JP2017129735A - Laser microdissector and laser microdissection method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a laser microdissector that enables accurate dissection of a sample around a target portion thereof.SOLUTION: A laser microdissector comprises: a stage 10 on which a sample 1 is placed; a microscope 20 configured to acquire a three-dimensional image of the sample 1; a target portion identifier 301 configured to identify three-dimensional coordinates of a target portion of the sample 1 from the three-dimensional image; a dissection laser light source 41 configured to emit a dissection laser beam for dissecting the sample 1; and a position control mechanism 47 configured to alter the incident position and incident angle of the dissection laser beam on the sample 1 on the basis of the three-dimensional coordinates of the target portion in order to dissect the sample 1 around the target portion.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a microfabrication technique, and relates to a laser microdie sector and a laser microdissection method.

  Pathological examination is a highly reliable examination method in diagnosis of diseases. For example, pathological diagnosis is often required in the diagnosis of cancer. Pathological examination is classified into histological examination and cytological examination. In particular, tissue examination is a useful examination method among pathological examinations because various information can be obtained from tissues. In histological examination, a pathological section obtained by slicing a tissue collected from the body to a thickness of several microns is observed under a microscope. However, even a pathological section is often composed of normal cells or cells not to be examined. Therefore, there are very few lesion cells to be examined, which are included in the pathological section. At present, the analysis method of a lesion cell is mainly observed under a microscope, but when there are very few lesion cells, the analysis of the lesion cells is difficult.

  Therefore, a method for diagnosing a disease by not only observing a diseased cell under a microscope but also excising and collecting the diseased cell from a tissue and analyzing the diseased cell using an analyzer such as a mass spectrometer is proposed. ing.

  Laser microdissection is used to excise and collect diseased cells from tissue. The laser microdissection method is a method for cutting out, collecting, and collecting a target lesion cell mass with a laser beam while observing a tissue section under a microscope using a laser irradiation apparatus connected to a microscope ( For example, see Patent Documents 1 to 3.) This method is useful for elucidating diseases and life phenomena because only a target cell group or a single cell can be collected from a complex mixed tissue sample. The laser microdissection method is used not only for medical use but also for cutting out a minute target portion from a sample such as an industrial product.

JP 2014-78008 A JP-T-2015-517669 JP 2013-63336 A

  An object of the present invention is to provide a laser microdissector and a laser microdissection method capable of accurately cutting around a target portion of a sample.

  According to an aspect of the present invention, (a) a stage on which a sample is placed, (b) a microscope that acquires a three-dimensional image of the sample, and (c) three-dimensional coordinates of a target portion of the sample are specified from the three-dimensional image. A target portion specifying portion; (d) a cutting laser light source that emits a cutting laser beam that cuts the sample; and (e) cutting on the sample so that the periphery of the target portion of the sample is cut based on the three-dimensional coordinates of the target portion. There is provided a laser micro-die sector comprising a position control mechanism for changing an incident position and an incident angle of a laser.

  In the above laser microdie sector, the incident angle of the cutting laser beam to the sample may be changed based on the cross-sectional shape of the target portion obtained from the three-dimensional image. You may change the incident angle of the cutting | disconnection laser beam with respect to a sample along the outline of the cross-sectional shape of a target part obtained from a three-dimensional image.

  In the laser micro-die sector, the position control mechanism may drive the cutting laser beam optical system to change the incident position and the incident angle of the cutting laser beam on the sample. The position control mechanism may change the incident angle of the cutting laser beam with respect to the sample by tilting the stage.

  In the laser micro-die sector described above, the incident angle of the cutting laser beam with respect to the sample may vary in the range of 0 ° to 90 °.

  In the above laser micro-die sector, the cutting laser beam may be a deep ultraviolet laser beam. The wavelength range of the cutting laser light may be 200 nm or more and 300 nm or less. The focal spot size of the cutting laser beam may be 400 nm or more and 10 μm or less.

  The laser micro-die sector may further include a manipulator that recovers the target portion isolated by the cutting laser light. The manipulator may include a capillary in which the liquid containing the target portion rises due to capillary action, or may include a cantilever that collects the target portion. The manipulator may recover the target portion using magnetic force, or may recover the target portion using intermolecular affinity.

