a biological sample embedded in wax.

[0011] The imaging method provided by the present invention is a method for complementary observation of the same measurement location on the same biological sample using an X-ray microscope and an optical microscope. The image data captured by these microscopes are provided as images with nearly identical orientations and minimal deformation of the sample. "Complementary observation" refers to observing tissue using the unique characteristics of an optical microscope and an X-ray microscope, respectively. For example, the characteristics or advantages of one microscope, an optical microscope, and an X-ray microscope, complement each other with those of the other microscope, which lack or lack such characteristics or advantages. As a result, the accuracy of pathological evaluation of biological tissue can be improved. The orientation error between the X-ray microscope image and the optical microscope image permitted by the method of the present invention is, for example, within 10°. In the examples, the orientation error is set to within 10° to achieve high accuracy, but is not limited to this and may be greater than 10° depending on the required precision. For example, Figure 17 shows an example of observation of the same measurement location on the same biological sample using an X-ray microscope and an optical microscope using images obtained by the present invention.

[0012] The present invention is characterized by including an X-ray microscope observation step within an optical microscope observation step. An example of an embodiment of the present invention is shown in FIG. 16 . In FIG. 16 , X-ray microscope observation can be incorporated between wax infiltration and microtome sectioning in a biological sample observation step in which a collected biological sample is chemically fixed, dehydrated, waxed, microtomed, sectioned, dewaxed, and stained, followed by optical microscope observation. Generally, preparation of a sample for optical microscope observation involves excising the biological sample, fixing it, and optionally degreasing, decalcifying, and dehydrating it. Next, wax infiltration is followed by microtoming, sectioning the sections, dewaxing, and staining, followed by optical microscope observation. In the present invention, by incorporating an X-ray microscope observation step into the process for optical microscope observation, X-ray microscope examination can be performed as part of a conventional optical microscope pathological examination. This series of steps allows X-ray microscope observation to be performed using a sample for optical microscope observation, eliminating the need to prepare a new sample for X-ray microscope examination. Furthermore, the area extracted using the X-ray microscope can be directly and pinpoint-examined using the optical microscope. Therefore, the compatibility of the X-ray microscope observation process with the conventional biological sample observation process using an optical microscope is significantly improved. The optical microscope observation process will be described in detail later.

[0013] The following describes an embodiment in which a biological sample is embedded in a wax block having an X-ray path length of 2 mm or less at a minimum and more than 2 mm at a maximum, and the same measurement location of the same biological sample is imaged with an X-ray microscope using X-rays with an energy of 4 to 12 keV, and an arbitrary position of the biological sample in the image imaged with the X-ray microscope is used as a reference to identify positional information of an observation target region for imaging the biological sample with an optical microscope, and the biological sample including the observation target region is sliced ​​based on the identified positional information, and the slice is imaged with an optical microscope.

[0014] 1. Preparation of a Block for X-Ray Imaging of a Biological Sample This section describes the steps for X-ray microscopy performed during the optical microscopy observation process. (1) Preparation of a Biological Sample In order to observe the three-dimensional structure of a biological sample at submicron resolution using an X-ray microscope, the present invention uses a modified version of the wax imaging method described in WO 2022 / 234844. The "wax imaging method" described in the above publication involves infiltrating a biological sample with wax such as paraffin to replace the water in the sample with wax, thereby utilizing the negative contrast effect to enhance X-ray contrast, embedding the wax-infiltrated biological sample in the wax to prepare a block, and irradiating the block with X-rays to image the embedded biological sample under an X-ray microscope.

[0015] Unstained biological samples generally have low X-ray contrast, making it difficult to observe them with cellular-level spatial resolution using an X-ray microscope. This is because the main component of an unstained biological sample, water, and other components have similar X-ray transmittance. Wax contrast imaging improves contrast by replacing the water in the biological sample with wax, which has higher X-ray transmittance, enabling X-ray microscopy at the cellular level. Wax contrast imaging is a type of technique commonly known as negative contrast imaging.

[0016] The method of the present invention uses a plate-shaped wax block with a very long maximum optical path length of X-rays, and does not remove excess wax around the biological sample to be examined, which differs from the wax imaging method described in WO2022 / 234844.

