JP2007033312A - Method and device for manufacturing segment sample for analysis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method and a device for manufacturing a segment sample for analysis that can easily sample many segment samples continuously without damage or loss, at a predetermined attitude, and in a predetermined order, and to allow efficient three-dimensional structural analysis. <P>SOLUTION: Segment samples E sequentially formed by cutting a solid S including an analyzed object with a knife 42 for cut are sequentially supplied onto a free surface of liquid L with an equivalent attitude. Liquid flows F1-F4 flowing along the free surface are produced in the liquid L by a peristaltic pump 61. The segment samples E are moved along the liquid flows in a moving course on the liquid free surface. At this time, the width of the moving course of the segment samples E is set larger than the minor diameter of the segment samples E and smaller than the longest diameter. The segment samples E moving in the moving course are adhered onto the upper surface of a sample base P and collected by a collecting means 12. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method and apparatus for producing a section sample by cutting a solid containing an analysis object. The section sample is two-dimensionally analyzed by an analyzer such as an infrared absorption analyzer (IR), an X-ray photoelectron spectrometer (XPS), a scanning probe microscope (SPM), a transmission electron microscope (TEM), and a scanning electron microscope (SEM). Used to get information. The two-dimensional information obtained by these is used to connect them to construct three-dimensional information.

  In general, a microtome is used as a means for preparing a section sample to be observed or photographed with an electron microscope or an optical microscope. In the microtome, in general, an analysis object is embedded in a resin or the like and then fixed to an arm. The arm is moved up and down and cut into a thin piece with a knife made of diamond or glass. Since the slice obtained by this cutting floats on the water surface stretched on a boat with a cutting blade, the slice can be scooped with a mesh or the like and used as a microscope sample such as a transmission electron microscope. In this way, the two-dimensional information obtained based on observation or imaging of the section sample continuously produced by cutting from the solid containing the analysis object is reconstructed to reconstruct the three-dimensional structure of the analysis object. The This method is used for three-dimensional structural analysis of biological samples and the like. Further, not only a sliced slice sample but also a block obtained as a residue from which a slice has been removed by cutting can be used as an analysis sample. For example, a microscope sample of an optical microscope or a scanning electron microscope Used as

  In order to reconstruct a three-dimensional structure of an analysis object based on two-dimensional information from a plurality of section samples obtained by continuous cutting, first, a three-dimensional region (including the analysis object) in which a solid including the analysis object is focused The region is cut (sliced) sequentially by a certain thickness. Then, it is necessary to obtain a large number of section samples thus obtained in order. The continuous collection of such section samples using a microtome requires a great deal of patience and effort. In addition, since the section sample is collected manually, there is a risk that a part of the section sample may be lost or the section sample may be wrinkled, which is inefficient. This is a high hurdle for executing the three-dimensional structural analysis by the continuous section method.

On the other hand, Patent Document 1 discloses a method of collecting and holding a slice sample floating on a water surface produced using a microtome and stretched on a boat, on a long tape.
Japanese Patent Publication No. 7-97069

Thus, in the method described in Patent Document 1, in order to affix the section samples to the long tape in order, a section separated from the solid is formed by cutting, and at the same time, the section is placed on the long tape. And pulling it up from the water surface. As described above, when the residence time of the section sample on the free water surface is short, it is affixed to the long tape with almost no flattening action due to the surface tension of the free water surface. The section formed by cutting is generally not flat but in a bent state. For this reason, the section attached to the long tape at the same time as cutting may be damaged by cracking or the like due to a sudden flattening force when it is pulled up.
The present invention provides a method and apparatus for preparing a section sample for analysis that can easily collect a large number of section samples continuously without damage or loss, and in a predetermined posture and in a predetermined order. The purpose is to do.

The method for preparing an analytical section sample of the present invention that achieves the above object is as follows.
Cutting a solid containing the analyte to form a section sample,
Forming a liquid flow,
The formed slice sample is conveyed by the liquid flow,
The section sample transported is collected on a sample stage.

More specifically, a method of cutting a solid containing an analysis object to produce a section sample for analysis,
A plurality of slice samples formed sequentially by cutting are sequentially supplied onto the free surface of the liquid in an equivalent posture,
Forming a liquid flow in the liquid that flows along a free surface of the liquid;
Moving the section sample on the free surface according to the liquid flow, setting a movement path of the section sample on the free surface, the width of the movement path being larger than the minor axis of the section sample and Set smaller than the longest diameter,
Collecting the section sample that has moved along the movement path on a sample stage;
It is.