  In the above laser microdissector, the microscope may be selected from the group consisting of a confocal laser microscope, a confocal Raman microspectroscope, an optical microscope, a fluorescence microscope, and an electron microscope.

  In the above laser microdie sector, the sample may be a biological sample or a biological tissue.

  According to an aspect of the present invention, (a) acquiring a three-dimensional image of a sample, (b) specifying the three-dimensional coordinates of a target portion of the sample from the three-dimensional image, and (c) cutting the sample Emitting a cutting laser beam, and changing an incident position and an incident angle of the cutting laser with respect to the sample so that the periphery of the target portion of the sample is cut based on the three-dimensional coordinates of the target portion. A method is provided.

  In the laser microdissection method described above, the incident angle of the cutting laser beam on the sample may be changed based on the cross-sectional shape of the target portion obtained from the three-dimensional image. You may change the incident angle of the cutting | disconnection laser beam with respect to a sample along the outline of the cross-sectional shape of a target part obtained from a three-dimensional image.

  In the laser microdissection method described above, the cutting laser beam optical system may be driven to change the incident position and the incident angle of the cutting laser beam on the sample. You may change the incident angle of the cutting laser beam with respect to a sample by inclining the stage on which a sample is mounted.

  In the laser microdissection method described above, the incident angle of the cutting laser beam with respect to the sample may vary within a range of 0 ° to 90 °.

  In the laser microdissection method described above, the cutting laser beam may be a deep ultraviolet laser beam. The wavelength range of the cutting laser light may be 200 nm or more and 300 nm or less. The focal spot size of the cutting laser beam may be 400 nm or more and 10 μm or less.

  The laser microdissection method may further comprise recovering the target portion isolated with the cutting laser light with a manipulator. The manipulator may include a capillary in which the liquid containing the target portion rises due to capillary action, or may include a cantilever that collects the target portion. The manipulator may recover the target portion using magnetic force, or may recover the target portion using intermolecular affinity.

  In the laser microdissection method described above, the microscope for acquiring the sample image is any one selected from the group consisting of a confocal laser microscope, a confocal Raman microspectroscope, an optical microscope, a fluorescence microscope, and an electron microscope. May be.

  In the laser microdissection method described above, the sample may be a biological sample or a biological tissue.

  According to the present invention, it is possible to provide a laser microdissector and a laser microdissection method capable of accurately cutting around a target portion of a sample.

It is a schematic diagram of the laser micro-die sector which concerns on the 1st Embodiment of this invention. It is a schematic diagram of the stage of the laser micro-die sector which concerns on the 1st Embodiment of this invention. It is typical sectional drawing of the sample before the cutting | disconnection which concerns on the 1st Embodiment of this invention. It is a typical sectional view of the cut sample concerning a 1st embodiment of the present invention. It is a typical sectional view of a sample before being cut with a laser micro die sector concerning a 1st embodiment of the present invention. It is typical sectional drawing of the sample cut | disconnected by the laser micro-die sector which concerns on the comparative example of the 1st Embodiment of this invention. It is typical sectional drawing of the sample cut | disconnected by the laser micro-die sector which concerns on the 1st Embodiment of this invention. It is typical sectional drawing of the sample cut | disconnected by the laser micro-die sector which concerns on the 1st Embodiment of this invention. It is a schematic diagram of the laser micro-die sector which concerns on the 2nd Embodiment of this invention. It is a schematic diagram of the laser micro-die sector which concerns on the 3rd Embodiment of this invention. It is a schematic diagram of the capillary which concerns on the 3rd Embodiment of this invention. It is a schematic diagram of the capillary which concerns on the 3rd Embodiment of this invention. It is a schematic diagram of the manipulator which concerns on the 3rd Embodiment of this invention. It is a schematic diagram of the laser micro-die sector which concerns on the 4th Embodiment of this invention. It is a schematic diagram of the cantilever which concerns on the 4th Embodiment of this invention. It is a schematic diagram of the magnet which concerns on other embodiment of this invention.

  Embodiments of the present invention will be described below. In the following description of the drawings, the same or similar parts are denoted by the same or similar reference numerals. However, the drawings are schematic. Therefore, specific dimensions and the like should be determined in light of the following description. Moreover, it is a matter of course that portions having different dimensional relationships and ratios are included between the drawings.