[0017] In other words, in the method described in WO 2022 / 234844, in order to obtain better spatial resolution, it was necessary to remove as much wax as possible from the tissue to be observed from the X-ray microscope specimen and specify the maximum optical path length of the X-rays as 2 mm or less. In contrast, in the method of the present invention, the biological specimen to be observed is embedded in a wax block, but the maximum optical path length of the X-rays exceeds 2 mm. If the maximum optical path length exceeds 2 mm, the spatial resolution will be slightly reduced, but by setting the minimum optical path length of the X-rays to 2 mm or less, submicron spatial resolution can be maintained.

[0018] Therefore, by using the block of the present invention for X-ray microscopy, compatibility with conventional biological sample observation processes using optical microscopy can be significantly improved while maintaining the spatial resolution required for complementary observation with optical microscopy. Therefore, the wax block of the present invention has a biological sample embedded therein such that the X-ray path length is 2 mm or less at a minimum and greater than 2 mm at a maximum. While there is no theoretical limit to the maximum X-ray path length of the block, for example, by setting it to 35 mm or less, taking into account the size of a typical embedding dish, compatibility with biological sample observation processes using optical microscopy can be further improved.

[0019] The biological sample used in the method of the present invention can be prepared according to the general process for preparing a sample for optical microscopy. Figure 1 shows a process diagram from excision of a biological sample to placing the biological sample in wax such as paraffin and infiltrating it with wax. In Figure 1, a sample (biological sample) obtained by excising cells or tissue from a living organism is fixed, for example, by chemical fixation using formalin, glutaraldehyde, alcohol, Bouin's solution, or the like. This is followed by dehydration using alcohol, xylene, or the like, and wax infiltration. Degreasing and decalcification are optional steps, and either or both can be performed as needed. Wax refers to a lipophilic compound that is solid at room temperature (20°C to 30°C) and has a melting point of 40°C to 80°C. Examples of wax include paraffin, other petroleum waxes, and synthetic waxes, with paraffin being preferred.

[0020] (2) Embedding dish for wax imaging to embed the biological sample to be examined After infiltration with wax such as paraffin, the biological sample to be examined is embedded. A schematic diagram of an embedding dish for embedding the biological sample to be examined is shown in Figure 2. Figure 2 shows an embedding dish with a rectangular opening and bottom as an example, and the vertical (longitudinal) direction L of the inner wall bottom surface is 1 / 4. １ is 3 to 32 mm, horizontal direction L ２ The plate has a length of 3 to 28 mm and a depth d of 2 mm or less.

[0021] However, in the present invention, the shape of the mouth (top surface) and bottom of the embedding dish may be rectangular, square, circular, or elliptical, and is not limited thereto. Furthermore, the side walls of the embedding dish may be shaped to extend in the normal direction from the bottom surface, i.e., a rectangular parallelepiped shape in which the areas of the bottom and top inner walls are the same, or may be shaped in which the area of ​​the top inner wall is larger than that of the bottom inner wall, i.e., the bottom surface of the inner wall is tapered when viewed from above (a shape in which the extension of the side wall toward the bottom surface is tapered).

[0022] The depth d of the embedding dish is 2 mm or less, but is preferably 1.0 to 1.5 mm, for example, 1.2 mm. This depth of the embedding dish corresponds to the thickness of the block in which the biological sample is embedded. The area of ​​the bottom surface of the embedding dish's inner wall, i.e., the area of ​​the surface of the block in which the biological sample is embedded that is molded by the bottom surface of the embedding dish's inner wall (defined as the upper surface of the wax block), is 9 to 900 mm ２ However, these areas are not limited to the above, and a person skilled in the art can appropriately determine the size of the block for X-ray microscope imaging. The embedding dish may be made of stainless steel or other metals, glass or ceramic, hard or soft resin, paper, or wood, but is not limited to these materials and may be made of other materials.

[0023] As shown in Figure 3, a biological sample is placed in an embedding dish with a depth of 2 mm or less, and wax is poured into the embedding dish to create a plate-shaped wax block with a thickness of 2 mm or less, in which the biological sample is embedded. For example, the biological sample is placed approximately in the center of the embedding dish (Figures 3a and 3b), and wax is poured in and allowed to solidify. The solidified wax is then removed to create a block (hereinafter simply referred to as "block") in which the biological sample is embedded (Figure 3c).

