JP2007212276A - Thin slice manufacturing apparatus and method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a thin slice manufacturing apparatus capable of manufacturing a thin slice by automatically exposing a living body sample suitable for observation from an embedding block in which a biosample is embedded, and a thin slice manufacturing method. <P>SOLUTION: The thin slice manufacturing apparatus 1 includes a feed means 8 capable of cutting the embedding block B in a predetermined thickness and moving a cutter 4 in a thickness direction Y with respect to the embedding block B, a cutting means 3 for moving the cutter 4 in a cutting direction X with respect to the embedding block B to cut it, an observation means 12 for observing the cut surface B2 of the embedding block B cut by the cutting means 3 to acquire observation data and a control part 9 for setting the moving quantity due to the feed means 8 to set feed quantity to successively cut the embedding block B by the cutting means 3 and judging the quality of the cut surface B2 on the basis of the acquired observation data and altering the moving quantity due to the feed means 8 to main cutting quantity when the cut surface B2 is determined to be good. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a thin-slice preparation device and a method for preparing a thin slice by cutting an embedded block in which a biological sample taken out of a human body, a laboratory animal, or the like is embedded.

  Conventionally, as one of the methods for inspecting and observing biological samples taken from human bodies and laboratory animals, ultrathin slices with a thickness of several μm are prepared from embedded blocks in which biological samples are embedded with embedding agents. After that, a method of performing a staining process and observing is known. As a detail of the process for producing such a thin slice, first, as a rough cutting process, the embedded block is cut to expose the biological sample embedded therein to the cutting surface. Then, it is confirmed whether the exposed biological sample is suitable for observation. This is repeated when it is determined that a biological sample more suitable for observation is embedded below the current cutting surface. When it is determined that a biological sample suitable for observation is exposed on the cutting surface, the biological sample is cut by a cutter so as to have a predetermined thickness, that is, about 3 to 5 μm, as the main cutting process. Can be prepared.

  Conventionally, the work of producing a thin section from such an embedding block has been left to manual work by a skilled worker because the thin section is extremely thin and difficult to handle. On the other hand, for example, in preclinical studies, it is necessary to prepare hundreds of embedded blocks per test and several thin sections per embedded block. Automation of a series of processes is desired.

As what automates the production of such a thin section, for example, a cutter, a cutter driving mechanism that moves the cutter so that the surface of the embedding block can be cut by an amount corresponding to the thickness of a predetermined thin section, There has been proposed a thin-slice manufacturing apparatus having a sample transport mechanism that sends an embedded block in a cutting direction with respect to a cutter (see, for example, Patent Document 1). According to such a sliced piece manufacturing apparatus, the cutter is automatically moved so as to have a predetermined thickness by the cutter driving mechanism, and if the embedded block is sent by the sample transport mechanism, the embedded block is cut by the cutter, It is said that a thin section can be automatically produced.
JP 2004-28910 A

  However, in the apparatus and method according to Patent Document 1, a biological sample is included in the prepared thin section only by automatically cutting the embedded block to a predetermined thickness, and the biological sample included It is not possible to judge whether is suitable for observation. For this reason, as a pre-process for slicing the embedding block using such an apparatus, it is necessary to manually perform the rough cutting process as described above to expose a suitable cutting surface, and as a result, the thin slice The production was not automated.

  The present invention has been made in view of the above-described circumstances, and it is possible to produce a thin section by automatically exposing a biological sample suitable for observation from an embedded block in which the biological sample is embedded. A thin slice production apparatus and a thin slice production method are provided.

In order to solve the above problems, the present invention proposes the following means.
The present invention is a thin-slice preparation device that cuts an embedded block in which a biological sample is embedded to prepare a thin slice, and the embedded block can be cut at a predetermined thickness so that the embedded block can be cut into the embedded block. A cutting means for cutting the embedded block by moving the cutter relative to the embedding block relative to the embedding block and moving the cutter relative to the embedding block relative to the embedding block. Observing the cutting surface of the embedding block cut by the cutting means, the observation means for obtaining observation data, and setting the movement amount by the feeding means to a predetermined set feeding amount, The embedding block is sequentially cut by the cutting means, the quality of the cutting surface is judged based on the acquired observation data, and when the cutting surface is judged to be good, The amount of movement, is characterized in that it comprises a control unit for cutting by changing the main cutting amount corresponding to the thickness of the thin section from the set feed amount.

  Further, in the method for producing a thin section of the present invention, the cutter for cutting the embedded block is relatively set by a predetermined feed amount in the thickness direction with respect to the embedded block in which the biological sample is embedded. A rough cutting step of cutting the embedded block by moving the cutter in the cutting direction relative to the embedded block after the movement, and a cutting surface of the embedded block cut in the rough cutting step An observation process for observing the observation data, an evaluation process for judging the quality of the cutting surface based on the observation data acquired in the observation process, and the thickness of the cutter with respect to the embedded block After moving in the direction by the main cutting amount corresponding to the thickness of the thin slice to be manufactured, the embedded block is cut by moving in the cutter cutting direction relative to the embedded block. Book In the evaluation step, if the cutting surface is determined to be negative, the roughing step, the pre-observation step, and the evaluation step are performed again, and if the cutting surface is determined to be good, It shifts to the said main cutting process, It is characterized by the above-mentioned.

  According to the thin-slice manufacturing device and the thin-slice manufacturing method according to the present invention, first, as the roughing process, the moving amount by the feeding means is set to the set feeding amount, and the cutter is moved in the thickness direction with respect to the embedding block. Move relative. Then, by moving the cutter in the cutting direction relative to the embedding block by the cutting means, the embedding block can be cut at a thickness corresponding to the set feed amount. Next, as an observation step, the cutting surface of the embedding block is observed by the observation means, and observation data relating to the cutting surface is acquired. Next, as an evaluation process, the control unit determines the quality of the cut surface based on the observation data. When the cutting surface is “No”, that is, when it is determined that the portion of the biological sample exposed to the cutting surface is not suitable for observation, the roughing process is performed again to cut the embedded block, Observation data is acquired and the quality of a cutting surface is judged again as an evaluation process. When it is determined that the cutting surface is “good”, that is, the part of the biological sample exposed to the cutting surface is suitable for observation, the process proceeds to the main cutting step. In other words, by changing the amount of movement by the feeding means to the actual cutting amount and cutting the embedded block into thin slices of a predetermined thickness, thin slices containing biological samples suitable for observation are automatically Can be produced.

