Method for observing biological sample

Abstract

To provide a method for collating and precisely matching images of biological samples.SOLUTION: A method for collating and matching images of a same measurement location of a same biological sample captured by an X-ray microscope and an optical microscope, comprises: acquiring an image of a biological sample embedded in a wax block captured by an X-ray microscope using X-rays with an energy of 4 to 12 keV; acquiring an image, captured by the optical microscope, of a portion of the biological sample included in the image captured by the X-ray microscope; selecting arbitrary observation target regions of the images of the biological sample from the acquired images from the X-ray microscope and optical microscope as position markers and collating and matching the image from the X-ray microscope and the image from the optical microscope using the position markers.SELECTED DRAWING: Figure 22

Description

Technical Field

[0001] The present invention relates to a method for precisely matching images of the same measurement location of the same biological sample captured by an X-ray microscope and an optical microscope.

Background Art

[0002] The pathological evaluation of biological tissues mainly focuses on the pathological evaluation in the plane (two-dimensional) cut out from pathological specimens. However, pathological changes progress three-dimensionally within the tissue. Also, when preparing pathological specimens, a part of the specimen is discarded and evaluation becomes impossible. From such a current situation, the development of a method for three-dimensionally evaluating specimens and a method for evaluating all specimens has been an important problem to be solved. Conventionally, as methods for observing biological samples, a method of imaging a biological sample using a microscopy method (Patent Document 1), a method of imaging the kidney (renal tubule) of a rat (Patent Document 2), a method of infiltrating a contrast agent into a biological sample to solidify and perform imaging (Patent Document 3), etc. are known.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Patent Document 2

Patent Document 3

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, at present, there is no practical observation method that can three-dimensionally evaluate the pathological changes of a specimen at the cellular level of spatial resolution. Although optical microscopes have relatively high two-dimensional resolution of about 0.2 μm and the advantage of accurate tissue identification by various staining methods, there are limitations in microscopic observation, such as sample deformation (destructive deformation) caused by physical forces during sample preparation and insufficient depth resolution due to a sample thickness of about 4 μm. On the other hand, although X-ray microscopes have the advantages of isotropic submicron three-dimensional resolution and non-destructiveness allowing sample reuse, since the shades of X-ray microscope images are determined by reflecting the electron density of relatively light elements that make up the biological sample, they provide gray-scale images with low contrast, resulting in difficulties in tissue identification. Therefore, there has been a demand for the development of a practical method for three-dimensionally observing the same measurement location of the same biological sample at the cellular level of spatial resolution by combining an X-ray microscope and an optical microscope to complement the above limitations and difficulties.

[0005] As a result of intensive studies to solve the above problems, the present inventor has succeeded in precisely matching the images of the same measurement location of the same biological sample captured by an X-ray microscope and an optical microscope by using cell nuclei and the like contained in the biological sample as position markers and performing orientation adjustment based on the rotation operation of the X-ray microscope image, thus completing the present invention.

Means for Solving the Problems

[0006] That is, the present invention is as follows. [1] A method for collating and matching images of the same measurement location of the same biological sample captured by an X-ray microscope and an optical microscope, comprising: a step of obtaining an image captured by an X-ray microscope using X-rays with an energy of 4 to 12 keV of a biological sample embedded in a wax block; a step of obtaining an image captured by an optical microscope of a part of the biological sample included in the image captured by the X-ray microscope; Selecting an arbitrary observation target area of the image in the biological sample as a position marker from the obtained X-ray microscope and optical microscope images, and collating and matching the X-ray microscope image and the optical microscope image using the position marker. The method including the above. [2] The method according to [1], wherein the image captured by the optical microscope is an image of the same measurement location of the same biological sample captured with an azimuth error within 10° of the image captured by the X-ray microscope. [3] The method according to [1], wherein the selection of the position marker is based on at least one of the size, structure, and type of cells or tissues, cell nuclei, and defective forms in the biological sample. [4] The method according to [3], wherein the defective form is at least one of cancer, fibrosis, calcification, calculus, and deposits. [5] The collation of the X-ray microscope image and the optical microscope image is performed by comparing the LM reference slice among the reference slices (referred to as "XRM reference slice" and "LM reference slice" respectively) including the observation target area (reference area) including the position marker commonly present in the X-ray microscope image and the optical microscope image, and the index slice (referred to as "XRM index slice") including the observation target area including the position marker included in the LM reference slice but not included in the XRM reference slice and appearing within the predetermined upper and lower ranges of the XRM reference slice, and is performed by azimuth adjustment based on the rotation operation of the XRM reference slice. The method according to [1]. [6] The method according to [5], wherein the rotation operation is performed by rotating the CT rotation angle and / or the tilt angle image in the X-ray microscope. [7] The method according to [1], wherein the collation of the X-ray microscope image and the optical microscope image further includes a correction step of the image of the optical microscope. [8] A microscope image processing apparatus, comprising: first specifying means for specifying an arbitrary observation target region included in a biological sample embedded in a wax block, from an X-ray microscope image of the biological sample imaged using X-rays having an energy of 4 to 12 keV; second specifying means for acquiring an image obtained by imaging a part of the biological sample included in the image captured by the X-ray microscope with an optical microscope image, and specifying a region corresponding to the observation target region specified by the first specifying means; means for collating information on the region specified by the first specifying means and information on the region specified by the second specifying means; output means for outputting the collation result; and the apparatus comprising the above. [9] The apparatus according to [8], wherein the first specifying means and the second specifying means specify the observation target region from the X-ray microscope image and the optical microscope image by a region extraction process associated with at least one of the size, structure, and type of cells or tissues, cell nuclei, and defective forms.

[10] The apparatus according to [8], further comprising means for associating the observation target region specified by the first specifying means with the observation target region by the second specifying means, using the position information of the observation target region specified by the first specifying means.

[11] The apparatus according to [8], wherein the first specifying means and the second specifying means specify the observation target region based on an observation target region designation operation by a user, a predetermined priority for the observation target region, and / or a priority preset by the user.

[12] further comprising display means; wherein the output means outputs information on the observation target region extracted from the optical microscope image and the X-ray microscope image to the display means; and the display means displays the information on the observation target region side by side or superimposed. The apparatus according to [8].

[13] The apparatus according to [8], comprising collating means for collating the optical microscope image and the X-ray microscope image by azimuth adjustment based on a rotation operation using a position marker when the observation target region specified by the first specifying means and the second specifying means includes the position marker.

[14] A computer First specifying means for specifying an arbitrary observation target region included in the biological sample from an X-ray microscope image of a wax-embedded biological sample imaged using X-rays with an energy of 4 to 12 keV Second specifying means for acquiring an image obtained by imaging a part of the biological sample included in the image captured by the X-ray microscope with an optical microscope image and specifying a region corresponding to the observation target region specified by the first specifying means Means for collating information on the region specified by the first specifying means and information on the region specified by the second specifying means, and Output means for outputting the collation result A microscope image processing program for causing the above to function.