[0024] However, the order in which the biological sample is placed in the embedding dish and the wax is poured is not limited to the above and can be any order. That is, the biological sample can be pre-filled in wax and then poured into the embedding dish together with the wax. Alternatively, the embedding dish can be pre-filled with wax and the biological sample can be placed in it. The biological sample placed in the embedding dish should be finely adjusted as needed so that it is positioned in the center of the block before the wax solidifies. In the present invention, the biological sample may be suspended in the wax, but it is preferable to place it so that it is in contact with the bottom of the embedding dish.

[0025] Furthermore, before the wax solidifies, a cassette for X-ray imaging (called the "embedding cassette") is placed on top of the embedding dish in which the biological sample is embedded, and the wax is allowed to solidify while the surface of the wax poured into the embedding dish is in contact with the embedding cassette.The block can then be removed from the embedding dish to produce a combination of the embedding cassette and the embedded block (Figure 4).

[0026] 2. Wax Contrast Embedding Cassette for Placing a Test Biological Sample In the present invention, the embedding cassette for placing a block in which a test biological sample is embedded has a strip-shaped or rectangular notch (space) through which X-rays can pass when irradiated, as shown in FIG. 5 . The block is placed in the cassette so that the area to be irradiated with X-rays, i.e., the embedded biological sample, is positioned within the notch area. The size of the notch in the cassette is larger than the imaging region of the test biological sample. In other words, the imaging region of the biological sample is adjusted to fit within the area of ​​the notch in the cassette. The notch is designed so that when X-ray imaging is performed using the cassette, the loss of rotation angle due to the cassette blocking X-rays is less than 30°, i.e., the X-rays are not blocked even when the imaging rotation angle range is 150° or more. Details of the imaging rotation angle range will be described later. The biological sample is placed in the embedding block so as to fill the imaging angle range of the X-ray microscope, which is 150° or more, in the embedding dish. However, factors that block X-rays include not only the blocking of X-rays by the cassette, but also other factors such as the blocking of X-rays by the thickness of the wax itself in which the biological sample is embedded.

[0027] Fig. 6 shows the embedded block placed so that the biological sample is positioned in the cutout area of ​​the cassette, and the block is positioned in the direction of X-ray irradiation in the X-ray microscope. As shown in Fig. 6, X-rays are irradiated toward the block from the X-ray generator of the X-ray microscope, and the X-rays pass through the cutout in the cassette (the strip-shaped cutout in Fig. 6) and irradiate the biological sample in the block placed in the cassette.

[0028] FIG. 7 shows a cassette with a block mounted thereon, fixed to a stage. In FIG. 7 , a block 702 containing an embedded biological sample 701 is attached to a cassette 703 and positioned so that the biological sample 701 fits within a cutout area 706 of the cassette. That is, the biological sample is placed within the cutout so that the imaging angle range (described later) of 150° or greater is satisfied. The cassette is then fixed to the stage so that the block faces the detector (the side opposite to the side onto which X-rays are incident). In FIG. 7 , the longitudinal direction of the biological sample is oriented in the x direction (horizontal), but it can also be oriented in the z direction (vertical). In this case, the cutout of the cassette may be oriented upward, or the longitudinal direction of the biological sample can be oriented vertically (z direction) as long as the biological sample 701 fits within the horizontal cutout area.

[0029] The cassette 703 is fixed on the stage 704 via a fixing member 705. As shown in FIG. 7 , the fixing member 705 can be fixed by providing a fitting jig with a recess on the stage and fitting the ends of the cassette into the fitting jig. While FIG. 7 shows a configuration in which both ends of the cassette 703 are fitted into the fixing member 705, the member for fixing the cassette to the stage is not particularly limited, and the cassette may be fixed by affixing it with double-sided tape or clay (not shown), or by using clips or screws. When using clips, the cassette is fixed by utilizing the elasticity of an object such as a leaf spring. When using screws, the cassette is fixed by using set screws or the like.