(First embodiment)
As shown in FIG. 1, the laser microdissector according to the first embodiment of the present invention includes a stage 10 on which a sample 1 is placed, a microscope 20 that acquires a three-dimensional image of the sample 1, and a three-dimensional image. Based on the target part specifying part 301 for specifying the three-dimensional coordinates of the target part of the sample 1, the cutting laser light source 41 for emitting a cutting laser light for cutting the sample 1, and the three-dimensional coordinates of the target part, A position control mechanism 47 that changes an incident position and an incident angle of the cutting laser with respect to the sample 1 so as to cut the periphery.

  The sample is, for example, a biological sample such as a cultured cell and a sliced biological tissue. For example, a stage driving mechanism that moves the stage 10 is connected to the stage 10. The stage drive mechanism moves the stage 10 in a direction parallel to the surface of the stage 10 (x direction and y direction) or in a direction perpendicular to the stage 10 surface (z direction). Further, the stage driving device tilts the stage 10 around the z direction as an axis.

  As the microscope 20, for example, a confocal Raman microspectroscopic device can be used. The microscope 20 includes an excitation light source 21. The excitation light source 21 is a laser that emits laser light that excites Raman scattered light. Although the wavelength of the laser beam which excites Raman scattered light is 532 nm, 633 nm, and 785 nm, for example, it is not limited to these. The excitation laser light emitted from the excitation light source 21 passes through the line filter 22, for example.

  The microscope 20 has an inverted excitation light path for irradiating the sample 1 with excitation laser light from below, a transmission type excitation light path for irradiating the sample 1 with excitation laser light from above, Both may be provided.

  In the inverted excitation light path, for example, the excitation laser light is reflected by the reflecting mirror 23 downward in the vertical direction with respect to the stage 10 and further reflected by the reflecting mirror 24 in a direction substantially parallel to the stage 10. The excitation laser light reflected by the reflecting mirror 24 is collected by the lens 25. The excitation laser beam condensed by the lens 25 is reflected toward the stage 10 by the half mirror 26 that reflects the excitation laser beam and transmits the Raman scattered light, and passes through the objective lens 27 and is reflected on the sample 1 on the stage 10. Focused.

  In the transmissive excitation light path, for example, the excitation laser light is reflected by the reflecting mirror 23 upward in the vertical direction with respect to the stage 10 and further reflected by the reflecting mirror 44 in a direction parallel to the stage 10. The excitation laser light passes through the xyθ aperture 45 and is collected by the lens 46, for example, enters the objective lens 48 via the position control mechanism 47 including a galvano scanner, and is focused on the sample 1. As will be described later, the transmissive excitation light optical path may at least partially overlap the optical path of the cutting laser light.

  The Raman scattered light generated by the sample 1 irradiated with the excitation laser light passes through the half mirror 26 through the objective lens 27 and is reflected by the reflecting mirror 28 in the direction parallel to the stage 10. The Raman scattered light reflected by the reflecting mirror 28 passes through a bandpass filter 29 that removes Rayleigh scattered light, is collected by a lens 30, and enters a Raman spectrometer 50.

  In the Raman spectroscope 50, the Raman scattered light is collected by the lens 51, and only the Raman scattered light generated at the focal point of the objective lens 27 passes through the pinhole 52. Further, the Raman scattered light is reflected by the reflecting mirror 53 and converted into parallel light by the concave reflecting mirror 54. The collimated Raman scattered light is dispersed by the diffraction grating 55, and the dispersed Raman scattered light is collected by the concave reflecting mirror 56. The condensed Raman scattered light is received by a detector 57 such as a CCD (charge coupled device) image sensor capable of detecting the dispersed Raman scattered light for each wavelength. The detector 57 photoelectrically converts the received Raman scattered light and outputs an electrical signal.

  The detector 57 of the Raman spectrometer 50 is connected to a central processing unit (CPU) 300. The CPU 300 includes an image generation unit 302. The image generation unit 302 receives an electrical signal from the detector 57 and generates a three-dimensional image of the sample 1. The three-dimensional image of the sample 1 includes three-dimensional coordinate (x, y, z) information. A display device 401 is connected to the CPU 300. The display device 401 displays a three-dimensional image of the sample 1.