Advantages of the Invention

[0007] According to the present invention, it has become possible to precisely match and collate images of the same measurement location of the same biological sample captured by an X-ray microscope and an optical microscope. The present invention provides precisely azimuth-matched images necessary for a method of practically three-dimensionally observing the same measurement location of the same biological sample at the cell-level spatial resolution by complementary use of an X-ray microscope and an optical microscope. By more precisely matching the azimuths of both the X-ray microscope image and the optical microscope image, complementary use of the X-ray microscope and the optical microscope becomes possible for the first time.

Brief Description of the Drawings

[0008]

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Embodiments for Carrying Out the Invention

[0009] The present invention relates to a method for collating and matching images of the same measurement location of the same biological sample imaged by an X-ray microscope and an optical microscope. The method of the present invention includes the following steps. A step of obtaining an image by imaging a biological sample embedded in a wax block with an X-ray microscope using X-rays with an energy of 4 to 12 keV. A step of obtaining an image by imaging a part of the biological sample included in the image captured by the X-ray microscope with an optical microscope. From the obtained images of the X-ray microscope and the optical microscope, an arbitrary observation target region of the image in the biological sample is selected as a position marker, and the X-ray microscope image and the optical microscope image are collated and matched using the position marker.

[0010] When observing the same measurement location of the same biological sample with an X-ray microscope and an optical microscope, the observation target region in the X-ray microscope image and the observation target region in the optical microscope image do not always match with a precision that allows for easy comparison. Therefore, the present invention is characterized in that the optical microscope image and the X-ray microscope image are collated, and azimuth adjustment based on the rotation operation of the X-ray microscope image is performed to precisely match the two microscope images.

[0011] An image obtained by imaging a biological sample embedded in a wax block with an X-ray microscope using X-rays with an energy of 4 to 12 keV, and an image obtained by imaging a part of the biological sample included in the image captured by the X-ray microscope with an optical microscope are obtained. From both images, an arbitrary observation target region of the two images in the biological sample is selected as a position marker, and a mode of collating and precisely matching the two images using the position marker is shown below.

[0012] 1. Preparation of a block for X-ray imaging of a biological sample (1) Preparation of a biological sample In order to observe the three-dimensional structure of a biological sample with sub-micron resolution using an X-ray microscope, in the present invention, a wax contrast method described in WO2022 / 234844 is used with some modifications. The "wax contrast method" described in the above publication is a method of enhancing the contrast against X-rays by utilizing the negative contrast effect obtained by infiltrating a wax such as paraffin into a biological sample and replacing the water in the sample with the wax. Further, this wax-infiltrated biological sample is embedded in the same wax to prepare a block, and the embedded biological sample is imaged by irradiating the block with X-rays using an X-ray microscope.

[0013] An unstained biological sample generally has low contrast against X-rays, and thus it is difficult to observe it at the cellular level of spatial resolution using an X-ray microscope as it is. The reason is that in an unstained biological sample, water, which is its main component, and other components have approximately the same X-ray transmittance. In the wax contrast method, the contrast is improved by replacing the water in the biological sample with a wax having a higher X-ray transmittance, enabling X-ray microscopic observation at the cellular level. The wax contrast method is generally a type of technique called negative contrast.

[0014] In the present invention, a plate-shaped wax block with a very large maximum optical path length of X-rays is used, and also, since the extra wax around the test biological sample is not removed, it is different from the wax contrast method described in WO2022 / 234844 in this regard.

[0015] That is, in the method described in WO2022 / 234844, in order to obtain better spatial resolution, it was necessary to remove as much wax around the tissue to be observed from the X-ray microscope observation sample as possible and specify the maximum optical path length of X-rays to be 2 mm or less. In contrast, in the present invention, the biological sample to be observed is embedded in a wax block, but the maximum optical path length of X-rays exceeds 2 mm. When the maximum optical path length exceeds 2 mm, the spatial resolution becomes slightly inferior, but by setting the minimum optical path length of X-rays to 2 mm or less, sub-micron spatial resolution can be maintained.

[0016] Therefore, by performing X-ray microscopic observation using the block of the present invention, while maintaining the spatial resolution necessary for complementary observation with an optical microscope, the compatibility with the conventional biological sample observation process for performing optical microscope observation can be significantly improved. "Complementary observation" means an observation in which the features and advantages of one of the optical microscope and the X-ray microscope complement the other microscope that does not have such features or advantages or has a low degree thereof. Therefore, the wax block of the present invention has a minimum value of 2 mm or less and a maximum value exceeding 2 mm for the optical path length of X-rays in which the biological sample is embedded. Although there is no theoretical limit for the maximum value of the maximum optical path length of X-rays of the block, for example, by setting it to 35 mm or less in consideration of the size of a general embedding dish, the compatibility with the biological sample observation process by an optical microscope can be further enhanced.

[0017] The preparation of the biological sample used in the present invention can follow the steps of preparing a sample for general optical microscope observation. FIG. 1 is a process diagram from cutting out a biological sample to pouring the biological sample into wax such as paraffin and performing wax penetration. In FIG. 1, for a sample (biological sample) cut out and collected from a living body, fixation, for example, chemical fixation using formalin, glutaraldehyde, alcohol, Bouin's solution, etc. is performed. Subsequently, dehydration using alcohol, xylene, etc. and wax penetration are performed. Defatting and decalcification are optional steps and either one or both can be performed as necessary. Wax means a lipophilic compound that is solid at normal 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, synthetic waxes, etc., but paraffin is preferred.

[0018] (2) Embedding dish for wax contrast for embedding the test biological sample After performing wax penetration of wax such as paraffin, the test biological sample is embedded. A schematic diagram of an embedding dish for embedding the test biological sample is shown in FIG. 2. FIG. 2 shows an embedding dish with a rectangular mouth and bottom as an example. The dish has a length in the vertical (longitudinal) direction L1 of the bottom surface of the inner wall of 3 to 32 mm, a length in the transverse direction L2 of 3 to 28 mm, and a depth d of 2 mm or less.

[0019] However, in the present invention, the shapes of the mouth (upper surface) and bottom of the embedding dish may be square, circular or elliptical in addition to rectangular, and are not limited. Also, the side wall of the embedding dish may have a shape extending in the normal direction from the bottom surface, that is, a shape of a rectangular parallelepiped with the same area of the bottom inner wall surface and the upper inner wall surface, or a shape with a larger area of the upper inner wall surface than the area of the bottom inner wall surface, that is, a shape in which the bottom inner wall is constricted when viewed from the top (a shape in which the extension line of the side wall toward the bottom is tapered).