[0030] After the cassette 703 is fixed on the stage 704, the stage 704 can be moved left and right, forward and backward (x or y direction), or up and down (z direction) using a position adjustment mechanism (details will be described later) so that approximately the center of the biological sample is positioned on the X-ray irradiation path (optical path) p1. Furthermore, the X-ray irradiation path p1 is normal to the block 702, but if a rotation axis q1 is provided at approximately the center of the biological sample 701 and normal to the xy plane, and the biological sample 701 is rotated around the rotation axis q1, p1 becomes oblique to the block 702. In other words, the X-ray optical path becomes oblique to the block 702. The rotation angle θ at this time is within the X-ray imaging rotation angle range, and can be rotated up to 150° or more, preferably 180° or more.

[0031] The relationship between the biological sample 701 and the X-ray irradiation direction is shown in Figure 8. In Figure 8, the left panel shows the minimum and maximum optical path lengths of X-ray imaging in a conventional test example. Conventionally, X-ray imaging was performed on a biological sample from which the wax on the surface had been removed after wax imaging, so the maximum and minimum optical path lengths roughly correspond to the thickness or length of the biological sample.

[0032] In contrast, in the present invention, X-rays are irradiated onto a plate-shaped wax block in which a biological sample with wax contrast is embedded, so the optical path length depends on the length or thickness of the block. Even if the block thickness is 2 mm or less, rotating the sample by a certain angle θ from the rotation axis of the sample increases the optical path length to a length close to the longitudinal direction of the block (Test Example of the Present Invention; Figure 8, right panel). In the present invention, the minimum X-ray optical path length of the block is 2 mm or less, and the maximum is greater than 2 mm. Here, there is no theoretical limit to the maximum X-ray optical path length of the block, but considering the size of a typical embedding dish, a value of 35 mm or less is preferable. The sample can be rotated by fixing the sample and rotating the X-ray source and camera relative to the sample, or by fixing the X-ray source and camera and rotating the sample. The rotation axis (direction) can be either the longitudinal or lateral direction of the sample.

[0033] FIG. 9 shows a position adjustment mechanism 90 for adjusting the xy directions (horizontal directions), z direction (height direction), and θ (rotation angle) of the stage 704 .

[0034] The position adjustment mechanism 90 is a mechanism for aligning the position of an observation target for optical microscope observation. It includes a support base 901 on which the stage 704 is placed, and an adjustment unit 902 for adjusting the x- and y-directions (horizontal directions), z-direction (height direction), and horizontal rotation angle of the support base 901. A protrusion (not shown) may be provided on the bottom surface of the stage 704, and the support base 901 may be provided with a fixing hole 903 into which the protrusion is fitted and fixed. The adjustment unit 902 includes an adjustment knob 902a for adjusting the x-direction of the stage 901, an adjustment knob 902b for adjusting the y-direction, an adjustment knob 902c for adjusting the z-direction, and a knob 902d for adjusting the horizontal rotation angle of the support base 901. It goes without saying that the x-direction, y-direction, z-direction, and rotation angle can also be adjusted in the negative direction (e.g., if the x-direction is rightward, then the x-direction is changed to leftward, and so on). The position adjustment mechanism 90 can be operated manually or as an electrically powered mechanism that can be automatically controlled by a computer program or the like.

[0035] 3. X-ray Imaging Wax imaging requires X-rays with an energy of 4 to 12 keV (e.g., using Rigaku Corporation's nano3DX X-ray microscope with Cu wavelength X-rays), and the sample size must be adjusted to allow sufficient penetration of the X-rays. Therefore, in the method of the present invention, a plate-shaped wax block of 2 mm or less in thickness, in which the biological sample to be examined is embedded, is placed so that the block surface is aligned with the rotation axis of the X-ray microscope, and X-ray imaging is performed. As described above, the block is mounted in the cassette of the present invention, and the cassette is further fixed to the stage with a fixing member or a removable adhesive material (e.g., double-sided tape or clay) for X-ray imaging. However, to improve the accuracy and reproducibility of the installation, a jig with an adjustment mechanism designed to mount the cassette on the observation stage at a constant sample height and tilt angle may be used.