  In one aspect of the present invention, the section sample is collected by moving the sample stage upward from the liquid through the free surface and placing the section sample on the surface of the sample stage. . In one aspect of the present invention, the direction in which the section sample is supplied onto the free surface of the liquid is the same as the direction of the movement path of the section sample on the free surface. In one aspect of the present invention, when the section sample is moved on the free surface according to the liquid flow, the width of the movement path is set smaller than the major axis in the direction perpendicular to the minor axis of the section sample. deep.

Furthermore, the analytical section sample preparation apparatus of the present invention that achieves the above-described object is as follows.
A tank containing liquid;
A cutting means having a cutting blade for cutting a solid containing the analysis object;
A liquid flow generating means for generating a liquid flow in the liquid to convey the section sample formed by the cutting means;
A collecting means for collecting the section sample conveyed by the liquid flow on a sample stage;
It is characterized by providing.

More specifically, an apparatus for cutting a solid containing an analysis object to produce a section sample for analysis,
A tank containing liquid;
A cutting means including a cutting knife disposed in the tank and a driving unit that drives the movement of the solid including the analysis object so as to be cut by the knife;
A liquid flow generating means for generating a liquid flow in the liquid along the free surface of the liquid;
A movement path setting means for setting the movement path and the width of the section sample on the free surface;
An apparatus for preparing a section sample for analysis, comprising: a collection means for collecting the section sample that has moved along the movement path.

  In one aspect of the present invention, the collecting means includes a sample stage holder for holding a sample stage on which the section sample is placed and attached, and a holder moving means for moving the sample stage holder in the vertical direction. In one aspect of the present invention, the cutting means supplies the section sample onto the free surface of the liquid, and the direction of the supply is the same as the direction of the movement path of the section sample on the free surface. In one aspect of the present invention, the liquid flow generating means causes the liquid to flow laterally along the free surface in the tank, to flow downward toward the tank bottom, and to flow laterally along the tank bottom. Furthermore, the circulating liquid flow is generated so as to flow upward toward the free surface. In one aspect of the present invention, the liquid flow generating means includes a peristaltic pump provided at the bottom of the tank.

  According to the present invention, a section sample can be easily collected without damage or loss.

  Furthermore, it is possible to collect the section sample that has moved along the movement path without causing rotation or change of order, and the section sample can be continuously collected in a predetermined posture without damage or loss. This enables efficient implementation of the three-dimensional structure analysis.

  Hereinafter, embodiments of a method and apparatus for preparing a section sample for analysis according to the present invention will be described with reference to the drawings.

  FIG. 1 is a schematic cross-sectional view showing an embodiment of an analytical section sample preparation apparatus according to the present invention, FIG. 2 is a plan view thereof, and FIGS. 3 and 4 are cross-sectional views taken along line AA ′ of FIG. It is a figure and BB 'sectional drawing.

  In these drawings, a liquid L is contained in a tank (boat) 2. The liquid L can be preferably used as long as it can cause buoyancy based on surface tension to act on the slice sample and does not cause a reaction such as a chemical reaction with the slice sample. An example of the body surface of such a liquid L is water.

  The production apparatus includes cutting means 4. The cutting means 4 includes a cutting knife 41 disposed at the upper edge of the tank 2 and a drive unit 42 that drives the movement of the solid S containing the analysis object at the time of cutting. The drive unit 42 can hold the solid S including the analysis target and move the solid S in the vertical direction (X direction) and the horizontal direction (Y direction). At the time of cutting, a motion combining a reciprocating movement in the X direction and an intermittent movement in a direction approaching the cutting knife (cutting blade) 41 in the Y direction is performed. Thereby, the solid S is sequentially cut, and the formed plurality of slice samples E are sequentially supplied onto the free surface of the liquid L in the tank in an equivalent posture. The configuration and operation of such cutting means 4 are the same as those of a known microtome.