[0020] The depth d of the embedding dish is 2 mm or less, preferably 1.0 to 1.5 mm, for example 1.2 mm. The depth of this embedding dish becomes the thickness of the block for embedding the biological sample. The area of the bottom surface of the inner wall of the embedding dish, that is, the area of the surface (defined as the upper surface of the wax block) of the block for embedding the biological sample that is shaped by the bottom surface of the inner wall of the embedding dish is 9 to 900 mm 2 However, these areas are not limited to the above, and those skilled in the art can appropriately set the size of the block for X-ray microscopic imaging. Examples of the material of the embedding dish include stainless steel or other metal, glass or ceramic, hard or soft resin, paper or wood, etc., but are not limited thereto, and other materials may be used.

[0021] As shown in FIG. 3, while placing a biological sample using an embedding dish with a depth of 2 mm or less, and pouring wax into the embedding dish, a plate-shaped wax block with a thickness of 2 mm or less embedding the test biological sample is produced. For example, a biological sample is placed approximately at the center of the embedding dish (FIGS. 3a and b), wax is poured, and the wax is solidified. By taking out the solidified wax, a block embedding the biological sample (hereinafter simply referred to as "block") can be produced (FIG. 3c).

[0022] However, the order of arranging the biological sample in the embedding dish and pouring the wax is not limited to the above and is arbitrary. That is, it is also possible to previously contain the biological sample in the wax and pour the biological sample together with the wax into the embedding dish. Alternatively, the embedding dish can be filled with wax in advance and the biological sample can be placed therein. The biological sample placed in the embedding dish may be appropriately finely adjusted so as to be located at 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 preferably arranged in contact with the bottom of the embedding dish.

[0023] Furthermore, an X-ray imaging cassette (referred to as an "embedding cassette") is placed on the embedding dish in which the biological sample has been embedded before the wax solidifies, and the wax is solidified with the surface of the wax poured into the embedding dish in contact with the embedding cassette. Then, by taking out the block from the embedding dish, a combination of the embedding cassette and the embedding block can be produced (Figure 4).

[0024] 2. Embedding Cassette for Wax Contrast Imaging for Installing a Test Biological Sample In the present invention, an embedding cassette for installing a block in which a test biological sample is embedded has a strip-shaped or rectangular cut (space) for allowing X-rays to pass through when irradiated with X-rays, as shown in FIG. 5. The block is installed in the cassette such that the region to be irradiated with X-rays, that is, the embedded biological sample, is located within the cut region. The size of the cut of the cassette is made larger than the imaging site of the test biological sample. In other words, the size of the imaging site of the biological sample is adjusted so as to fit within the area of the cut of the cassette. And the above cut is designed such that when performing X-ray imaging using the cassette, even if the loss rotation angle range caused by the cassette blocking the X-rays is less than 30°, that is, the imaging rotation angle range is 150° or more, the X-rays are not blocked. Details of the imaging rotation angle range will be described later. Note that the biological sample is arranged in the embedding block in the embedding dish so as to satisfy an imaging possible angle range of 150° or more of the X-ray microscope. However, as factors for blocking X-rays, 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 for embedding the biological sample may be considered.

[0025] FIG. 6 is a diagram showing that an embedding block is installed such that a biological sample is located in the cut region of the cassette and is arranged in the X-ray irradiation direction in the X-ray microscope. As shown in FIG. 6, X-rays are irradiated from the X-ray generator of the X-ray microscope toward the block, and the X-rays pass through the cut of the cassette (the cut cut out in a strip shape in FIG. 6) and irradiate the biological sample in the block installed in the cassette.

[0026] FIG. 7 is a diagram showing that the cassette with the block installed is fixed to the stage. In FIG. 7, a block 702 in which a biological sample 701 is embedded is provided in a cassette 703 and is positioned such that the biological sample 701 fits within the range of the cut region 706 of the cassette. That is, the biological sample is arranged within the cut so as to satisfy an imaging possible angle range of 150° or more, which will be described later. And the cassette is fixed on the stage such that the block is located on the detector side (the side opposite to the side where the X-rays enter). In FIG. 7, the longitudinal direction of the biological sample is in the x direction (horizontal), but it can also be in the z direction (vertical). In that case, the cut of the cassette may be arranged to face upward, and as long as the biological sample 701 fits within the horizontally long cut region, it is also possible to arrange the longitudinal direction of the biological sample vertically (z direction).

[0027] Cassette 703 is fixed on stage 704 via fixing member 705. As shown in FIG. 7, the fixing member 705 can be provided on the stage with a fitting jig having a recess, and the end of the cassette can be fitted and fixed to the fitting jig. In FIG. 7, both ends of cassette 703 are fitted to fixing member 705, but the member for fixing the cassette to the stage is not particularly limited, and it may be fixed by pasting with double-sided tape or clay (not shown), or may be fixed by a clip or screwing. When using a clip, it is fixed by utilizing the elasticity of an object such as a leaf spring. When using screwing, it is fixed by utilizing a thumb screw or the like.

[0028] After fixing cassette 703 on stage 704, it can be moved in the left-right or front-back direction (x or y direction), or in the vertical direction (z direction) by the position adjustment mechanism of stage 704 (details will be described later) so that the approximate center of the biological sample is located on the X-ray irradiation path (optical path) p1. Also, although the X-ray irradiation path p1 is in the normal direction with respect to block 702, if a rotation axis q1 is provided at the approximate center of biological sample 701 in the normal direction with respect to the xy plane and rotated around rotation axis q1, p1 will be in an oblique direction with respect to block 702. That is, the X-ray optical path is in an oblique direction with respect to block 702. The rotation angle θ at this time is within the X-ray imaging rotation angle range, and it can be rotated by 150° or more, preferably 180° or more.

[0029] The relationship between biological sample 701 and the X-ray irradiation direction is shown in FIG. 8. In FIG. 8, the left panel shows the minimum and maximum optical path lengths of X-ray imaging in a conventional test example. Conventionally, since X-ray imaging was performed on a biological sample from which the wax on the surface had been removed after wax imaging, the maximum optical path length and the minimum optical path length approximately match the thickness or length of the biological sample.

[0030] In contrast, in the present invention, since X-ray irradiation is performed on a plate-shaped wax block embedding a wax-imaged biological sample, the optical path length depends on the length or thickness of the block. Even if the thickness of the block is 2 mm or less, when the sample is rotated by a certain angle θ from the rotation axis of the sample, the optical path becomes longer up to a length close to the longitudinal direction of the block (test example of the present invention; right panel of FIG. 8). In the present invention, the minimum value of the X-ray optical path length of the block is 2 mm or less, and the maximum value is larger than 2 mm. Here, there is no theoretical limit for the maximum value of the X-ray maximum optical path length of the block, but it is preferably 35 mm or less in consideration of the size of a general embedding dish. Note that for the rotation of the sample, the sample may be fixed and the X-ray source and the camera may be rotated with respect to the sample, or the X-ray source and the camera may be fixed and the sample may be rotated. Also, the rotation axis (direction) may be either the longitudinal direction or the short-side direction of the sample.

