JP4849405B2 - Automatic slicing device and automatic slicing method - Google Patents

Description

  The present invention relates to an automatic slicing apparatus and an automatic slicing method used when preparing a sliced specimen used for physics and chemistry experiments and microscopic observation.

Thin slice specimens used for physics and chemistry experiments and microscopic observations are obtained by fixing a thin slice having a thickness of several μm (for example, 3 μm to 5 μm) on a substrate such as a slide glass.
This thin-section specimen is prepared from an embedded block in a block state prepared by substituting a paraffin-substituted biological sample such as a formalin-fixed organism or animal and further solidifying the periphery with paraffin. That is, first, the embedding block is set on a microtome, which is a dedicated slicing device, and rough cutting is performed. By this rough cutting, the surface of the embedding block is made smooth, and the embedded biological sample which is the object of experiment and observation is exposed on the surface. After finishing this rough cutting, the cutting blade of the microtome performs the main cutting to slice the embedding block into the above-mentioned thickness and obtains a thin slice.

  By the way, the prepared thin slice specimen is observed after the staining process. However, when a lesion or the like is found in the biological sample contained in the thin section, it is necessary to further investigate the biological specimen. Come out. That is, it is necessary to perform additional observation on a biological sample, for example, by performing a staining process such as staining only a specific protein or staining RNA. At that time, a new thin section specimen must be prepared separately from the thin section specimen used at present, and therefore, the thin specimen sample block once removed from the cutting table is set on the cutting table again. In addition, it is necessary to collect a plurality of continuous thin sections from the already cut surface of the sample block.

  Conventionally, as one method of collecting a plurality of thin slices from a sample block from which a thin slice has been removed from the cutting table, the thin sample collected sample block is set again on the microtome cutting table and actually The cutting surface of the sample block is changed to the virtual cutting surface of the knife by manually changing the inclination of the sample block holder through the holder inclination angle varying means while slicing with a knife and observing the shape of the cutting surface. The so-called surface alignment, which is arranged so as to be parallel, is performed, and then the thin slice is cut.

  However, with such a method of sampling a thin section, a preliminary thin cutting (rough cutting) of about several hundred μm is required to obtain a predetermined cutting surface, and the position is several hundred μm away from the existing cutting surface. There was a problem that only thin sections could be collected. In addition, since the mating is basically performed manually, it takes time for a worker who cannot work, and it is very painful.

In order to deal with such problems, when the feed block is fed to the cutting surface via the power drive device and the sample block surface comes into contact with the knife, the feed stand is moved to the direction of the surface sensor via the power drive device. An automatic slicing device is disclosed that detects when the surface of the sample block and the surface sensor are in contact with each other, and determines the feed amount of the sample block based on these two detection values (for example, Patent Document 1). reference.).
As another countermeasure, a sample holder that can be oriented and aligned (an automatic slicing device that indicates the position of the sample block holder is disclosed (for example, see Patent Document 2).

Special Table 2002-539424 JP 2004-354389 A

  However, the technique described in Patent Document 1 is a method in which a knife or a surface sensor is brought into contact with the already-cut surface of the sample block for detection, and the already-cut surface of the sample block may be damaged. Moreover, in the technique described in Patent Document 2, when a sample block from which a thin section has been collected is set in the apparatus again, although it can be relatively easily matched, for example, roughing and main cutting are performed. There is a problem that face-to-face matching is not possible when set in different devices, such as using different devices.

  The present invention has been made in view of the above circumstances, and an automatic slicing apparatus capable of continuously collecting a thin slice having a predetermined thickness from a cutting surface even if a sample block is once removed from a cutting table, and An object is to provide an automatic slicing method.

The present invention employs the following means in order to solve the above problems.
An automatic slicing device according to the present invention includes a cutting unit that cuts a thin section from a rough- cut sample block in which a sample is embedded, and cutting that places the sample block and tilts the sample block with respect to a horizontal plane. A table, a tilt angle control unit that instructs the cutting table to control a tilt angle of the sample block with respect to the horizontal plane, a moving unit that relatively moves the cutting unit and the cutting table, and the cut sample block a measuring unit for measuring the coordinate position of a point on the cutting surface of the current, from the measured values of the coordinate position of the at least three points located in two or more straight line crossing each other on the cutting surface of the current, the rough calculating a relative position of the cutting surface of the current with respect to the previously stored virtual plane as a cutting surface at the end, for controlling the tilt angle control unit to match the cutting surface of the current to the virtual plane calculating unit , Characterized in that it comprises.