[0036] In the present invention, X-ray microscopy may be performed on a bare wax block without an embedding cassette, as described in the Examples. In this case, the wax block after X-ray microscopy observation is brazed to the top surface of a wax block without a biological sample attached to a standard embedding cassette without a notch (Figure 15). The combination of the wax block without a biological sample and the unnotched cassette is called a two-part combination. In this two-part combination, the wax constituting the wax block without a biological sample is preferably the same as the wax constituting the wax block of the two-part combination, but a similar wax may also be used. "Brazing" refers to bonding the wax block and the wax block without a biological sample using a hot-melt wax called brazing wax as an adhesive, and then allowing the wax to solidify at room temperature to bond the two blocks together. A wax block containing a biological sample after X-ray microscopy observation is brazed to the top surface of the wax block in the two-part combination, resulting in a combination of the two-part combination and the wax block containing a biological sample. This combination is called a three-part combination. The wax used for brazing the three-component assembly is preferably the same as the wax used to form the wax block or the wax block without the biological sample, but a similar wax may also be used. The brazed wax block can be handled in the same way as a biological sample embedded in wax and attached to a standard embedding cassette. The wax block contained in the brazed wax block remains solid throughout the entire brazing process and maintains its orientation before and after brazing, so the positional information of the wax block is largely preserved, and deformation of the embedded biological sample is minimized. Optical microscopy observation is then performed according to the steps described below.

[0037] 4. Acquisition of position information and observation with an optical microscope Figure 10 shows the process from X-ray microscope photography to optical microscope photography. After photography with the X-ray microscope, position information to be evaluated in detail with an optical microscope is acquired from the captured three-dimensional image.

[0038] Figure 11 shows an overview of how to obtain positional information from a three-dimensional image. In Figure 11, panel A shows a biological sample embedded in a wax block. The three-dimensional image obtained after X-ray imaging is displayed in a format in which CT slices, which are two-dimensional images of cross sections of the wax block containing the biological sample cut along an arbitrary plane, are stacked continuously in a direction perpendicular to the slice plane. Of these, any position on the biological sample can be used as the reference plane; for example, the surface that contacts the top of the biological sample can be used as the reference plane. In this case, for example, when viewed from the top surface of the block (the surface formed by a1-a2-a3-a4 in FIG. 11A ), a plane U (a plane parallel to the plane including the block top surface, a distance p away from the plane formed by a1-a2-a3-a4 in FIG. 11A , a plane tangent to the top of the biological sample (the plane formed by b1-b2-b3-b4)) is used as a reference plane. Continuous cutting along planes parallel to the reference plane (x-y direction in the figure) results in a series of CT slice images from the top to bottom of the biological sample (Panel B). The thickness of each CT slice can be set as desired, so the number of CT slice images obtained from a biological sample of a given size is determined. Therefore, the region to be observed (the observation target region) is searched for within the series of CT slice images. For example, in Panel B of FIG. 11 , the thickness of each slice can be set as desired, so the position (d) of the reference plane U (reference image) of the biological sample can be determined. ０ ) to the nth image, d ｎ Therefore, when observing with an optical microscope, the biological sample is cut with a microtome until the reference plane appears, and then d ｎ The specimen is sliced ​​to a thickness of microns to obtain slices up to 100 mm thick. The specimen is then stretched, dewaxed, and stained according to the steps shown in FIG. 10 , and photographed under an optical microscope. In FIG. 11 , the reference plane is a plane that includes the top end of the biological specimen as viewed from the top side of the block and is parallel to the plane including the top surface of the block. However, this is not limited to this, and any plane including the bottom end of the biological specimen or the top and bottom surfaces of the block may also be used as the reference plane. Furthermore, the reference plane may be a plane that is parallel to the plane including the block surface, or a plane that is not parallel to it.

[0039] In the stretching process, the thinly sliced ​​sections are picked up with tweezers, floated in warm water to stretch them, and then scooped onto a glass slide and adhered. In the dewaxing process, the glass slide with the sections attached is dried and dewaxed by immersing it in xylene or the like. In the staining process, staining is performed according to the purpose of observation using methods such as HE (hematoxylin-eosin) staining, which stains cell nuclei and other tissues separately, or PAS (periodic acid Schiff's reagent) staining, which clearly stains the basement membrane. In the optical microscope photographing process, various stained biological samples are photographed with an optical microscope according to the positional information photographed with an X-ray microscope, and digital images are obtained.