[0031] FIG. 9 shows a position adjustment mechanism 90 for adjusting the xy direction (horizontal direction), z direction (height direction), and θ (rotation angle) of the stage 704.

[0032] The position adjustment mechanism 90 is a mechanism for aligning the observation target position for optical microscope observation, and includes a support base 901 on which the stage 704 is placed, and an adjustment unit 902 for adjusting the xy direction (horizontal direction), z direction (height direction), and the horizontal rotation angle of the support base 901. Protrusions (not shown) may be provided on the bottom surface of the stage 704, and fixing holes 903 and the like for fitting and fixing the protrusions may be provided on the support base 901. 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. Needless to say, the minus directions (for example, if the x direction is rightward, then leftward, and the same applies to the others) of the x direction, y direction, z direction, and rotation angle can also be adjusted. In addition, the position adjustment mechanism 90 can be an electric mechanism that can be automatically controlled by a computer program or the like in addition to manual operation.

[0033] 3. X-ray imaging For wax imaging, X-rays with an energy of 4 to 12 keV are used (for example, the X-ray microscope nano3DX of Rigaku Corporation is used with X-rays of Cu wavelength), and it is necessary to adjust the sample size so that the X-rays can pass through the sample sufficiently. Therefore, in the present invention, a plate-shaped wax block with a thickness of 2 mm or less in which the test biological sample is embedded is installed so that the block plate surface is aligned with the rotation axis of the X-ray microscope for X-ray imaging. As described above, the block is mounted on the cassette of the present invention, and the cassette is further fixed to the stage with a fixing member or a detachable adhesive material (such as double-sided tape or clay) for X-ray imaging. However, in order to improve the installation accuracy and reproducibility, a jig with an adjustment mechanism designed to attach the cassette to the observation stage with a constant sample height and tilt angle may be used.

[0034] In the present invention, X-ray microscopic observation may be performed on a bare plate-shaped wax block that is not combined with an embedding cassette. In this case, after the X-ray microscopic observation, the plate-shaped wax block is waxed onto the upper surface of a wax block that does not contain a biological sample and is combined with a general embedding cassette without a notch (FIG. 16). A combination of both in which a wax block that does not contain a biological sample is placed in a cassette without a notch is referred to as a two-piece combination. In the two-piece combination, it is desirable that the wax constituting the wax block that does not contain the biological sample is the same as the wax constituting the plate-shaped wax block, but another similar wax may also be used. "Waxing" means using a heat-melted wax called waxing wax as an adhesive to bond the plate-shaped wax block and the wax block that does not contain the biological sample, and then allowing the waxing wax to solidify by standing at room temperature, thereby bonding both blocks. By waxing the biological sample-embedded wax block after X-ray microscopic observation onto the upper surface of the wax block in the two-piece combination, a combination of the two-piece combination and the biological sample-embedded wax block can be obtained. This combination is referred to as a three-piece combination. It is desirable that the wax of the waxing wax when producing the three-piece combination is the same as the wax constituting the plate-shaped wax block or the wax block that does not contain the biological sample, but another similar wax may also be used. The wax block after waxing can be handled in the same manner as a biological sample embedded in wax and combined with a general embedding cassette. The plate-shaped wax block contained in this wax block after waxing maintains a solid state throughout the entire waxing process and maintains the corresponding relationship of orientation before and after waxing, so the position information of the plate-shaped wax block is substantially preserved, and deformation of the embedded biological sample is suppressed. Thereafter, optical microscopic observation is performed according to the steps described later.

[0035] 4. Acquisition of position information and observation with an optical microscope FIG. 10 is a diagram showing the process until optical microscopic photography is performed after X-ray microscopic photography is completed. After photographing with an X-ray microscope, position information to be evaluated in detail with an optical microscope is acquired from the three-dimensional image thus photographed.

[0036] FIG. 11 is a diagram showing an overview for acquiring position information from a three-dimensional image. In FIG. 11, panel A shows a biological sample embedded in a wax block. The three-dimensional image after X-ray imaging is displayed in a format in which CT slices, which are two-dimensional images of cross-sections obtained by cutting the wax block containing the biological sample in an arbitrary plane, are continuously stacked in a direction perpendicular to the slice plane. Among these, an arbitrary position of the biological sample is used as a reference plane, for example, a plane in contact with the upper end of the biological sample can be used as the reference plane. In this case, for example, when viewed from the side of the upper surface of the block (the surface formed by a1-a2-a3-a4 in FIG. 11A), a plane U (a plane formed by b1-b2-b3-b4 that is parallel and separated by a distance p from the surface formed by a1-a2-a3-a4 and is in contact with the upper end of the biological sample) parallel to the reference plane is used as the reference plane, and when continuously cutting with a plane parallel to the reference plane (in the xy direction in the figure), a series of CT slice images from the upper end to the lower end of the biological sample can be obtained (panel B). Since the thickness of one CT slice can be set as appropriate, the number of CT slice images obtained from a biological sample of a predetermined size is determined. Therefore, an area to be observed (observation target area) is searched for among the series of CT slice images. For example, in panel B of FIG. 11, since the thickness of one slice can be arbitrarily set, the distance d n from the position (d0) of the reference plane U (reference image) of the biological sample to the nth image can be obtained. Therefore, at the time of optical microscope observation, the sample is cut with a microtome until the reference plane of the biological sample appears, and from there, d nSlice it thinly in micron thickness so that slices up to [a certain point] can be obtained. Then, perform stretching, dewaxing, staining, and optical microscopy imaging according to the process shown in FIG. 10. In FIG. 11, the plane including the upper end of the biological sample and parallel to the upper surface of the block when viewed from the upper surface side of the block was used as the reference plane, but it is not limited to this, and any plane including the lower end of the biological sample or the upper and lower surfaces of the block may be used as the reference plane. Also, the reference plane may be a plane parallel to the plane including the block surface or a non-parallel plane.

[0037] In the stretching process, pick up the thinly sliced section with tweezers, float the section in warm water to stretch it, and then scoop it up onto a slide glass and adhere it. In the dewaxing process, dry the glass with the section attached and perform dewaxing treatment by immersing it in xylene or the like. In the staining process, use methods such as HE (hematoxylin - eosin) staining to differentiate between cell nuclei and other tissues, or PAS reagent (periodic acid Schiff’s reagent) staining that clearly stains the basement membrane, and perform staining according to the observation purpose. In the optical microscopy imaging process, photograph variously stained biological samples with an optical microscope along the position information photographed with an X - ray microscope to obtain digital images.

[0038] For example, regarding the acquisition of position information, as shown in FIG. 18A, reslice the X - ray microscope image in a direction perpendicular to the surface of the wax block (changing the slice direction of the CT image; for example, using the reslice tool of ImageJ). If the position of the upper end of the sample is taken as the reference (0.0μm), the exact depth of any object in the sample can be specified. In the examples of FIGS. 18B and C, the renal corpuscle 1 is at a position 115.8μm from the upper end of the sample. Regarding the observation with an optical microscope, the sample after X - ray imaging is directly sectioned with a microtome in a general method without wax re - embedding for optical microscope observation, stained in a general method, and observed using a general optical microscope.