  The production apparatus also includes liquid flow generating means 6 for generating a liquid flow that flows along the free surface of the liquid L. The liquid flow generating means 6 generates a liquid flow that circulates in the tank. In the tank 2, the liquid L flows sideways along the free surface (arrow F1), flows downward toward the tank bottom (arrow F2), flows sideways along the tank bottom (arrow F3), and further on the free surface. Flow upward (arrow F4). For this reason, the liquid flow path forming member 8 is disposed in the tank 2, and the liquid flow circulates around the liquid flow path forming member 8. The liquid flow generating means 6 further includes a peristaltic pump 61 provided at the bottom of the tank 2. That is, the bottom of the tank 2 is made of a flexible sheet 611, and a liquid channel that circulates between the upper surface of the flexible sheet 611 and the liquid channel forming member 8 is formed. Below the flexible sheet 611, a driving roller 612 connected to a driving source (not shown) and a belt 614 wound around a free roller 613 are arranged. A protrusion 614 ′ is formed on the outer surface of the belt 614. When the driving roller 612 rotates, the belt 614 travels and the protrusion 614 ′ moves while pushing the flexible sheet 611 upward. As a result, the position at which the flexible sheet 611 is pressed against the lower surface of the liquid flow path forming member 8 (contact position between the protrusion 614 ′ and the flexible sheet 611) moves, and the circulating liquid flow is generated.

  The production apparatus also includes a movement path setting means 10 for setting the movement path and the width of the section sample E on the free surface of the liquid L. The moving path setting means 10 has a pair of path width adjusting plates 101 and 102 arranged so as to cover a part of the free surface of the liquid L in the upper part of the tank 2. A moving path 103 of the section sample E on the free surface of the liquid L is formed between the linear edges of the path width adjusting plates 101 and 102 facing each other. Adjustment screws 101 'and 102' are attached to the path width adjusting plates 101 and 102, respectively. By operating the adjusting screws 101 'and 102' outside the tank, the path width adjusting plates 101 and 102 can be moved closer to or away from each other, and the distance between the path width adjusting plates 101 and 102, that is, the moving path width. W can be set appropriately.

  The production apparatus also includes a collection means 12 for collecting the section sample E that has moved along the movement path 103. The collection means 12 includes a sample stage holder 121 for holding a plurality of sample stands P on which each of the section samples E is mounted and attached, and a holder moving means 122 for moving the sample stage holder in the vertical direction. Yes. The holder moving means 122 is formed by fitting a vertical drive screw 122 ′ supporting the sample stage holder 121 from below with a female screw 122 ″ provided at the bottom of the tank 2. Drive outside the tank. By manipulating the screw 122 ′, the sample stage holder 121 can be moved up and down, and the sample stage P can be moved upward.In the tank 2, the sample is around the drive screw 122 ′. A bellows 123 is provided so as to connect between the base holder 121 and the bottom of the tank 2. Further, in the tank 2, a plurality of sample stands P are arranged in an appropriate vertical direction. A guide frame 124 is formed.

  Next, an embodiment of the production apparatus of the present embodiment, that is, an embodiment of the method for producing an analytical section sample of the present invention will be described.

  First, the solid S containing the analysis object is held by the drive unit 42 of the cutting means 4. The solid S is obtained by embedding an analysis object in a resin or the like by a known method, for example. Then, while the drive unit 42 is reciprocated in the X direction, a required pitch (corresponding to the thickness of the slice sample to be formed) in the Y direction toward the cutting knife 41 in the vicinity of the top dead center of the reciprocation. To move intermittently. Eventually, the cutting of the solid S by the cutting knife 41 is started. This cutting is performed by contacting the cutting knife 41 when the solid moves downward. The slice samples E sequentially formed by cutting are sequentially supplied onto the free surface of the liquid L in an equivalent posture.

  The position of the section sample supply corresponds to the starting point of the moving path 103 of the section sample E on the free surface of the liquid L. From here, the section sample E moves away from the cutting knife 41 according to the liquid flow on the liquid free surface. Moving. The direction (Y direction) of the moving path of the slice sample E on the liquid free surface is the same as the direction in which the slice sample E is supplied onto the liquid free surface.

  The width W of the movement path 103 is set so as to have a specific relationship with the short diameter and the longest diameter of the slice sample E. 5 and 6 are views of the slice sample S as viewed in the thickness direction, and are diagrams showing the short diameter d1, the long diameter d2, and the longest diameter d3 of the slice sample E. FIG. That is, the short diameter d1 indicates the outer diameter when viewed in a specific direction so that the outer diameter becomes the smallest, the long diameter d2 indicates the outer diameter when viewed in a direction orthogonal to the specific direction, and the longest diameter d3. Indicates the outer diameter when the outer diameter is viewed to be the largest. In the present invention, the width W of the moving path 103 is preferably set larger than the short diameter d1 and smaller than the longest diameter d3 of the section sample E. In this way, the rotation and overtaking of the section sample E by 180 ° or more in the movement path 103 is prevented, and the section sample E moves with a uniform direction without being in the opposite direction. More preferably, the width W of the moving path 103 is set to be larger than the short diameter d1 of the slice sample E and smaller than the long diameter d2. By doing in this way, the permissible rotation angle of the section sample E in the movement path 103 is further reduced, and the section sample E moves with a more uniform directionality.