  Further, in the above-described thin section manufacturing apparatus, the observation unit includes an illumination system that illuminates the cutting surface of the embedded block, and an imaging optical system that photographs the cutting surface illuminated by the illumination system, More preferably, the observation data is image data photographed by the photographing optical system.

  According to the thin-section manufacturing apparatus according to the present invention, image data can be acquired as observation data by photographing the cutting surface illuminated by the illumination system with the photographing optical system. Since the color of the portion of the biological sample exposed to the cutting surface and the portion that is the embedding agent that embeds the biological sample is different, the biological sample exposed to the cutting surface is identified based on the image data, and the cutting surface is identified. Can be judged.

  In the thin-section manufacturing apparatus, the illumination system of the observation unit reflects the parallel light emitted from the light source and the light substantially parallel to the cutting surface. It is more preferable that the imaging optical system is provided behind the half mirror so as to be able to receive reflected light that is reflected by the cutting surface and transmitted through the half mirror. Yes.

  Further, in the method for producing a thin slice, the observation step includes reflecting parallel light irradiated from a light source by a half mirror to irradiate the cutting surface substantially orthogonally, and behind the half mirror, It is more preferable that the cut surface is photographed by receiving reflected light that is reflected by the cut surface and transmitted through the half mirror, and image data is acquired as the observation data.

  According to the thin-slice manufacturing apparatus and thin-slice manufacturing method according to the present invention, in the observation step, the parallel light irradiated from the light source of the illumination system is reflected by the half mirror, and is irradiated substantially orthogonally to the cutting surface. Illuminated as so-called epi-illumination. Since the photographing optical system is provided behind the half mirror, it is possible to photograph the cutting surface by receiving the reflected light from the epi-illumination. For this reason, the image data captured and acquired by the photographing optical system is an image in which the contrast between the portion where the biological sample is exposed and the portion which is the embedding agent is remarkable, and the biological sample is exposed more clearly. The part can be identified.

  Further, in the above-described thin-slice manufacturing apparatus, the control unit determines whether the cutting surface is good or bad based on brightness data obtained by binarization processing based on luminance information of the image data acquired by the observation unit. It is more preferable to judge this.

  Further, in the above method for producing a sliced piece, the evaluation step generates a light / dark data by binarization processing based on the luminance information of the image data acquired in the observation step, and the light / dark data is stored. In addition, it is more preferable to judge the quality of the cutting surface.

  According to the thin-slice manufacturing device and the thin-slice manufacturing method according to the present invention, in the observation process, based on the luminance information of the image data acquired by the observation means, binarization processing is performed to create light / dark data. By this, it is possible to more clearly identify the portion of the cutting surface where the biological sample is exposed and the portion that is the other embedding agent.

  Moreover, in the above-described thin-slice preparation device, the control unit is a tissue area that is an area of a portion of the cutting surface where the biological sample is exposed, based on the observation data acquired by the observation unit. It is said that it is more preferable to calculate the quality of the cutting surface based on the tissue area.

  Further, in the above method for producing a thin section, the evaluation step is a tissue area that is an area of a portion of the cutting surface where the biological sample is exposed, based on the observation data acquired in the observation step. It is said that it is more preferable to calculate the quality of the cutting surface based on the tissue area.

  According to the thin-slice manufacturing device and the thin-slice manufacturing method according to the present invention, in the evaluation step, the control unit calculates the tissue area that is the area of the portion of the cutting surface where the biological sample is exposed. The quality of the cut surface can be determined by quantitatively evaluating the size of the exposed part of the sample.

  In the thin-section manufacturing apparatus, the control unit includes the observation data acquired this time and the observation data acquired last time out of the observation data acquired every time the cutting unit cuts. It is said that it is more preferable to judge the quality of the cutting surface by comparison.

  Further, in the above method for producing a thin section, the evaluation step compares the observation data acquired in the current observation step with the observation data acquired in the previous observation step, and performs the cutting. It is more preferable to judge the quality of the surface.

  According to the thin-slice manufacturing device and the thin-slice manufacturing method according to the present invention, the observation data acquired this time in the observation process and the observation data acquired last time are compared each time the cutting means cuts in the rough cutting process. Thereby, the quality of a cutting surface can be judged relatively in the thickness direction of an embedding block. Therefore, the biological sample embedded in the embedding block is different in size and type for each embedding block, but can be determined relatively in the thickness direction of the embedding block, For each embedding block, it is possible to determine a cutting surface suitable for producing a thin section in the main cutting process.

  Moreover, in the above-described thin-slice preparation device, the control unit is a tissue area that is an area of a portion of the cutting surface where the biological sample is exposed, based on the observation data acquired by the observation unit. It is more preferable to judge whether the cutting surface is good or bad by comparing the tissue area calculated this time with the tissue area calculated last time.

  Further, in the above method for producing a thin section, the evaluation step may include a tissue area that is an area of a portion of the cutting surface where the biological sample is exposed from the observation data acquired in the observation step. It is more preferable to calculate and judge the quality of the cutting surface by comparing the tissue area calculated this time with the tissue area calculated last time.

  According to the thin-slice preparation device and thin-slice preparation method according to the present invention, in the evaluation step, the control unit calculates the tissue area, which is the area of the portion of the cutting surface where the biological sample is exposed, and is calculated this time. The calculated tissue area can be compared with the previously calculated tissue area. For this reason, the magnitude | size of the part which the biological sample has exposed can be evaluated comparatively and quantitatively in the thickness direction of an embedding block. That is, even if the size and type of the biological sample embedded in each embedded block used is different, a thin slice is prepared at a position where the tissue area increases in the thickness direction of the embedded block. Can do.