[0039] As a result, X-ray microscope images and optical microscope images of the same measurement location of the same biological sample can be easily obtained in a form where the orientations are substantially the same. The orientation error between the two images in this state is, for example, within 10°, but is not limited thereto, and may be 10° or more depending on the required precision.

[0040] When slicing with a microtome to a constant sample thickness, if the sections where the upper end of the sample appears are recorded, it is possible to calculate which section contains the object of interest. In the examples of FIGS. 18B and C, if the section thickness is 4 μm, the optical microscope image corresponding to the X-ray microscope image of the renal corpuscle 1 can be obtained from approximately the 30th section from the upper end of the sample.

[0041] 5. Matching of X-ray Microscope Image and Optical Microscope Image When X-ray microscope images and optical microscope images with substantially the same orientation are obtained as input images in the initial state, the two can be matched and precisely aligned by adjusting the orientation of the X-ray microscope image by the following method.

[0042] (1) Selection as a position marker for an arbitrary observation target region in the biological sample First, an arbitrary observation target region that is included in the biological sample and can be used as a position marker for orientation adjustment is selected from the images of the biological sample captured by the X-ray microscope and the optical microscope. The "image" means both the data of the image itself (data displayed in pixels, etc.) and numerical data such as coordinates. The form of the arbitrary observation target region is not limited, but for example, the size, structure, and type of cells or tissues, cell nuclei, and defective forms can be selected as position markers.

[0043] The defective form is any one of cancer, fibrosis, calcification, stones, and other deposits, or a combination thereof. In the examples, cell nuclei were used as position markers, one reference region serving as a reference for orientation adjustment was selected, and ten index regions for evaluating the degree of coincidence were selected. In the examples, the selection of the position markers was performed manually, but it may be automated using AI or the like.

[0044] (2) Step of collating and precisely matching the X-ray microscope image and the optical microscope image According to the flowchart of the orientation adjustment (details will be described later), it is performed by rotating the image of the CT rotation angle and / or the tilt angle in the X-ray microscope (using the rotation tool of ImageJ, etc.).

[0045] (3) Flowchart of orientation adjustment The step of collating and precisely matching the X-ray microscope image and the optical microscope image will be described in more detail. Consider the case of precisely matching the optical microscope (LM) image and the X-ray microscope (XRM) image. In Fig. 12A, (a) is a perspective schematic diagram of the LM image, and (b) is a perspective schematic diagram of the XRM image. The figure of the flowchart of the orientation adjustment is shown in Fig. 12B.

[0046] (3-1) First, find one observation target region containing position markers (such as cell nuclei) that commonly exist in the LM image and the XRM image, and set this region as the reference region (Fiducial region) (step S1). The slice where the reference region exists is called the "reference slice (Fiducial slice)". Note that the reference region may contain a plurality of position markers.

[0047] (3-2) Next, select several regions in the observation target regions containing position markers (such as cell nuclei) that exist in the LM image but do not exist in the reference slice of the XRM image but exist within approximately 20 slices before and after (step S2). This region is called the "marker region", and the slice where the marker region exists is called the "marker slice". In the present invention, the number of selected marker regions is not limited, but 8 to 20 locations, preferably about 10 locations, are selected. Note that one marker region may contain a plurality of position markers.

[0048] In FIG. 12A, (b) shows a mode in which the XRM image is shifted by an angle of θ1 counterclockwise from the x - coordinate or y - coordinate of the reference slice plane around the reference region of the reference slice, and is shifted by an angle of θ2 counterclockwise around the y - axis passing through the center of the reference region. In the present invention, θ1 is referred to as the CT rotation angle, and θ2 is referred to as the tilt angle. Note that the CT rotation angle may be a clockwise rotation angle or a counterclockwise rotation angle with respect to the rotation axis. Also, the rotation axis of the tilt angle may be the x - axis or the y - axis. When the angles of θ1 and θ2 are shifted, the position markers in the regions other than the reference region of the LM image become invisible in the XRM image, but they will exist in some slices above and below the reference slice (for example, within 10 slices or 20 slices above and below).

[0049] FIG. 12A shows a mode in which three regions (M1, M2, and M3) existing in the LM image are selected as index regions including position markers (such as cell nuclei) in the XRM image. For the sake of simplicity of explanation, the number of index regions is set to three. In FIG. 12A(b), the slice where the index region M1 exists is defined as index slice 1, and the slices where the index regions M2 and M3 exist are defined as index slice 2.

[0050] Next, when these index regions are z - projected onto the reference slice, the index region M1 of index slice 1 and the index regions M2 and M3 of index slice 2 are projected onto the reference slice (FIG. 12A(c), step S3). When selecting the index regions, it is preferable that when the index regions are z - projected onto the reference slice, the projected index regions (projected index regions) are scattered as evenly as possible over the entire reference slice.

[0051] (3 - 3) The projection index area is a virtual existence. Since the actual XRM image is shifted by θ1 and θ2 from the reference slice of the LM image, there is no physical position marker in the projection index area of the reference slice. Therefore, azimuth adjustment is performed using a rotation tool such as ImageJ (step S4) so that a position marker appears in the projection index area of the reference slice. Step S4 includes steps S5 to S10. The azimuth adjustment is performed as follows.

[0052] First, calculate the rotation angle such that a position marker appears in the projection index area of the reference slice. When an index area is added, calculate only for that added part. For a certain index area, let the distances in the x and y directions (in pixel units) between the reference area and the projection index area be dx (=x_index - x_reference) and dy (=y_index - y_reference), and the distance between the reference slice and the index slice be dz (=z_index - z_reference). Then,

[0053] I) Select the axis that is closer to being parallel to the straight line connecting the projection index area and the reference area among the x or y axes (in the order of selection), and calculate the z-angle such that the axis and the straight line are parallel (step S5). z-angle = -tan -1 (dy / dx), or z-angle = tan -1 (dx / dy) For example, in Fig. 12A(c), since the axis closer to being parallel to the straight line connecting M1 and the reference area is the x-axis, the x-axis is selected, and thus the z-angle is calculated by the former of the above two formulas.

[0054] II) Next, calculate the y-angle or x-angle such that a position marker appears in the projection index area of the reference slice (using the following formula) (step S6). For example, perform angle adjustment of θ2 (the tilting angle around the x-axis or y-axis) so that the cell nucleus selected as the position marker included in the index area appears on the reference slice. That is, y-angle = {if dx > 0 tan -1 (dz / sqrt(dx 2+dy 2 )) else -tan -1 (dz / sqrt(dx 2 +dy 2 ))}、または x-angle={if dy>0 -tan -1 (dz / sqrt(dx 2 +dy 2 )) else tan -1 (dz / sqrt(dx 2 +dy 2 ))} For example, for M1 in FIG. 12A(c), since the x-axis is selected, the y-angle is calculated by the former of the above two expressions. The sign is determined by the value of dx.