  FIG. 7 shows how the slice sample E moves along the movement path 103. FIG. 7A shows a case where the movement path width W is set within the above range by the path width adjusting plates 101 and 102 of the movement path setting means 10. In this case, the same applies to the case of FIG. 2, but the section sample E moves in the same direction with the same directionality in the cutting order. On the other hand, FIG. 7B shown for comparison shows a conventional type that does not have a movement path setting means. In this case, since the slice sample E can freely move on the free surface of the liquid, the slice sample E can meander or rotate to move in a disjointed posture, and further, the arrangement of the cutting order becomes unclear.

  Next, the section sample E that has moved along the movement path 103 is sequentially collected by the collecting means 12. In this collection, first, as shown in FIG. 1 and FIG. 8 (a), the uppermost one of the plurality of sample stands P arranged in a stack is placed on a part of the upper surface (cutting knife). The portion where the distance from 4 is large) is exposed from the liquid free surface. When the section sample E arrives there, the tip portion thereof rides on the upper surface of the sample stage P as shown in FIG. Next, when the sample stage holder 121 is raised by the holder moving means 122, the section sample E is placed on the upper surface of the sample stage P and attached, as shown in FIG. Next, as shown in FIG. 8D, the sample stage P to which the section sample E is attached is collected. At this time, the section sample E extends on the sample stage P without any wrinkles due to the action of the surface tension of the liquid L. Thereafter, when the sample stage P with the section sample E is naturally dried, the section sample E is flatly and firmly adhered and fixed on the sample stage P with no gap as the liquid evaporates.

  The formation of the slice sample E by cutting by the cutting means 4, the generation of the liquid flow by the liquid flow generating means 6, and the collection of the slice sample E by the collecting means 12 are related to each other as follows. Can be done. That is, the formation of the section sample E by cutting, the movement of the section sample E along the movement path 103, and the collection of the section sample E are performed for each required number to be attached to one sample stage P. The required number can be set as appropriate based on the major axis of the section sample E and the length of the sample stage P in the Y direction. Only one section sample E may be attached to one sample stage P. First, after the required number of slice samples E formed by cutting are arranged in a line in the cutting order in the movement path 103, the cutting is stopped. Then, a liquid flow is generated by the liquid flow generating means 6, and a required number of sections of the sample E are moved to the collecting means 12. After this movement is completed, the liquid flow generation is stopped. Then, a required number of slice samples E are collected by the collecting means 12. Such an operation can be performed under a microscope.

  In the above embodiment, even if the slice sample E formed by cutting is in a bent state, it is based on the surface tension of the liquid L until it is collected by moving along the movement path 103 on the liquid free surface. It is gradually extended on the free surface of the liquid. Therefore, as shown by a1 in FIG. 9, the section sample E can be easily and reliably attached to the upper surface of the sample stage P without being damaged during the collection. After that, when the section sample E on the sample stage P is naturally dried as shown by a2 in FIG. 9, the section sample E is fixed flatly and firmly on the sample stage P without any gap as the moisture evaporates. On the other hand, in the case of a section prepared by a conventional dry method, as shown in b1 and b2 of FIG. 9, the section sample E ′ often has wrinkles and curls. When placed, there is a gap between the sample stage P and the slice sample E ′. Therefore, when IR measurement is performed in such a state, interference occurs in the spectrum and a satisfactory waveform cannot be obtained. In order to prevent this, a method of mechanically affixing a slice by pressing it from the top is usually used, but in this case, deformation causes tissue deformation and composition diffusion, so accurate information Was not obtained.

  The section sample E collected as described above and attached to the sample stage P includes an infrared absorption analyzer (IR), an X-ray photoelectron spectrometer (XPS), a scanning probe microscope (SPM), and a transmission electron microscope. (TEM) and a scanning electron microscope (SEM) are used to acquire two-dimensional information by an analyzer. Then, the two-dimensional information obtained in this way is used to construct three-dimensional information by connecting them by image processing. The sample stage P is appropriately selected depending on the type of analysis from an insulator such as glass, a semiconductor such as silicon, or a conductor such as metal.