[0040] For example, in the examples, position information is acquired by reslicing the X-ray microscope image in a direction perpendicular to the wax block surface (by changing the slice direction of the CT image; for example, using the reslice tool in ImageJ), as shown in FIG. 12A , and using the position of the top of the sample as the reference (0.0 μm), thereby allowing the accurate depth of any object in the sample to be specified. In the example of FIGS. 12B and 12C , the renal corpuscle 1 is located 115.8 μm from the top of the sample. Regarding observation by optical microscope, the sample after X-ray imaging is microtomed in a conventional manner without being re-embedded in wax for optical microscope observation, stained in a conventional manner, and observed using a conventional optical microscope.

[0041] As a result, it is possible to easily obtain X-ray and optical microscope images of the same measurement point on the same biological sample with nearly identical orientations. In this state, the orientation error between the two images is, for example, within 10°, but is not limited thereto and may be 10° or more depending on the required precision.

[0042] When using a microtome to slice a sample at a constant thickness, if the slice at which the top of the sample appears is recorded, it is possible to calculate which slice contains the target object. In the example of Figures 12B and 12C, if the slice thickness is 4 μm, the optical microscope image corresponding to the X-ray microscope image of renal corpuscle 1 will be obtained on approximately the 30th slice from the top of the sample.

[0043] 5. Microscope Image Processing Device The microscope image processing device provided by the present invention comprises a means for acquiring positional information of a biological sample from an X-ray microscope image acquired by irradiating a biological sample with X-rays and photographing the biological sample, using an arbitrary position of the biological sample as a reference, for observation with an optical microscope, and a means for outputting the positional information. The device of the present invention is used for analyzing biological samples.

[0044] FIG. 13 shows an overview of a microscopic image processing device 100 provided in the present invention. In FIG. 13, the microscopic image processing device 100 includes an X-ray microscopic imaging device 10, an optical microscopic imaging device 30, and an information processing device 40. These devices may be connected via a communication network 20 as shown in FIG. 13. Alternatively, the X-ray microscopic imaging device 10, the optical microscopic imaging device 30, and the information processing device 40 may be installed independently without being connected to a network. In this case, only necessary information such as image data may be imported and processed into the independent information processing device.

[0045] The X-ray microscopy apparatus 10 writes basic information such as patient information from which the sample originates, examination information, and image ID into the header of an image file in accordance with, for example, the DICOM (Digital Imaging and Communications in Medicine) standard, and transmits the image file to the information processing device 40. In the present invention, the X-ray microscopy apparatus 100 preferably uses nano3DX (Rigaku Corporation).

[0046] The optical microscope photographing device 30 is a commonly used microscope such as a stereo microscope, an inverted microscope, or a metallurgical microscope, but it may also be a digital microscope equipped with a high-resolution digital camera instead of an eyepiece. By using a digital microscope, the photographed microscopic image can be saved as a digital image. In this case, the optical microscope photographed image information is sent to the information processing device 40.

[0047] The information processing device 40 is a computer device that analyzes images transmitted from the X-ray microscopy device 10 and the optical microscopy device 30, supports the examination of biological samples, and outputs the image information. When an X-ray microscopy image is acquired by irradiating the biological sample with X-rays and capturing the image, the information processing device 40 acquires positional information of the biological sample for observation with an optical microscope, using an arbitrary position of the biological sample as a reference. Once the positional information has been acquired, the information processing device outputs the positional information.

[0048] 14, the information processing device 40 includes a control unit 101, a communication unit 102, an operation unit 103, a display unit 104, a storage unit 110, etc. The control unit 101 is composed of a CPU, a ROM, a RAM, etc., and comprehensively controls the processing operations of each unit of the information processing device 40. Specifically, the CPU reads out various processing programs stored in the ROM, loads them into the RAM, and performs various processes in cooperation with the programs.

[0049] The information processing device 40 also includes a storage unit 110 provided in a ROM or RAM, and the storage unit 110 includes an X-ray microscope information database (DB) 111, an optical microscope information DB 112, and an additional information DB 113. The additional information DB stores the name of the human or animal tissue from which the biological sample is derived, its ID, the date of sample collection, the reference plane, the thickness of the section, and the like.