[0055] (3-4) Furthermore, among the combinations of the two projection index regions, select a pair in which the angle sandwiching the reference region is closer to a right angle (step S7). For example, in FIG. 12A(c), among the angles formed by (i) M1 - reference region - M2, (ii) M2 - reference region - M3, and (iii) M1 - reference region - M3, select the pair in which the angle is closer to a right angle. If the angle of (i) is 89°, the angle of (ii) is 80°, and the angle of (iii) is 180°, the pair in which the angle is closer to a right angle is the pair of M1 and M2 of (i).

[0056] As an example, in FIG. 21, in the pair of M4 and M9, since the angle formed by M4 - F - M9 is 87.1° and is close to a right angle, this pair is selected.

[0057] Calculate the average z-angle for the selected pair of projection index regions (the following formula) (step S8). That is, z-angle=(z-angle_1+z-angle_2) / 2 As an example, in Fig. 21, since the z-angles of M4 and M9 are 40.6° and 37.6° respectively, the average z-angle is 39.1°. Conversely, by selecting pairs of target regions with close z-angles, pairs that are nearly orthogonal can be found. When additional target regions are added, in order to improve the matching degree, the pairs of target regions that are closer to the orthogonal relationship may be reselected based on the z-angle.

[0058] (3-5) For the pair of projection target regions selected in (3-4) above, input the values of x-angle, y-angle, and z-angle into the ImageJ TransformJ Rotate plugin to perform a rotation operation (the following command) (step S9). That is, run(“TransformJ Rotate”, “z-angle=z-angle y-angle=y-angle x-angle=x-angle interpolation=Linear background=0.0 adjust resample anti-alias”); For example, in Fig. 21, since the z-angle (average) is 39.1°, the y-angle (M4) is 2.58°, and the x-angle (M9) is 2.62°, these values are input.

[0059] (3-6) If necessary, search for (x and y)-angles around the current orientation (step S10).

[0060] (3-7) If the matching number is equal to or greater than the evaluation criterion, the orientation adjustment ends. If the matching number is less than the evaluation criterion, return to the step (step S2) of (3-2) to add a target region.

[0061] (3-8) Evaluation of orientation adjustment based on the matching number of target regions As a result of performing orientation adjustment according to the above flowchart, for a certain index region, when a position marker appears in the projected index region of the reference slice, it is evaluated that the images of the X-ray microscope and the optical microscope match for that index region. The evaluation criterion for orientation adjustment is set to 6 to 8 matching index regions. That is, orientation adjustment is completed when 6 or more, 7 or more, or 8 or more index regions match, but 8 or more matches are desirable. In addition, in three examples where 8 or more index regions matched and orientation adjustment was successful, the average residual of the x-angle or y-angle of these matching index regions was 0.21° to 0.34°. Therefore, it can be considered that the precision of precise matching obtained by the method of the present invention is generally within 0.5°.

[0062] Note that the optical microscope image includes deformations caused by sample preparation, and there may be local structural differences between the X-ray microscope image and the optical microscope image. Therefore, the comparison of the X-ray microscope image and the optical microscope image may further include a correction process (such as Rigid Registration in Fiji) of the image data of the optical microscope. Software for automating the above flowchart may be designed.

[0063] 6. Microscope Image Processing Apparatus, Program, and Recording Medium The present invention further provides a microscope image processing apparatus and a computer program for microscope image processing.

[0064] The apparatus of the present invention includes: first specifying means for specifying an arbitrary observation target region included in a wax block-embedded biological sample imaged using X-rays with an energy of 4 to 12 keV from an X-ray microscope image of the wax block-embedded biological sample; second specifying means for acquiring an image of a part of the biological sample included in the image captured by the X-ray microscope with an optical microscope image and specifying a region corresponding to the observation target region specified by the first specifying means; means for comparing information on the region specified by the first specifying means and information on the region specified by the second specifying means; It includes output means for outputting the collation result. The device of the present invention is used for the analysis of biological samples.

[0065] Also, the program of the present invention causes a computer to from an X-ray microscope image of a wax block-embedded biological sample imaged using X-rays with an energy of 4 to 12 keV, first specifying means for specifying an arbitrary observation target region included in the biological sample, acquire an image obtained by imaging a part of the biological sample included in the image captured by the X-ray microscope with an optical microscope image, and second specifying means for specifying a region corresponding to the observation target region specified by the first specifying means, means for collating the information of the region specified by the first specifying means and the information of the region specified by the second specifying means, and output means for outputting the collation result, It is a microscope image processing program for causing it to function as such. The program of the present invention is used for the analysis of biological samples.

[0066] FIG. 13 shows an overview of a microscope image processing apparatus 100 provided in the present invention. In FIG. 13, the microscope image processing apparatus 100 includes an X-ray microscope imaging apparatus 10, an optical microscope imaging apparatus 30, and an information processing apparatus 40. These apparatuses may be connected via a communication network 20 as shown in FIG. 13. Also, the X-ray microscope imaging apparatus 10, the optical microscope imaging apparatus 30, and the information processing apparatus 40 can be installed independently without connecting to a network. In this case, only necessary information such as image data can be taken into an independent information processing apparatus and processed.

[0067] The X-ray microscope imaging device 10 writes basic information such as patient information, examination information, and image ID from which the sample is derived into the header of the image file and transmits it to the information processing device 40 in accordance with, for example, the DICOM (Digital Imaging and Communications in Medicine) standard. In the present invention, the X-ray microscope imaging device 100 preferably uses nano3DX (Rigaku Corporation).

[0068] The optical microscope imaging device 30 is a generally used microscope such as a stereomicroscope, an inverted microscope, or a metallurgical microscope, but may also be a digital microscope equipped with a high-resolution digital camera instead of an eyepiece lens. By using a digital microscope, the captured microscope image can be saved as a digital image. In this case, the optical microscope imaging image information is transmitted to the information processing device 40.

[0069] The information processing device 40 is a computer device that analyzes the images transmitted from the X-ray microscope imaging device 10 and the optical microscope imaging device 30, supports the examination of the biological sample, and outputs this image information. When an X-ray microscope image is obtained by irradiating the biological sample with X-rays and imaging, the information processing device 40 acquires the position information of the biological sample for observation with an optical microscope based on an arbitrary position of the biological sample. Once the position information is acquired, the information processing device outputs the position information.

[0070] As shown in FIG. 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, and the like. The control unit 101 is composed of a CPU, a ROM, a RAM, etc., and comprehensively controls the processing operations of each part of the information processing device 40. Specifically, the CPU reads out various processing programs stored in the ROM and expands them in the RAM, and performs various processes in cooperation with the programs.