  Further, in the above-described thin-slice preparation device, the control unit is configured when an area increase rate that is a ratio of the tissue area calculated this time to the previously calculated tissue area is equal to or less than a predetermined set increase rate. It is more preferable to judge that the cutting surface is good.

  Further, in the above method for producing a thin section, the evaluation step is performed when the area increase rate that is a ratio of the tissue area calculated this time to the tissue area calculated last time is equal to or less than a predetermined set increase rate, It is more preferable to judge that the cutting surface is good.

  According to the thin-slice manufacturing apparatus and the method for manufacturing a thin-section according to the present invention, in the evaluation process, the control unit calculates an area increase rate that is a ratio of the tissue area calculated this time to the tissue area calculated last time. Can do. Since the biological sample embedded in the embedding block is obtained by excising a tissue such as a human body or an experimental animal, the shape thereof is a rounded lump. For this reason, the tissue area of the biological sample exposed to the cutting surface gradually increases from one end surface toward the center in the thickness direction, reaches the maximum tissue area at a certain position, and gradually decreases toward the other end surface. Go. Further, the area increase rate of the tissue area gradually decreases from the one end surface in the thickness direction toward the position where the tissue area becomes the maximum, and the area increase rate of the tissue area becomes zero at the position where the tissue area becomes the maximum. For this reason, by calculating the area increase rate of the tissue area by the control unit and judging whether it is equal to or less than a predetermined set increase rate, a thin slice is produced at a position close to the position where the maximum tissue area is obtained. be able to.

  Further, in the above-described thin-slice preparation device, the control unit is configured such that an area increase amount that is a difference of the tissue area calculated this time with respect to the tissue area calculated last time is equal to or less than a predetermined set increase amount. The cutting surface may be determined to be good.

  Further, in the above-described method for producing a thin section, the evaluation step is performed when an area increase amount that is a difference of the tissue area calculated this time with respect to the tissue area calculated last time is equal to or less than a predetermined set increase amount. The cutting surface may be determined to be good.

  According to the thin-slice preparation device and the method for preparing a thin-section according to the present invention, in the evaluation step, the control unit calculates an area increase amount that is a difference of the tissue area calculated this time with respect to the tissue area calculated last time. Can do. As described above, the area increase amount of the tissue area gradually decreases from one end surface in the thickness direction toward the position where the tissue area becomes the maximum, and the area increase amount of the tissue area becomes zero at the position where the tissue area becomes the maximum. Become. For this reason, by calculating the area increase amount of the tissue area by the control unit and judging whether it is equal to or less than a predetermined set increase amount, a thin slice is produced at a position close to the position where the maximum tissue area is obtained. be able to.

  In the thin-section manufacturing apparatus, the set feed amount includes a first set feed amount and a second set feed amount smaller than the first set feed amount. When the amount of movement by the feeding means is set to the first set feed amount to determine whether the cutting surface is good or not, and when the cutting surface is determined to be good, the amount of movement by the feeding means is set to the first amount. If the cutting surface is determined to be good or bad again and the cutting surface is determined to be good again, the amount of movement by the feeding means may be changed to the main cutting good. More preferred.

  In the thin slice manufacturing method, the roughing step includes a first roughing step in which the set feed amount is a first set feed amount, and the set feed amount is smaller than the first set feed amount. The second roughing process with a second set feed amount, and the evaluation process corresponds to the first evaluation process corresponding to the first roughing process and the second roughing process. A first evaluation step, wherein the first evaluation step performs the first roughing step and the observation step, and the quality of the cutting surface is determined based on the observation data acquired in the observation step. When the cutting surface is determined to be negative, the first roughing step, the observation step, and the first evaluation step are performed again, and when the cutting surface is determined to be good, Shift to the second roughing process and the second evaluation If the cutting surface is judged good or bad based on the observation data acquired in the observation step by performing the second rough cutting step and the observation step, When the second rough cutting step, the observation step, and the second evaluation step are performed and the cutting surface is determined to be good, it is more preferable to move to the main cutting step.

  According to the thin-slice manufacturing apparatus and the thin-slice manufacturing method according to the present invention, first, the moving amount by the feeding means is set to the first set feed amount that is larger than the second set feed amount, and the first roughing step By repeating the observation process and the first evaluation process, the embedded block can be cut efficiently. If the cutting surface is determined to be good in the first evaluation step, the amount of movement by the feeding means is set to a second set feed amount that is smaller than the first set feed amount, and the second roughing step, observation By repeating the process and the second evaluation process, a cutting surface suitable for observation of a biological sample can be confirmed with high accuracy. In the second roughing step, the second set feed amount, which is a movement amount smaller than the first set feed amount, is set, that is, the thickness of the embedding block to be cut in one cutting is made thinner. By setting, cutting resistance can be made smaller than in the first rough cutting step. For this reason, it can be set as the favorable cutting surface without fear of dropping-off of a structure | tissue, etc., and a more favorable thin section can be produced in the next main cutting process.

According to the thin-section manufacturing apparatus of the present invention, the rough cutting is sequentially performed by including an observation unit that observes the cutting surface of the embedding block cut by the cutting unit and a control unit that determines the quality of the cutting surface based on the observation data. , And a thin slice can be automatically produced with a cutting surface containing a biological sample suitable for observation.
Further, according to the method for producing a thin section of the present invention, the cutting surface of the embedding block cut in the roughing process is observed in the observation process, and the quality of the cutting surface is determined in the evaluation process based on the observation data. A cutting surface containing a biological sample suitable for observation can be automatically selected, and a thin slice can be produced in the main cutting process.