It is typical sectional drawing which shows one Embodiment of the preparation apparatus of the section | slice sample for analysis of this invention. It is a top view of the preparation apparatus of FIG. It is A-A 'sectional drawing of FIG. It is B-B 'sectional drawing of FIG. It is a figure which shows the short diameter, long diameter, and longest diameter of a section | slice sample. It is a figure which shows the short diameter, long diameter, and longest diameter of a section | slice sample. It is a figure which shows the mode of a movement of a section | slice sample. It is a figure which shows the process of collecting a section | slice sample by a collection means. It is a figure which shows the characteristic of the slice sample by the difference in a collection means.

Explanation of symbols

2 Tank (boat)
DESCRIPTION OF SYMBOLS 4 Cutting means 41 Cutting knife 42 The drive part which drives the motion of the solid containing an analysis target object 6 Liquid flow generation means 61 Peristaltic pump 611 Flexible sheet 612 Drive roller 613 Free roller 614 Belt 614 'Protrusion 8 Liquid flow path formation member 10 Moving path setting means 101, 102 Path width adjusting plates 101 ′, 102 ′ adjusting screw 103 Section sample moving path 12 Section sample collecting means 121 Sample stage holder 122 Holder moving means 122 ′ Drive screw 122 ”Female screw 123 Bellows 124 Guide Frame L Liquid S Solid containing the analyte E Section sample E 'Dry collected section sample P Sample stage

Claims (10)

A method for preparing a section sample for analysis,
Cutting a solid containing the analyte to form a section sample,
Forming a liquid flow,
The formed slice sample is conveyed by the liquid flow,
A method for producing a section sample for analysis, wherein the section sample transported is collected on a sample stage. A method for producing a section sample for analysis by cutting a solid containing an analysis object,
A plurality of slice samples formed sequentially by cutting are sequentially supplied onto the free surface of the liquid in an equivalent posture,
Forming a liquid flow in the liquid that flows along a free surface of the liquid;
Moving the section sample on the free surface according to the liquid flow, setting a movement path of the section sample on the free surface, the width of the movement path being larger than the minor axis of the section sample and Set smaller than the longest diameter,
A method for producing a section sample for analysis, wherein the section sample that has moved along the movement path is collected on a sample stage. The analytical section according to claim 1 or 2, wherein the sample stage is moved upward through a free surface of a liquid, the section sample is placed on the surface of the sample stage, and the collection is performed. Sample preparation method. The analysis according to any one of claims 1 to 3, wherein a direction in which the section sample is supplied onto the free surface of the liquid is the same as a direction of a movement path of the section sample on the free surface. Method for preparing a section sample. When moving the section sample according to the liquid flow on the free surface, the width of the movement path is set smaller than the major axis in the direction orthogonal to the minor axis of the section sample. A method for preparing a section sample for analysis according to any one of 1 to 4. An apparatus for preparing a section sample for analysis,
A tank containing liquid;
A cutting means having a cutting blade for cutting a solid containing the analysis object;
A liquid flow generating means for generating a liquid flow in the liquid to convey the section sample formed by the cutting means;
A collecting means for collecting the section sample conveyed by the liquid flow on a sample stage;
An apparatus for preparing a section sample for analysis, comprising: An analytical section sample preparation apparatus for preparing an analytical section sample by cutting a solid containing an analysis object,
A tank containing liquid;
A cutting means including a cutting knife disposed in the tank and a driving unit that drives the movement of the solid including the analysis object so as to be cut by the knife;
A liquid flow generating means for generating a liquid flow in the liquid along the free surface of the liquid;
A movement path setting means for setting the movement path and the width of the section sample on the free surface;
An apparatus for preparing a section sample for analysis, comprising: a collection means for collecting the section sample that has moved along the movement path on a sample stage. The analysis means according to claim 6 or 7, wherein the collecting means includes a sample stage holder for holding the sample stage and a holder moving means for moving the sample stage holder in the vertical direction. Section sample preparation device. 9. The cutting means supplies the section sample onto the free surface of the liquid, and the direction of the supply is the same as the direction of the movement path of the section sample on the free surface. An apparatus for preparing a section sample for analysis according to any one of the above. The liquid flow generating means causes the liquid to flow laterally along the free surface in the tank, to flow downward toward the tank bottom, to flow laterally along the tank bottom, and further toward the free surface. The apparatus for preparing a section sample for analysis according to any one of claims 6 to 9, which generates the circulating liquid flow that flows upward.

Images