[0071] Further, the information processing apparatus 40 includes a storage unit 110 provided in a ROM or a RAM, and the storage unit 110 includes an X-ray microscope information database (DB) 111, an optical microscope information DB 112, and an attached information DB 113. The attached information DB stores the names, IDs, sample collection dates, reference planes, slice thicknesses, reference regions, index regions, etc. of the human or animal tissues from which the biological samples are derived.

[0072] The control unit 101 executes a process of acquiring X-ray microscope photographing image information and optical microscope photographing image information, and thereby, in cooperation with the display unit 104, exhibits an image display function on a monitor or the like (not shown). The communication unit 102 is composed of a network interface or the like, and performs data transmission and reception with an X-ray microscope photographing apparatus, an optical microscope photographing apparatus connected via a communication network, and external devices connected to the information processing apparatus. The operation unit 103 includes a keyboard having various input keys and function keys, and a mouse or the like, and outputs an operation signal input by a key operation on the keyboard or a mouse operation to the control unit 101. Further, the operation unit 103 can also be composed of a touch panel, and in that case, outputs an operation signal to the control unit 101 according to a touch operation by the user. The display unit 104 is configured to include a monitor such as an LCD (Liquid Crystal Display), and displays various screens according to an instruction of a signal input from the control unit 101.

[0073] FIG. 15 is a flowchart showing an azimuth adjustment process by the microscope image processing apparatus of the present invention. First, the control unit 101 of the information processing apparatus 40 receives an input of an X-ray microscope image and sets it as a target image 1. The control unit 101 specifies an observation target region that can be used as a position marker for azimuth adjustment and is included in the biological sample from the target image 1 (first specifying means). The control unit 101 executes image analysis processing based on this target image (step S11). The image analysis processing is executed in cooperation with the control unit 101 and a program stored in the storage unit 110.

[0074] Next, position information to be evaluated in detail with an optical microscope is extracted from the target image 1 (step S12). As shown in FIG. 11, since the thickness of one slice can be arbitrarily set, the control unit 101 calculates the distance d from the position (d0) of the reference plane U (reference image) of the biological sample to the nth image. n The slice thickness may be input by the user or may be preset in advance.

[0075] Next, the control unit 101 receives the input of the optical microscope image and sets it as the target image 2 (step S13). The control unit 101 selects an observation target region that can be used as a position marker for azimuth adjustment from among the target image 1 and the target image 2 (step S14). That is, the control unit 101 specifies an observation target region corresponding to the observation target region specified by the first specifying means from the image obtained by imaging a part of the biological sample included in the image captured by the X-ray microscope with an optical microscope image (second specifying means).

[0076] The form of the observation target region can be selected by pre-training the form in the form identifier and automatically selecting a form that matches or approximates the form, or by receiving the input of the form selected by the user. In step S14, for example, for each slice of the microscope image, image feature amounts such as density, shape, texture, and multi-resolution features are extracted on a pixel-by-pixel basis. One type of image feature amount may be extracted, or a plurality of types may be extracted.

[0077] The first and second specifying means specify the observation target region based on an observation target region designation operation by the user, a predetermined priority for the observation target region, and / or a priority preset by the user. The "priority" is the order for specifying the observation target region and indicates in which order the observation target regions are specified. For example, when selecting an observation target region as a position marker for azimuth adjustment of an X-ray microscope image of a renal biopsy sample embedded in a wax block, the cell nucleus has the first priority, and the particulate defect form (such as fibrosis) has the second priority.

[0078] In addition, the azimuth error between the optical microscope image and the X-ray microscope image is within 10°, but is not limited thereto, and may be 10° or more according to the required precision.

[0079] Next, the control unit 101 executes the azimuth adjustment process shown in FIG. 12B (step S15) in order to collate the information of the observation target area specified by the first specifying means and the information of the observation target area specified by the second specifying means. The control unit 101 selects one reference area and several index areas from the observation target area including the position marker according to the azimuth adjustment process shown in FIG. 12B. During the operation of step S15, when the control unit 101 receives an input to change the designation of the reference area and the index area from the user, or when an index area is added, the control unit 101 calculates the CT rotation angle θ1 and the tilt angle θ2 of the index area from the XRM images and displays them on the display unit 104 (step S16).

[0080] Subsequently, the control unit 101 calculates a projected index area obtained by z-projecting the index area onto the reference slice, and selects a specific area from among the plurality of projected index areas. For example, when three projected index areas (M1, M2, and M3) are selected as shown in FIG. 12A(c), two straight lines formed by one projected index area - reference area - the other projected index area are extracted, and it is calculated which combination of projected index areas makes the angle formed by these straight lines closest to a right angle, and a candidate pair of projected index areas is displayed (step S17). Next, the control unit 101 displays the form of the projected index area (step S18). Then, the control unit 101 calculates the rotation angles x-angle, y-angle, and z-angle for azimuth adjustment (step S19).

[0081] The control unit 101 inputs the values of x-angle, y-angle, and z-angle for a pair of projection index regions to the storage unit 113 to perform a rotation operation, or displays the rotation angle for the user to perform a rotation operation on the display unit 104 (step S20). As a result of the orientation adjustment, if a position marker appears in the projection index region of the reference slice for a certain index region, it is evaluated that the images of the X-ray microscope and the optical microscope match for that index region. If necessary, a search for (x and y)-angle is performed around the current orientation (step S21). If the number of matching index regions reaches or exceeds the evaluation criterion, the orientation adjustment is terminated. If the number of matches is less than the evaluation criterion, an index region is added and the process returns to step S16. The evaluation criterion is set to the default value of 8 and can be appropriately changed within the range of 6 to 8.

[0082] The program of the present invention can be stored in a computer-readable recording medium. Therefore, a computer-readable recording medium storing the program of the present invention is also included in the present invention. Examples of the recording medium or storage means include, but are not limited to, magnetic media (such as flexible disks and hard disks), optical media (such as CDs and DVDs), magneto-optical media, and flash memories.

[0083] Examples Hereinafter, the present invention will be described more specifically with reference to examples. However, the scope of the present invention is not limited by these examples. [Example 1]

[0084] Method An experiment was conducted using a wax (paraffin)-contrasted mouse renal biopsy simulation sample to determine whether it is possible to observe the same measurement location of the same biological sample with an X-ray microscope and an optical microscope. Kidneys from a kidney disease model mouse created by the partial nephrectomy method described in the literature (Kunishima et al. (2022) Scientific Reports 12, 9436) were used in the experiment. Next, a wax-contrasted sample (wax block) was prepared from the kidneys of the same disease model mouse using the method described in the same literature. Next, an X-ray microscope observation sample was prepared from the same wax block using the direct mounting method described in the same literature. This sample was attached to an X-ray microscope nano3DX (Rigaku Corporation) (CCD detector), and X-ray projection image data was taken under the conditions of Cu-target (40 kV / 30 mA), L0270-bin1-XD2 (0.27 μm / voxel), Step scan (60 s exposure per sheet for 3400 sheets) (required time: 57.7 hours).