In the automatic slicing method according to the present invention, the rough- cut sample block once removed from the cutting table is attached to the cutting table again, and thin sections are continuously collected from the already-cut surface of the sample block by the cutting unit. An automatic slicing method, measuring a coordinate position of at least three points located on two or more straight lines intersecting each other on the current cutting surface of the sample block reattached to the cutting table; A cutting surface position calculating step for calculating a relative position of the current cutting surface with respect to a virtual plane stored in advance as a cutting surface at the end of the rough cutting from the measured values of the coordinate positions of the at least three points, and the current cutting surface And a cutting surface adjustment step of matching the virtual plane with the virtual plane.

  In the present invention, when the cutting surface is aligned when the sample block once removed from the cutting table is mounted on the cutting table again, the coordinate position of at least three points on the cutting surface after mounting is measured by the measuring unit. The coordinate position of the current cutting surface is calculated. And the coordinate position of the cutting surface before and behind removal is compared, and the difference is calculated. As a result, even if the coordinate position of the cutting surface is deviated before and after removal, the position of the cutting surface before and after cutting is matched by tilting the cutting table with the tilt angle control unit based on the instruction from the calculation unit for the difference. Can be made.

Moreover, the automatic slicing apparatus according to the present invention is the automatic slicing apparatus, wherein the measurement unit includes a non-contact displacement meter.
According to the present invention, when measuring the position coordinates of a point on the cutting surface, it can be measured without damaging the cutting surface.

  The automatic slicing device according to the present invention is the automatic slicing device, wherein the two or more straight lines are a first straight line along a relative movement direction of the cutting part and the cutting table, and the first straight line. And measuring the coordinate positions of a plurality of points along the straight line.

  According to the present invention, the rotation angle around the first straight line and the rotation angle around the second straight line of the cutting surface can be calculated, and the inclination state of the cutting surface with respect to the horizontal plane can be calculated more accurately.

  According to the present invention, even if the sample block is once removed from the cutting table, a thin slice having a predetermined thickness can be continuously collected from the cutting surface.

An embodiment according to the present invention will be described with reference to FIGS. 1 to 5. In the present embodiment, a biological tissue collected from a laboratory animal such as a spider will be described as an example of the biological sample.
The automatic slicing apparatus 1 according to the present embodiment includes a sample block B in which the living tissue S is embedded in a packing agent (as a sample block, a new sample block, a sample block from which a thin section has been collected, or another part In this device, a sheet-like thin section having a predetermined thickness is cut out from a sample block or the like that has been completed up to rough cutting. The XYZ axis direction is defined as the direction shown in FIG.

  As shown in FIGS. 1 to 3, the automatic slicing apparatus 1 places a sample block B via a knife (cutting unit) 2 for cutting a thin section from a sample block B and a cassette 3, and then the sample block The cutting table 5 for inclining B with respect to the horizontal plane, the tilt angle control unit 6 for instructing the cutting table 5 to control the tilt angle of the sample block B with respect to the horizontal plane, the knife 2 and the cutting table 5 in the X-axis direction An X-axis guide rail (moving part) 7 for relative movement, a measuring part 8 for measuring the coordinate position of an arbitrary point on the cutting surface P of the cut sample block B, and first straight lines orthogonal to each other on the cutting surface P From the measured values of the coordinate positions of at least three points located on L1 and the second straight line L2, the relative position of the cutting surface P with respect to the virtual plane stored in advance is calculated, and the cutting surface P is inclined so as to coincide with the virtual plane. Calculation unit 1 for controlling the angle control unit 6 It has a, and. Here, the direction in which the first straight line L1 extends is defined as the X-axis direction, and the direction in which the second straight line L2 extends is defined as the Y-axis direction. That is, the horizontal plane refers to the XY plane.

  The sample block B is obtained by replacing the moisture in the formalin-fixed biological tissue S with paraffin, and then fixing an embedding block whose periphery is solidified with an embedding agent such as paraffin on the embedding cassette. . Thereby, the biological tissue S is embedded in paraffin.

  The knife 2 is provided with a cutting surface 2a provided along a horizontal plane.

  The cutting table 5 is mounted on the Z stage 12 that moves in the Z-axis direction by the operation of the lifting motor 11 and the operation of the roll motor 13 along an arc surface centered on an axis extending in the X-axis direction. The roll stage 15 made movable by (the inclination of the lateral direction can be adjusted with reference to the moving direction with respect to the knife 2), and an arc attached to the roll stage 15 and centered on the axis extending in the Y-axis direction A pitch stage 17 which can be moved along the surface by the operation of the pitch motor 16 (the inclination in the vertical direction can be adjusted with reference to the moving direction relative to the knife 2), and a sample block mounted on the pitch stage 17 Holder 18.