[0050] The control unit 101 executes a process for acquiring X-ray microscopy image information and optical microscopy image information, thereby working with the display unit 104 to display images on a monitor or the like (not shown). The communication unit 102 is configured with a network interface or the like and transmits and receives data between an X-ray microscopy apparatus, an optical microscopy apparatus, and external devices connected to the information processing device via a communication network. The operation unit 103 includes a keyboard with various input keys and function keys, a mouse, or the like, and outputs operation signals input by key operations on the keyboard or mouse to the control unit 101. The operation unit 103 can also be configured with a touch panel, in which case it outputs operation signals to the control unit 101 in response to touch operations by the user. The display unit 104 is configured with a monitor such as an LCD (Liquid Crystal Display) and displays various screens according to instructions of signals input from the control unit 101.

[0051] EXAMPLES The present invention will be explained in more detail below with reference to examples, although the scope of the present invention is not limited to these examples.

[0052] Methods: To obtain useful information for kidney pathological diagnosis using X-ray microscopy, it is necessary to observe the internal structure of the glomerulus. To visualize the interior of the glomerulus, for example, a Cu wavelength X-ray must be used, and the sample size must be adjusted to allow sufficient X-ray penetration. Therefore, we conducted an experiment to examine whether the internal structure of the glomerulus, the functional center of the kidney, can be observed by imaging a 1.2 mm thick wax (paraffin) contrast mouse kidney sample, positioned so that the sample plate surface was aligned with the rotation axis of the X-ray microscope. First, a portion containing a biological sample from a wax (paraffin) block prepared for a standard optical microscope was trimmed to 7 mm x 5 mm using a single-edged razor. Next, the surface of the block was horizontally cut to a thickness of 1.2 mm using a diamond wire saw. Next, the wax block sample (7 mm x 5 mm x 1.2 mm) was brazed to the top of a 3 mm diameter cylindrical metal rod, aligning the longitudinal axis of the sample plate with the metal rod, to prepare the sample for X-ray microscopy. The sample was mounted on a nano3DX X-ray microscope (Rigaku Corporation; CMOS detector), and X-ray projection image data were acquired (16.7 hours) under the following conditions: Cu-target (40 kV / 30 mA), L1080-bin1-XD3 (0.74 μm / voxel), continuous scan (2000 frames, 30 s exposure per frame). The projection image data were then processed with ring artifact correction, drift correction, and standard noise reduction filters (median1 and Gaussian1), followed by CT reconstruction using software based on a standard FBP algorithm.

[0053] Results: Figure 12 shows a series of CT slices obtained by reslicing an X-ray microscopy image of a wax block embedded with a mouse kidney sample to the same perspective as the optical microscopy image. By using the CT slice containing the top edge of the sample, i.e., a plane containing the top edge of the sample and parallel to the top surface of the wax block, as the reference plane, detailed positional information for three individual renal corpuscles was obtained (Figure 12A). Furthermore, the X-ray microscopy image confirmed the cellular microstructure, such as the state of cell aggregation within the glomerulus, which is essential for evaluating kidney pathology (Figure 12B). The spatial resolution was approximately 0.9 μm. Figures 12A and 12B show the results using all captured data (rotation angle range 0°-180°). Similar results were obtained when the data used was reduced to 150° (rotation angle range 15°-165°) (Figure 12C). Therefore, when using the embedding cassette of the present invention for X-ray microscopy, the loss of rotation angle due to the cassette blocking X-rays should be less than 30° arcmin, i.e., the imaging rotation angle should be 150° arcmin or greater. Optical microscopy images corresponding to the X-ray microscopy images can be easily obtained by further observing the wax plate block under an optical microscope after X-ray microscopy. For example, Figure 17 shows an example of observation of the same measurement location of the same biological sample (mouse kidney sample) using an X-ray microscope (top) and an optical microscope (bottom). From these results, it can be said that the method of the present invention was able to obtain X-ray microscopy images for observing the same measurement location of the same biological sample using both an X-ray microscope and an optical microscope.