[0085] After processing this projection image data with ring artifact correction, drift correction, and phase recovery (Paganin method; δ / β = 100), CT reconstruction was performed using software based on the general FBP algorithm. Furthermore, the renal biopsy simulation sample after X-ray microscope observation was re-embedded in a wax (paraffin) block, serially sectioned to a thickness of 4 μm with a microtome, stained with PAS reagent (periodic acid Schiff’s reagent) to create an optical microscope observation sample, and observed using an optical microscope for serial section observation Nano Zoomer C9600-03 (Hamamatsu Photonics). Coarse alignment of the X-ray microscope image and the optical microscope image was manually performed using general display software (ImageJ and Drishti). Then, alignment was performed according to the alignment process of the present invention. The alignment of the present invention was performed using ImageJ according to the procedures described in (3-1) to (3-8) above.

[0086] Results: As shown in FIGS. 19 and 20, a characteristic cell nucleus, which is a position marker common to the LM (optical microscope) image and the original XRM (X-ray microscope) image, was found and used as a reference region. The slices of the XRM image and the LM image containing the reference region were defined as the XRM reference slice and the LM reference slice, respectively. In this example, the cell nucleus (the 481st out of 1791) in the blood vessel in the renal corpuscle to be observed was used as the reference region (arrow in the figure). Next, as shown in FIG. 21, out of a number of characteristic cell nuclei, which are position markers present in the LM reference slice, ten cell nuclei that do not exist in the XRM reference slice but exist within approximately 20 slices before and after were selected and used as index regions. When the region obtained by z-projecting the index region onto the XRM reference slice was defined as the projected index region, the projected index region was made to be scattered as evenly as possible over the entire XRM reference slice without bias. At this point, there are no cell nuclei in the projected index region of the XRM reference slice (the number of matches is 0).

[0087] Next, as shown in FIG. 22, the orientation was adjusted using the rotation tool of ImageJ so that cell nuclei, which are position markers, appeared in the projected index region of the XRM reference slice. In this example, when rotated by 4.0° around the horizontal axis within the screen and 0.6° around the vertical axis within the screen, cell nuclei appeared in the projected index regions of all ten locations of the XRM reference slice. That is, since the number of matches in the index region became 10, which is equal to or greater than the evaluation criterion, the orientation adjustment was completed. As a result of the above experiment, it was shown that it is possible to precisely match the images of the same measurement location of the same biological sample captured by the X-ray microscope and the optical microscope by collating them using the method of the present invention.

Explanation of Reference Signs

[0088] 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: Cut region of cassette 90: Position adjustment mechanism, 901: Support base, 902: Adjustment unit

Claims

1. A method for collating and matching images of the same measurement location of the same biological sample captured by an X-ray microscope and an optical microscope, comprising: obtaining an image captured by an X-ray microscope using X-rays with an energy of 4 to 12 keV of a biological sample embedded in a wax block; obtaining an image captured by an optical microscope of a part of the biological sample included in the image captured by the X-ray microscope; selecting an arbitrary observation target region of the image in the biological sample as a position marker from the obtained images of the X-ray microscope and the optical microscope, and collating and matching the X-ray microscope image and the optical microscope image using the position marker; The method as described above, including the above steps.

2. The method according to claim 1, wherein the image captured by the optical microscope is an image captured at the same measurement location of the same biological sample with an azimuth error within 10° of the image captured by the X-ray microscope.

3. The method according to claim 1, wherein the selection of the position marker is based on at least one of the size, structure, and type of cells or tissues, cell nuclei, and defect morphology in the biological sample.

4. The method according to claim 3, wherein the defect morphology is at least one of cancer, fibrosis, calcification, calculus, and deposits.

5. The collation of the X-ray microscope image and the optical microscope image is performed by comparing an LM reference slice (referred to as "XR-M reference slice" and "LM reference slice" respectively) including an observation target region (reference region) including a position marker commonly present in the X-ray microscope image and the optical microscope image, and an index slice (referred to as "XR-M index slice") including an observation target region including a position marker included in the LM reference slice but not included in the XR-M reference slice and appearing within a predetermined range above and below the XR-M reference slice, and is performed by azimuth adjustment based on the rotation operation of the XR-M reference slice. The method according to claim 1.

6. The method according to claim 5, wherein the rotation operation is performed by rotating the CT rotation angle and / or the tilt angle image in the X-ray microscope.

7. The method according to claim 1, wherein the collation of the X-ray microscope image and the optical microscope image further includes a correction step of the image of the optical microscope.

8. A microscope image processing apparatus, comprising: first specifying means for specifying an arbitrary observation target region included in a biological sample from an X-ray microscope image of a wax block-embedded biological sample imaged using X-rays having an energy of 4 to 12 keV; second specifying means for acquiring an image obtained by imaging a part of the biological sample included in the image imaged by the X-ray microscope with an optical microscope image, and specifying a region corresponding to the observation target region specified by the first specifying means; means for collating information on the region specified by the first specifying means and information on the region specified by the second specifying means; output means for outputting the collation result; the apparatus comprising the above.

9. The apparatus according to claim 8, wherein the first specifying means and the second specifying means specify the observation target region from the X-ray microscope image and the optical microscope image by region extraction processing associated with at least one of the size, structure and type of cells or tissues, cell nuclei, and defect morphologies.

10. The apparatus according to claim 8, further comprising means for associating the observation target region specified by the first specifying means with the observation target region by the second specifying means using the position information of the observation target region specified by the first specifying means.

11. The apparatus according to claim 8, wherein the first specifying means and the second specifying means specify the observation target region based on a user's operation of designating the observation target region, a preset priority for the observation target region, and / or a priority preset by the user.

12. further comprising display means, wherein the output means outputs information on the observation target region extracted from the optical microscope image and the X-ray microscope image to the display means, and the display means displays the information on the observation target region side by side or superimposed. The apparatus according to claim 8.

13. The apparatus according to claim 8, comprising collation means for collating the optical microscope image and the X-ray microscope image by azimuth adjustment based on a rotation operation using a position marker when the observation target region specified by the first specifying means and the second specifying means includes the position marker.

14. A computer, first specifying means for specifying an arbitrary observation target region included in a biological sample from an X-ray microscope image of a wax block-embedded biological sample imaged using X-rays having an energy of 4 to 12 keV, An image obtained by imaging a part of the biological sample included in the image captured by the X-ray microscope with an optical microscope image, and a second specifying means for specifying a region corresponding to the observation target region specified by the first specifying means, A means for collating the information of the region specified by the first specifying means and the information of the region specified by the second specifying means, and An output means for outputting the collation result, A microscope image processing program for functioning as such.

Images