(First embodiment)
1 to 5 show a first embodiment according to the present invention. A thin-section preparation apparatus 1 shown in FIG. 1 prepares an ultrathin thin section having a thickness of about 3 to 5 μm from an embedded block B in which a biological sample S is embedded, and inspects the biological sample S contained in the thin section. In the process of observing, this is an apparatus for automatically roughing the embedded block B and further performing main cutting to produce a thin slice from the embedded block B. The biological sample S is, for example, a sample exhibiting a lump shape excised from a tissue such as an organ taken out of a human body or a laboratory animal, and is appropriately selected in the medical field, pharmaceutical field, food field, biological field, and the like. . The embedding block B is obtained by embedding the biological sample S as described above with the embedding agent B1, that is, covering and solidifying the periphery. In more detail, such an embedding block B is produced as follows. First, the mass of the biological sample S is immersed in formalin, and the protein constituting the biological sample S is fixed. And after making a structure | tissue solid, it cut | disconnects to a suitable magnitude | size. Finally, it is produced by embedding and solidifying a material obtained by replacing the moisture in the cut biological sample S with the embedding agent B1. Here, the embedding agent B1 is a material that can be easily liquefied and cooled and solidified as described above, and is dissolved by being immersed in an organic solvent, such as a resin or paraffin. Hereinafter, the configuration of the thin-slice manufacturing apparatus 1 will be described.

  As shown in FIGS. 1 and 2, the thin-section preparation apparatus 1 includes a sample stage 2 that positions and holds an embedding cassette C that fixes the embedding block B, and a cutting means 3 that cuts the embedding block B. It has. The cutting means 3 includes a cutter 4 and an X stage 5 that moves the sample table 2 in the cutting direction X with respect to the cutter 4. The cutter 4 is fixed to the cutter table 7 by a holder 6. For this reason, by driving the X stage 5, the embedded block B can be cut by moving the cutter 4 relative to the embedded block B. The thin-slice manufacturing apparatus 1 includes a Y stage 8 that is a feeding unit that moves the sample stage 2 by a predetermined movement amount in the thickness direction Y of the thin slice to be manufactured. By driving the Y stage 8, the cutter 4 can be moved relative to the embedded block B in the thickness direction Y, and the thickness when the embedded block B is cut by the cutter 4 is set. be able to.

  The thin-slice manufacturing apparatus 1 includes a computer 10 and a stage controller 11 as a control unit 9, and the X stage 5 of the cutting unit 3 and the Y stage 8 of the feeding unit are controlled by the computer 10. 11 is movable at a predetermined amount and at a predetermined speed. The moving speed of the X stage 5 that is the cutting speed of the embedding block B by the cutter 4 is preset in the computer 10. Further, the computer 10 has a main cutting corresponding to the set feed amount P suitable for roughing the embedding block B and the thickness of the thin slice at the time of main cutting as the amount of movement of the Y stage 8 per one time. The quantity Q is predetermined. In the present embodiment, for example, a thin slice having a thickness of 5 μm is manufactured, the main cutting amount Q is 5 μm, and the set feed amount P is 20 μm.

  Further, the thin-slice manufacturing device 1 includes an observation unit 12 that observes the cutting surface B2 of the embedding block B formed by cutting with the cutting unit 3 and acquires image data D as observation data. The observation means 12 includes an illumination system 13 that illuminates the cutting surface B2 of the embedding block B, and a photographing optical system 14 that photographs the cutting surface B2 illuminated by the illumination system 13. The illumination system 13 includes a surface emitting source 15 that is a light source capable of irradiating parallel light L, and a half mirror 16 that has a reflecting surface 16 a and is disposed above the embedding block B. The parallel light L irradiated from the surface light source 15 is reflected by the reflecting surface 16a of the half mirror 16, and is irradiated substantially orthogonally to the cutting surface B2, so that the cutting surface B2 can be illuminated as a so-called epi-illumination. 15 and the position of the half mirror 16 are set. The photographing optical system 14 includes a camera 17 provided behind the half mirror 16 and an image acquisition circuit 18 connected to the camera 17. That is, the parallel light L irradiated to the cutting surface B2 of the embedding block B by the illumination system 13 is reflected by the cutting surface B2, a part of which passes through the half mirror 16, and is received by the camera 17. For this reason, the camera 17 can photograph the cutting surface B2 of the embedding block B by epi-illumination, and can convert the image data D into data as image data D by the image acquisition circuit 18. To the computer 10. Then, the computer 10 of the control unit 9 determines the quality of the cutting surface B2 of the embedding block B based on the image data D, and produces a thin slice in the main cutting process based on the determination result. The details will be described below.

  As shown in FIG. 1, first, the embedding block B fixed to the embedding cassette C is placed on the sample stage 2. Then, as shown in FIG. 2, a rough cutting step S1 is performed. In the rough cutting step S1, first, the Y controller 8 is driven by the stage controller 11 under the control of the computer 10 of the controller 9, and the embedded block B is moved in the Y direction by the set feed amount P (20 μm). (Step S1a). Further, the X stage 5 is driven by the stage controller 11 to move the embedding block B in the X direction, that is, toward the cutter 4 at a predetermined moving speed. Thereby, the embedded block B can be cut by the cutter 4 by a thickness (20 μm) corresponding to the set feed amount P, and a cutting surface B2 is formed in the cut portion (step S1b).

  Next, as the total cutting amount confirmation step S2, the computer 10 of the control unit 9 confirms whether the total cutting amount Ps of the embedded block B is equal to or less than the cutting limit value Pc. Here, the total cutting amount Ps is an amount representing the total thickness of the embedding blocks B cut by repeating the rough cutting step S1. Further, the cutting limit value Pc is a value determined from, for example, the height of the embedding block B, and is suitable for observation when it is cut beyond this cutting limit value Pc. This is a numerical value representing the limit at which a thin slice containing the biological sample S cannot be produced, and is a value set in advance in the computer 10 of the control unit 9. That is, if the total cutting amount Ps does not exceed the cutting limit value Pc, it is determined that the embedded block B is in a state where cutting is possible, and the process proceeds to the next step. When the total cutting amount Ps exceeds the cutting limit value Pc, it is determined that if the cutting is further performed, it becomes impossible to produce a thin slice including the biological sample S, and the whole process is completed. .