[0054] 10: X-ray microscope imaging device, 20: communication network, 30: optical microscope imaging device, 40: information processing device, 100: microscope image processing device 101: control unit, 102: communication unit, 103: operation unit, 104: display unit, 110: storage unit 701: biological sample, 702: block, 703: cassette, 704: stage, 705: fixing member, 706: notch area of ​​cassette 90: position adjustment mechanism, 901: support base, 902: adjustment unit

Claims

1. A method for observing the same measurement location of a biological sample using an X-ray microscope and an optical microscope, using a wax block in which the biological sample is embedded and which has a minimum value of the optical path length of X-rays of 2 mm or less and a maximum value exceeding 2 mm, the method comprising: an imaging step of imaging the biological sample with an X-ray microscope using X-rays having an energy of 4 to 12 keV; a step of specifying position information of an observation target region for imaging the biological sample with an optical microscope, based on an arbitrary position of the biological sample in the image captured by the X-ray microscope; and a step of slicing the biological sample including the observation target region based on the specified position information and imaging the slice with an optical microscope.

2. The wax block is prepared by a process including chemical fixation, dehydration, and wax infiltration, and a biological sample is placed in an embedding dish having a bottom area of 9 to 900 mm ２ , with a depth of 2 mm or less, and wax is poured into the embedding dish to form a plate-like shape having a thickness of 2 mm or less. The method according to claim 1.

3. The method according to claim 1, wherein the arbitrary position belongs to a surface (reference surface) in contact with the upper end of the biological sample.

4. The method according to claim 1, wherein the wax block is installed in a cassette having a cut.

5. The method according to claim 4, wherein the size of the cut is larger than the imaging site of the biological sample.

6. The method according to claim 5, wherein the wax block is installed such that a region to be irradiated with X-rays is located within the cut region of the cassette.

7. The method according to claim 4, wherein the shape of the cut is strip-shaped or rectangular.

8. The observation by the X-ray microscope is incorporated between the wax infiltration and the microtome sectioning in a biological sample observation step of performing chemical fixation, dehydration, wax infiltration, microtome sectioning, stretching of the section, dewaxing and staining treatment on the collected biological sample and then performing optical microscope observation. The method according to claim 1.

9. The method according to claim 1, wherein the wax block is installed in a cassette having a cut through which X-rays can pass, and the imaging step by the X-ray microscope is performed in a state where the wax block and the cassette are combined.

10. A step of installing a wax block not containing a biological sample in a cassette without a cut to produce a combined body (two-body combined body) of the two, and further installing, by adhering with heat-melted wax as an adhesive, a biological sample-embedded wax block after X-ray microscope observation in which the biological sample is embedded, on the upper surface of the wax block in the two-body combined body, to produce a combined body (three-body combined body) of the two-body combined body and the biological sample-embedded wax block. The method according to claim 1.

11. An embedding dish for embedding a biological sample in wax, the embedding dish having an area of 9 to 900 mm ２ and a depth of 2 mm or less.

12. A method for manufacturing a plate-shaped biological sample embedding block, comprising the steps of placing a biological sample prepared by a process including chemical fixation and dehydration in the embedding dish according to claim 11 so as to satisfy an imaging angle range of 150° or more of an X-ray microscope, and pouring wax into the embedding dish.

13. A cassette for installing a wax block in which a biological sample is embedded and which has a minimum value of the optical path length of X-rays of 2 mm or less and a maximum value exceeding 2 mm, and which is provided with a notch for irradiating the biological sample with X-rays, for realizing the method according to any one of claims 1 to 9.

14. A holding device for holding the cassette according to claim 13 for X-ray microscope imaging, the device comprising a stage on which the cassette is placed and a fixing member holder for fixing the placed cassette.

15. The device according to claim 14, further comprising a position adjustment mechanism for moving the stage in the vertical or horizontal direction.

16. A microscope image processing apparatus comprising means for acquiring position information of a biological sample for observation with an optical microscope with reference to an arbitrary position of the biological sample from an X-ray microscope image obtained by irradiating the biological sample with X-rays and taking a photograph, and means for outputting the position information.

17. The apparatus according to claim 16, wherein the arbitrary position belongs to a surface (reference surface) in contact with the upper end of the biological sample.

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