The X-axis guide rail 7 is provided in a state extending in the X-axis direction to a position beyond the knife 2 so as to go to the fixed knife 2.
The Z stage 12 is controlled to reciprocate on the X-axis guide rail 7 by the stage control unit 19. Further, by operating the lifting motor 11 in synchronism with this reciprocating motion, the Z stage 12 is controlled to rise in the Z-axis direction by a certain distance every time the X-axis guide rail 7 is reciprocated once. Yes.

  In the present embodiment, the configuration is such that the sample block B is cut by fixing the knife 2 and moving the cutting table 5 side with respect to the knife 2, but this is not restrictive. For example, the cutting table 5 may be fixed and the knife 2 may be moved with respect to the cutting table 5, or the cutting table 5 and the knife 2 may be moved together.

  The tilt angle control unit 6 sends command signals to the lift motor 11, the roll motor 13, and the pitch motor 16, respectively, so that the tilt of the sample block holder 18 with respect to the horizontal plane and Z The height in the axial direction is arbitrarily set.

  The measurement unit 8 includes an X-axis measurement unit (not shown) that measures the amount of movement of the Z stage 12 that moves along the X-axis guide rail 7, and a Y-axis guide that extends in the Y-axis direction above the cutting table 5. A Y-axis measuring unit (not shown) that measures the amount of movement of the Y stage 21 provided so as to be movable along the rail 20, and a laser displacement meter (non-contact displacement meter) 22 provided on the Y stage 21 are provided. Yes. The laser displacement meter 22 measures the height in the Z-axis direction with respect to the horizontal plane of the cutting surface P by being arranged to face the cutting surface P. The Y stage 21 is controlled by the stage control unit 19 so as to reciprocate on the Y-axis guide rail 20.

Next, the operation of the automatic slicing apparatus 1 according to the present embodiment will be described together with an explanation of the automatic slicing method.
In this automatic slicing method using the automatic slicing device 1, the sample block B once removed from the cutting table 5 is attached to the cutting table 5 again, and thin sections are continuously cut from the already cut surface P of the sample block B by the knife 2. An automatic slicing method for sampling, a measuring step of measuring the coordinate positions of the first straight line L1 and the second straight line L2 on the cutting surface P of the sample block B reattached to the cutting table 5, and the coordinate position A cutting surface position calculating step of calculating a relative position of the cutting surface P with respect to a virtual plane (cutting surface at the end of rough cutting) stored in advance from a measurement value; a cutting surface adjustment step of matching the cutting surface P with the virtual plane; It has.

First, the operator transports the pre-cooled sample block B to the automatic slicing apparatus 1 side by a transport device (not shown) and places it on the cutting table 5.
Subsequently, the cutting block 5 is moved along the X-axis guide rail 7 to cut the sample block B. Cutting includes rough cutting and main cutting. First, rough cutting with a large depth of cut is performed. When this is completed, the knife 2 is replaced and main cutting is performed.

  Here, the sample block B, which has been completely roughed by this device or another device, is set on the cutting table 5 in advance, and the main cutting is continuously performed from the already-cut surface P of the sample block B, and the thin slice is obtained. It may be collected. In this case, the sample block B for which rough cutting has been completed is placed and fixed on the sample block holder 18 of the cutting table 5 by hand or by automatic operation. The following steps are also performed when the sample block B from which the thin slice has been removed once from the cutting table 5 is set on the cutting table 5 again and a thin section is continuously collected from the already-cut surface P of the sample block B. Can be applied.

Before performing the main cutting, a measurement process and a cutting surface adjustment process are performed.
In the measurement process, first, while moving the Z stage 12 along the X axis guide rail 7 with the Y stage 21 fixed, the amount of movement is measured by the X axis measurement unit. At the same time, the Z coordinate of the cutting surface P is measured at any time by the laser displacement meter 22. Thus, as shown in FIG. 4, the inclination θ around the straight line L1 is obtained as a measurement result.

  Next, while moving the Y stage 21 along the Y axis guide rail 20 with the Z stage 12 fixed, the amount of movement is measured by the Y axis measurement unit. At the same time, the Z coordinate of the cutting surface P is measured at any time by the laser displacement meter 22. Thus, as shown in FIG. 5, the inclination φ around the straight line L2 is obtained as a measurement result.