  Next, as an observation step S3, the cutting surface B2 is observed by the observation means 12. That is, the parallel light L is illuminated from the surface light source 15 that is the illumination system 13 onto the cutting surface B2 of the embedding block B, and is photographed by the camera 17 of the photographing optical system 14. The image obtained by photographing is photographed in a colored portion of the cutting surface B2 where the biological sample S is exposed, and in a white portion of the portion that is the embedding agent B1 covering the periphery. At this time, by providing the half mirror 16 as the illumination system 13, the illumination of the surface light source 15 can be used as epi-illumination, and a part of the reflected light transmitted through the half mirror 16 can be received and photographed. For this reason, the acquired image can be an image having a remarkable contrast between the portion where the biological sample S is exposed and the portion which is the embedding agent B1. Then, the captured image is transmitted to the image acquisition circuit 18, converted into data as image data D in the image acquisition circuit 18, and input to the computer 10 of the control unit 9.

  Next, as the evaluation step S4, the computer 10 of the control unit 9 determines whether the cutting surface B2 is good or not based on the input image data D. First, in the acquired image data D, a portion where the biological sample S is exposed is identified. At this time, light / dark data is created by performing binarization processing based on the luminance information of the image data D. Thereby, the portion where the biological sample S is exposed is “black”, the portion which is the embedding agent B1 is “white”, and the portion where the biological sample S is exposed can be identified more clearly. . Based on the brightness data, first, it is confirmed whether or not there is a portion where the biological sample S is exposed (step S4a). At this stage, if the portion where the biological sample S is exposed cannot be confirmed, the portion including the biological sample S has not been cut yet, so the roughing step S1 is performed again. If the portion where the biological sample S is exposed can be confirmed, the tissue area A, which is the area of the portion where the biological sample S is exposed, is calculated and stored in the computer 10 (step S4b).

 Next, the area increase rate R of the tissue area A is calculated, and it is confirmed whether the area increase rate R is equal to or less than a predetermined set increase rate Rc (step S4c). The area increase rate R is a ratio of the tissue area A calculated this time to the tissue area A calculated last time and stored in the computer 10. When a portion where the biological sample S is first exposed is confirmed, there is no data of the tissue area A calculated last time, so the process automatically returns to the roughing step S1, and again the roughing step S1, The total cutting amount confirmation step S2, the observation step S3, and the evaluation step S4 are repeated. Since the previous tissue area A is stored in the computer 10 after the second time, the calculated area increase rate R is compared with the set increase rate Rc in step S4c. Then, when the calculated area increase rate R is larger than the set increase rate Rc, the rough cutting step S1 is performed again.

  FIG. 3 shows a cross-sectional view of the embedding block B, and FIG. 4 shows an example of the relationship between the calculated tissue area A and the total cutting amount Ps when the rough cutting step S1 is repeatedly performed by the above flow. It is a graph to represent. As shown in FIG. 3, since the biological sample S embedded in the embedded block B is obtained by excising a tissue such as a human body or a laboratory animal, the shape thereof is a rounded lump. Therefore, as shown in FIGS. 3 and 4, the tissue area A of the biological sample S exposed to the cutting surface B2 has a total cutting amount Ps of Ps1, Ps2 in the thickness direction Y from the one end surface B3 toward the center. , Ps3, and Ps4, the tissue area A gradually increases to A1, A2, A3, and A4, and reaches a certain position (the maximum tissue area Amax at the total cutting amount Psmax. The tissue area A gradually decreases toward the end surface B4, and the area increase rate R of the tissue area A gradually decreases from the one end surface B3 to the maximum tissue area Amax in the thickness direction Y. The area increase rate R of the tissue area A is zero at the position where the tissue area Amax is reached.

  That is, when the calculated area increase rate R is equal to or less than a predetermined set increase rate Rc, the tissue area A in the cutting surface B2 means a value close to the maximum tissue area A. . For this reason, the control unit 9 determines that the cutting surface B2 is “good” when the area increase rate R is equal to or less than the set increase rate Rc, so that the tissue area is relatively in the thickness direction Y of the embedded block B. By increasing A, a thin slice containing a biological sample S suitable for observation can be produced. The set increase rate Rc is a numerical value that can be freely set as with the set feed amount P. By reducing the set increase rate Rc and the set feed amount P, the set amount of increase Rc depends on the position where the biological sample S has the maximum tissue area. Thin sections can be produced at close positions.

  When the area increase rate R is smaller than the set increase rate Rc, the formed cutting surface B2 is determined as “good”, and the process proceeds to the main cutting step S5. In the main cutting step S5, the moving amount of the Y stage 8 is changed from the set feed amount P to the main cutting amount Q by the computer 10 of the control unit 9, and the embedding block B is moved in the Y direction. Then, as shown in FIG. 5, by moving the X stage 5, the tissue area A is increased by slicing the embedding block B into a thin slice T having a predetermined thickness by the cutter 4. Thus, a thin slice T containing a biological sample S suitable for observation can be produced.

  As described above, according to the thin section manufacturing apparatus 1, the cutting surface B2 is based on the observation means 12 for observing the cutting surface B2 of the embedding block B cut by the cutting means 3 and the image data D that is the observation data. By providing the control unit 9 for judging whether the quality is good or bad, the rough cutting step S1 is sequentially performed, the cutting surface B2 is observed in the observation step S3, and the cutting surface is evaluated in the evaluation step S4 based on the image data D which is the observation data. The quality of B2 can be determined. For this reason, a thin slice can be produced automatically with the cutting surface B2 containing the biological sample S suitable for observation. Further, by using the observation data as the image data D photographed by the photographing optical system 14, based on the fact that the color of the portion where the biological sample S is exposed and the portion which is the embedding agent B 1 are different. The sample S can be identified, and further can be identified more accurately by using the binarized light and dark data. Further, every time cutting is performed in the rough cutting step S1, the tissue area A is calculated, and the area increase rate R is calculated, so that the tissue area A calculated last time can be compared. The quality of the cutting surface B2 can be determined relatively and quantitatively in the thickness direction Y. For this reason, the biological sample S embedded in the embedded block B is different in size and type for each embedded block B, but the tissue area A is increased as much as possible for each embedded block B. Thus, the cutting surface B2 is automatically determined, and the thin slice T including the biological sample S suitable for observation can be produced.

(Second Embodiment)
1 and 6 show a second embodiment according to the present invention. In this embodiment, members that are the same as those used in the above-described embodiment are assigned the same reference numerals, and descriptions thereof are omitted.

  As shown in FIG. 1, in the thin-slice manufacturing apparatus 20 of this embodiment, the computer 10 has a first set feed amount P1 and a second set feed amount P1 as the set feed amount P of the Y stage 8 when roughing is performed. A set feed amount P2 is set. The second set feed amount P2 is set to a movement amount smaller than the first set feed amount P1, and in the present embodiment, the first set feed amount P1 is set to 100 μm, and the second set feed amount is set to 100 μm. P2 is set to 20 μm. The roughing process includes a first roughing process S11 performed at the first set feed amount P1 and a second roughing process S15 performed at the second set feed amount P2. The evaluation process also includes a first evaluation process S14 corresponding to the first roughing process S11 and a second evaluation process S18 corresponding to the second roughing process S15. Further, the computer 10 of the control unit 9 determines the first set increase rate Rc1 when determining the quality of the cutting surface B2 in the first evaluation step S14 and the quality of the cutting surface B2 in the second evaluation step S18. A second set increase rate Rc2 for determination is set, and the second set increase rate Rc2 is set to a smaller value than the first set increase rate Rc1.

  That is, as shown in FIG. 6, first, as the first roughing step S11, the Y stage 8 is driven by the stage controller 11 under the control of the computer 10 of the control unit 9, and the embedding block B is moved to the first. Is moved in the Y direction by the set feed amount P1 (100 μm) (step S11a). Further, by driving the X stage 5 by the stage controller 11, the embedded block B is cut by the cutter 4 by a thickness (100 μm) corresponding to the first set feed amount P1. (Step S1b). At this time, since the moving amount is larger than the set feed amount P (20 μm) in the first embodiment, the embedded block B can be efficiently cut to expose the biological sample S.

  Next, as the total cutting amount confirmation step S12, the computer 10 of the control unit 9 confirms whether the total cutting amount Ps of the embedded block B is equal to or less than the cutting limit value Pc. If the total cutting amount Ps does not exceed the cutting limit value Pc, it is determined that the embedded block B is in a state where cutting is possible, and the process proceeds to the next step. When the total cutting amount Ps exceeds the cutting limit value Pc, it is determined that the thin slice including the biological sample S cannot be produced if further cutting is performed, and the entire process ends.

  Next, as the observation step S <b> 3, the cutting surface B <b> 2 is observed by the observation unit 12, and the acquired image data D is input to the computer 10 of the control unit 9. Next, as the first evaluation step S14, the computer 10 of the control unit 9 determines whether the photographed cutting surface B2 is good or not based on the input image data D. As in the first embodiment, binarization processing is performed based on the luminance information of the acquired image data D to create light / dark data. Based on the light / dark data, first, it is confirmed whether or not there is a portion where the biological sample S is exposed (step S14a). At this stage, when the portion where the biological sample S is exposed cannot be confirmed, the first roughing step S11 is performed again. If the portion where the biological sample S is exposed can be confirmed, the tissue area A, which is the area of the portion where the biological sample S is exposed, is calculated and stored in the computer 10 (step S14b).

  Next, the area increase rate R of the tissue area A is calculated, and it is confirmed whether or not the area increase rate R is equal to or less than a predetermined first set increase rate Rc1 (step S14c). When a portion where the biological sample S is exposed is confirmed first, there is no data of the tissue area A calculated last time, so the process automatically returns to the first roughing step S11 and again the first The rough cutting step S11, the total cutting amount confirmation step S12, the observation step S13, and the first evaluation step S14 are repeated. Since the previous tissue area A is stored in the computer 10 after the second time, the calculated area increase rate R is compared with the first set increase rate Rc1 in step S14c. When the calculated area increase rate R is larger than the first set increase rate Rc1, that is, when it is determined that the cutting surface B2 is “No”, the rough cutting step S1 is performed again. In the first evaluation step S14, when the calculated area increase rate R is smaller than the first set increase rate Rc1, that is, the cutting surface B2 is determined to be “good”, the second roughing step S15. Migrate to

  In the second rough cutting step S15, the movement amount of the Y stage 8 is reset to the second set feed amount P2 (20 μm), and the embedded block B is cut. At this time, since the second set feed amount P2 (20 μm) is set to a movement amount smaller than the first set feed amount P1 (100 μm), the embedded block B can be cut with high accuracy. . Similarly, it is confirmed whether the total cutting amount Ps does not exceed the cutting limit value Pc in the total cutting amount confirmation step S16. If not, the cutting surface B2 is observed in the observation step S17. Next, as the second evaluation step S18, brightness / darkness data is created from the image data D by the computer 10 of the control unit 9, and it is confirmed whether or not there is a portion where the biological sample S is exposed (step S18a). When the portion where the biological sample S is exposed can be confirmed, the tissue area A that is the area of the portion where the biological sample S is exposed is calculated and stored in the computer 10 (step S18b).

  Next, the area increase rate R is calculated from the calculated tissue area A and the previously calculated tissue area A, and compared with the second set increase rate (step S18c). In the first second evaluation step S18, the tissue area A calculated in the last first evaluation step S14 is calculated as the previous tissue area A. When the calculated area increase rate R is larger than the second set increase rate Rc2, the cutting surface B2 is determined as “No”, and the second roughing step S15, the total cutting amount confirmation step S16, The observation step S17 and the second evaluation step S18 are repeated. When the calculated area increase rate R is smaller than the second set increase rate Rc2, the cutting surface B2 is determined to be “good” and the process proceeds to the main cutting step S5.