Subsequently, the process proceeds to a cutting surface position calculation step.
Here, the calculation unit 10 calculates the plane data of the cutting surface P from a measurement result obtained in the measurement process by a known method. Then, the relative position of the cutting surface P is calculated from the obtained plane data and the virtual plane data stored in advance.

And it transfers to a cutting surface adjustment process.
Here, the difference from the virtual plane is calculated from the relative position of the cutting surface P. Then, the difference is transmitted from the calculation unit 10 to the tilt angle control unit 6. Thus, the inclination angle control unit 6 drives and controls the elevating motor 11, the roll motor 13, and the pitch motor 16 with a predetermined amount of rotation, respectively, and the cutting surface P is inclined at a predetermined height at a predetermined angle to virtually Match with the plane.

  Thereafter, the process proceeds to the main cutting operation, and a thin slice is produced.

  According to the automatic slicing apparatus 1 and the automatic slicing method, it is assumed that the coordinate position of the cutting surface P is displaced when the cutting surface P is aligned and removed from the cutting table 5 and reattached. At that time, the inclination angle control unit 6 tilts the cutting table 5 at a predetermined angle and moves it to a predetermined height based on an instruction of the calculation unit 10 with respect to the difference, so that the cutting surface P before and after cutting is moved. The positions can be matched. Therefore, even if the sample block B is once removed from the cutting table 5, a thin slice having a predetermined thickness can be continuously collected from the cutting surface P.

  Moreover, since the laser displacement meter 22 is used when measuring the cutting surface P, the cutting surface P is not touched directly, and measurement can be performed without damaging the cutting surface. At this time, since the coordinate positions are measured along the X-axis direction and the Y-axis direction which are the rotation center axis directions in the cutting table 5, the inclination state of the cutting surface P with respect to the horizontal plane can be calculated more accurately and easily. it can.

The technical scope of the present invention is not limited to the above embodiment, and various modifications can be made without departing from the spirit of the present invention.
For example, the points to be measured are not limited to the X-axis direction and the Y-axis direction. That is, it is only necessary to measure the coordinate positions of at least three points located on two straight lines intersecting on the cutting plane P.

It is a schematic diagram showing an automatic slicing device according to an embodiment of the present invention. It is a perspective view which shows the cutting stand of the automatic slicer which concerns on one Embodiment of this invention. It is a perspective view which shows a sample block. It is a graph which shows the measurement result of the cutting surface obtained by the automatic slicing device concerning one embodiment of the present invention. It is a graph which shows the measurement result of the cutting surface obtained by the automatic slicing device concerning one embodiment of the present invention.

Explanation of symbols

1 Automatic slicer 2 Knife (cutting part)
5 Cutting table 6 Inclination angle control unit 7 X-axis guide rail (moving unit)
8 Measurement unit 10 Calculation unit 22 Laser displacement meter (non-contact displacement meter)
L1 First straight line L2 Second straight line

Claims (4)

A cutting section for cutting a thin section from a rough- cut sample block in which a sample is embedded;
A cutting table for placing the sample block and inclining the sample block with respect to a horizontal plane;
An inclination angle control unit for instructing the cutting table to control an inclination angle of the sample block with respect to the horizontal plane;
A moving unit that relatively moves the cutting unit and the cutting table;
A measuring unit for measuring the coordinate position of a point on the current cutting surface of the cut sample block;
The current cutting surface with respect to a virtual plane stored in advance as a cutting surface at the end of the rough cutting from the measured values of the coordinate positions of at least three points located on two or more straight lines intersecting each other on the current cutting surface A calculation unit that controls the inclination angle control unit so as to match the current cutting surface with the virtual plane;
An automatic slicing device characterized by comprising:   The automatic slicing device according to claim 1, wherein the measurement unit includes a non-contact displacement meter. The two or more straight lines are a first straight line along a relative movement direction of the cutting part and the cutting table,
A second straight line orthogonal to the first straight line,
2. The automatic slicing device according to claim 1, wherein coordinate positions of a plurality of points are measured along the straight line. An automatic slicing method in which a rough cut sample block once removed from a cutting table is attached to the cutting table again, and a thin section is continuously collected from the already-cut surface of the sample block by a cutting unit,
A measuring step of measuring coordinate positions of at least three points located on two or more straight lines intersecting each other on the current cutting surface of the sample block reattached to the cutting table;
A cutting surface position calculating step for calculating a relative position of the current cutting surface with respect to a virtual plane stored in advance as a cutting surface at the end of the roughing from the measured values of the coordinate positions of the at least three points;
A cutting surface adjustment step of matching the current cutting surface with the virtual plane;
An automatic slicing method characterized by comprising:

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