  As described above, in the thin-slice manufacturing device 20 of the present embodiment, the embedded block can be efficiently cut by the first set feed amount as the first roughing process. Then, when it is determined that the cutting surface B2 is “good” in the first evaluation step S14, the second setting is the second set amount of movement smaller than the first set feed amount P1 as the second rough cutting step S15. By resetting the feed amount P2, the cutting surface B2 suitable for observation of the biological sample S can be confirmed with high accuracy. In addition, by setting the second set feed amount, which is a movement amount smaller than the first set feed amount, the thickness of the embedding block cut in one cutting can be reduced, and the cutting resistance Becomes smaller. For this reason, it can be set as the favorable cutting surface without fear of dropping-off of a structure | tissue, etc., and a more favorable thin section can be produced in the next main cutting process.

  As mentioned above, although embodiment of this invention was explained in full detail with reference to drawings, the concrete structure is not restricted to this embodiment, The design change etc. of the range which does not deviate from the summary of this invention are included.

  In this embodiment, the quality of the cutting surface B2 is determined by comparing the area increase rate R, which is the ratio of the tissue area A calculated before and after, and the set increase rate Rc, but is not limited thereto. It is not a thing. For example, when the area increase amount that is the difference between the tissue area A calculated this time and the tissue area A calculated last time is equal to or less than a predetermined set increase amount, the control unit 9 determines that the cutting surface is “good”. It is good also as what judges. Similarly to the set increase rate Rc, the area increase amount of the tissue area A gradually decreases from the one end surface in the thickness direction toward the position where the maximum tissue area Amax is reached, and the tissue area at the position where the maximum tissue area Amax is reached. The area increase amount of A becomes zero. For this reason, the control unit 9 calculates the area increase amount of the tissue area A and compares it with a predetermined increase amount, thereby producing a thin slice at a position close to the position where the maximum tissue area A is obtained. be able to. Moreover, it is good also as what judges the quality of the cutting surface B2 based on the area change rate which remove | divided said area increase amount by setting feed amount. In this case, even if the set feed amount is changed in the middle, there is an advantage that it can be continuously determined based on the same determination criterion. Further, the above-described method for determining the cutting surface based on the area increase rate, the area increase amount, and the like is based on a comparison of the front and back tissue areas. For example, the absolute amount of the required tissue area is specified. When necessary, it may be determined by directly comparing the prescribed amount with the calculated tissue area.

  In addition, as described above, the control unit 9 determines whether the cutting surface B2 is good or not based on the tissue area A of the portion of the image data D where the biological sample S is exposed. Not. For example, pattern matching may be performed on the exposed part of the biological sample S using the image data D obtained before and after. In this case, the acquired image data D is divided into a certain size, and it is determined whether or not the images of the preceding and following image data D match for each divided portion. Then, it is determined whether or not there is a portion where the images do not coincide with each other, and if the ratio is within a certain value, the cutting surface B2 is “good”, and if it exceeds a certain value, What is necessary is just to judge cutting surface B2 as "no".

  In the observation means, the light source of the illumination system is the surface light source 15. However, for example, light from a point light source may be converted into parallel light through a pinhole and a collimator lens. Further, not only the parallel light but also the observation of the cutting surface B2 by irradiating the scattered light can determine the quality of the cutting surface B2. The cutting means 3 includes an X stage 5 and moves the embedding block B in the cutting direction X with respect to the cutter 4. The feeding means includes a Y stage 8 and the embedding block with respect to the cutter 4. Although B is assumed to be moved in the thickness direction Y, both have the same effect even when the cutter 4 is moved.

1 is an overall view of a thin-slice manufacturing apparatus according to a first embodiment of the present invention. It is a flowchart of thin section preparation in the thin section preparation apparatus of 1st Embodiment of this invention. It is sectional drawing of the embedding block of 1st Embodiment of this invention. It is a graph which shows the relationship between the structure | tissue area of 1st Embodiment of this invention, and cutting total amount. It is explanatory drawing in the main cutting process of 1st Embodiment of this invention. It is a flowchart of thin section preparation in the thin section preparation apparatus of 2nd Embodiment of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1,20 Thin section production apparatus 2 Sample stand 3 Cutting means 4 Cutter 5 X stage 8 Y stage (feed means)
DESCRIPTION OF SYMBOLS 9 Control part 12 Observation means 13 Illumination system 14 Imaging optical system 15 Surface light source 16 Half mirror A Tissue area B Embedded block B2 Cutting surface P Set feed amount P1 First set feed amount P2 Second set feed amount Q book Cutting amount R Area increase rate Rc Set increase rate Rc1 First set increase rate Rc2 Second set increase rate S Biological sample T Thin section X Cutting direction Y Thickness direction S1 Roughing step S3 Observation step S4 Evaluation step S5 Main cutting step S11 First roughing step S13 Observation step S14 First evaluation step S15 Second roughing step S17 Observation step S18 Second evaluation step S19 Main cutting step

Claims (19)

A thin-section preparation device that cuts an embedded block in which a biological sample is embedded to prepare a thin section,
A feeding means for moving the cutter relative to the embedded block by a predetermined amount of movement in the thickness direction so that the embedded block can be cut at a predetermined thickness;
Cutting means for moving the cutter relative to the embedding block in a cutting direction to cut the embedding block;
Observation means for observing the cutting surface of the embedded block cut by the cutting means, and obtaining observation data;
The moving amount by the feeding unit is set to a predetermined set feeding amount, and the embedded block is sequentially cut by the cutting unit, and the quality of the cutting surface is determined based on the obtained observation data. And when the cutting surface is determined to be good, a control unit for cutting by changing the moving amount by the feeding means from the set feeding amount to a main cutting amount corresponding to the thickness of the thin section; A thin-slice manufacturing apparatus comprising: In the thin-slice preparation device according to claim 1,
The observation means includes an illumination system that illuminates the cutting surface of the embedded block;
A photographing optical system for photographing the cutting surface illuminated by the illumination system;
The thin slice preparation apparatus, wherein the observation data is image data photographed by the photographing optical system. In the thin-slice preparation device according to claim 2,
The illumination system of the observation means includes a light source capable of irradiating parallel light,
A half mirror that reflects the parallel light emitted from the light source and irradiates the cutting surface substantially orthogonally;
The thin-section manufacturing apparatus, wherein the photographing optical system is provided behind the half mirror so as to be able to receive reflected light reflected by the cutting surface and transmitted through the half mirror. In the thin-section preparation apparatus according to claim 2 or claim 3,
The control unit determines whether the cutting surface is good or not based on brightness data obtained by binarization processing based on luminance information of the image data acquired by the observation unit. Production device. In the thin-slice preparation device according to any one of claims 1 to 4,
The control unit calculates a tissue area that is an area of a portion of the cutting surface where the biological sample is exposed, based on the observation data acquired by the observation unit, and based on the tissue area A thin-slice manufacturing apparatus that determines whether the cutting surface is good or bad. In the thin-slice preparation device according to any one of claims 1 to 4,
The control unit compares the observation data acquired this time with the observation data acquired last time out of the observation data acquired every time the cutting unit cuts, and determines the quality of the cutting surface. A thin-slice manufacturing apparatus characterized by: The thin-section preparation apparatus according to claim 6,
Based on the observation data acquired by the observation means, the control unit calculates a tissue area that is an area of a portion of the cutting surface where the biological sample is exposed, and the tissue calculated this time A thin-slice manufacturing apparatus that compares the area with the previously calculated tissue area to determine whether the cutting surface is good or bad. In the thin-section preparation apparatus according to claim 7,
The control unit determines that the cutting surface is good when an area increase rate, which is a ratio of the tissue area calculated this time to the previously calculated tissue area, is equal to or less than a predetermined set increase rate. A thin-section preparation apparatus characterized by the above. In the thin-section preparation apparatus according to claim 7,
The control unit determines that the cutting surface is good when an area increase amount which is a difference between the tissue area calculated this time and the tissue area calculated last time is equal to or less than a predetermined set increase amount. A thin-section preparation apparatus characterized by the above. In the thin-slice preparation device according to any one of claims 1 to 9,
The set feed amount is composed of a first set feed amount and a second set feed amount smaller than the first set feed amount,
The control unit sets the amount of movement by the feed unit to the first set feed amount, determines whether the cutting surface is good, and if the cutting surface is determined to be good, When the amount of movement is reset to the second set feed amount, the quality of the cutting surface is judged again, and when the cutting surface is judged to be good again, the amount of movement by the feeding means is reduced to the main cutting. A thin-slice preparation device characterized by changing the amount. After moving the cutter for cutting the embedded block relative to the embedded block in which the biological sample is embedded by a predetermined set amount in the thickness direction, Roughing step of cutting the embedded block by moving the cutter relative to the cutting direction;
An observation step of observing the cutting surface of the embedded block cut in the rough cutting step to obtain observation data;
Based on the observation data acquired in the observation step, an evaluation step for determining the quality of the cutting surface;
The cutter is moved relative to the embedded block in the thickness direction by a main cutting amount corresponding to the thickness of the thin slice to be manufactured, and then relative to the embedded block in the cutter cutting direction. And a main cutting step of cutting the embedded block by moving to,
In the evaluation step, when it is determined that the cut surface is not, the roughing step, the pre-observation step, and the evaluation step are performed again.
A thin slice manufacturing method, wherein when the cut surface is judged good, the process proceeds to the main cutting step. The method for producing a thin section according to claim 11,
In the observation step, parallel light irradiated from a light source is reflected by a half mirror to irradiate the cutting surface substantially orthogonally, and is reflected by the cutting surface behind the half mirror and transmitted through the half mirror. A method for producing a thin slice, comprising: capturing the cut surface by receiving reflected light, and obtaining image data as the observation data. The method for producing a thin section according to claim 12,
The evaluation step creates light and dark data by binarization processing based on the luminance information of the image data acquired in the observation step, and determines the quality of the cutting surface based on the light and dark data A method for producing a thin section characterized by the following. In the method for producing a thin section according to any one of claims 11 to 13,
In the evaluation step, based on the observation data acquired in the observation step, a tissue area that is an area of a portion of the cutting surface where the biological sample is exposed is calculated, and the tissue area is calculated based on the tissue area. A method for producing a thin section, wherein the quality of a cut surface is judged. In the method for producing a thin section according to any one of claims 11 to 13,
The evaluation step compares the observation data acquired in the current observation step with the observation data acquired in the previous observation step, and determines whether the cutting surface is good or bad. Thin section preparation method. The method for producing a thin section according to claim 15,
The evaluation step calculates, from the observation data acquired in the observation step, a tissue area that is an area of a portion of the cutting surface where the biological sample is exposed, and the tissue area calculated this time A method for producing a thin slice, wherein the quality of the cut surface is judged by comparing the tissue area calculated last time. The method for producing a thin section according to claim 16,
The evaluation step determines that the cutting surface is good when an area increase rate that is a ratio of the tissue area calculated this time to the tissue area calculated last time is equal to or less than a predetermined set increase rate. A method for producing a thin slice. The method for producing a thin section according to claim 16,
The evaluation step determines that the cutting surface is good when an area increase amount that is a difference of the tissue area calculated this time with respect to the tissue area calculated last time is equal to or less than a predetermined set increase amount. A method for producing a thin slice. In the method for producing a thin section according to any one of claims 11 to 18,
The rough cutting step includes a first rough cutting step in which the set feed amount is set as a first set feed amount, and a second set feed amount in which the set feed amount is set to be a second set feed amount smaller than the first set feed amount. It consists of a roughing process and
The evaluation step includes a first evaluation step corresponding to the first roughing step, and a second evaluation step corresponding to the second roughing step,
The first evaluation step performs the first rough cutting step and the observation step, performs the quality of the cutting surface based on the observation data acquired in the observation step, and determines that the cutting surface is not acceptable. In that case, the first roughing step, the observation step and the first evaluation step are performed again, and when it is determined that the cutting surface is good, the process proceeds to the second roughing step,
The second evaluation step performs the second rough cutting step and the observation step, performs the quality of the cutting surface based on the observation data acquired in the observation step, and determines that the cutting surface is not acceptable. In that case, the second roughing step, the observation step, and the second evaluation step are performed again, and when the cutting surface is judged to be good, the process proceeds to the main cutting step. Thin section preparation method.

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