  An input device 402 is further connected to the CPU 300. Using the input device 402 such as a mouse, the user traces the outline of an arbitrary target portion of the three-dimensional image of the sample 1 displayed on the display device 401 with a mouse pointer, and surrounds the target portion in a three-dimensional shape. The target portion specifying unit 301 included in the CPU 300 specifies the three-dimensional coordinates of the three-dimensional shape that is the locus of the mouse pointer, and stores them in the storage device 403 as the three-dimensional coordinates of the contour of the target portion of the sample 1. Or the target part specific | specification part 301 may extract automatically the three-dimensional coordinate of the outline of a target part by pattern recognition, discrimination | determination of brightness, etc. FIG.

  The cutting laser light source 41 emits, for example, deep ultraviolet (DUV) laser light having a wavelength range of 200 nm to 300 nm as cutting laser light. The cutting laser light emitted from the cutting laser light source 41 passes through the line filter 42, for example. The cutting laser light is reflected by the reflecting mirror 43 upward in the vertical direction with respect to the stage 10, and further reflected by the reflecting mirror 44 in a direction parallel to the stage 10. The cutting laser light passes through the xyθ aperture 45 and is collected by the lens 46, for example, enters the objective lens 48 through the position control mechanism 47 including a galvano scanner, and is collected at the cutting position of the sample 1.

  When the cutting laser light is DUV light, the focal size (diameter) of the cutting laser light is focused to, for example, 400 nm or more and 10 μm or less by the lens 46 or the like. Thereby, it becomes possible to cut | disconnect a sample more correctly. Note that the focal spot size of the cutting laser beam may be changed according to the size of the target portion.

  The position control mechanism 47 drives the galvano scanner, for example, based on the cross-sectional shape of the target portion obtained from the three-dimensional image of the sample 1 to change the incident position and the incident angle of the cutting laser with respect to the sample 1. For example, the position control mechanism 47 reads information on the three-dimensional coordinates of the contour of the target portion of the sample 1 from the storage device 403, so that the focal point of the cutting laser beam moves along the contour of the target portion of the sample 1. Drives mirrors and the like. Here, if the outline of the cross-sectional shape of the target portion of the sample 1 is perpendicular to the surface of the stage 10, the position control mechanism 47 causes the cutting laser light to enter the sample 1 perpendicularly. Further, if the contour of the cross-sectional shape of the target portion of the sample 1 is inclined with respect to the surface of the stage 10, the position control mechanism 47 causes the cutting laser light to enter the sample 1 obliquely.

  A stage drive mechanism for moving the stage 10 may be included in the position control mechanism 47. As shown in FIG. 2, the incident angle of the cutting laser beam with respect to the sample 1 can be changed also by tilting the stage 10 by the stage driving mechanism. As a combination of the stage 10 and the stage driving mechanism, a gonio stage or the like can be used. The galvano scanner and the stage drive mechanism may cooperate to change the incident position and the incident angle of the cutting laser with respect to the sample 1. The incident angle of the cutting laser beam with respect to the sample 1 varies within a range of 0 ° to 90 °, for example.

  For example, as shown in FIG. 3, the outline of the target portion observed in the two-dimensional top surface image of the sample may change in the depth direction of the sample. When the contour of the target part increases in the depth direction of the sample, if the sample is cut along the contour of the target part observed in the two-dimensional top image of the sample, the part with the larger contour in the depth direction is collected. Can not.

  In addition, if the sample is cut so as to include a wide range of surrounding parts by taking a margin from the outline of the target part observed in the two-dimensional upper surface image of the sample, unnecessary parts that are not target parts are not necessary around the collected target parts. The part adheres. For example, if the target part is an abnormal cell surrounded by normal cells, and the normal cells are attached to the collected abnormal cells, the protein from the normal cells is analyzed when analyzing proteins and nucleic acids contained in the abnormal cells. And nucleic acids are mixed, and proteins and nucleic acids of abnormal cells cannot be analyzed accurately.

  On the other hand, as shown in FIG. 4, even in the depth direction of the target portion, the target portion can be accurately cut out from the sample by cutting the sample with the cutting laser light along the outline of the target. .

  Further, for example, as shown in FIG. 5, even when the target portion is buried in the cross-sectional direction of the sample, when the sample is cut vertically with the cutting laser light, the target is placed above and below the target portion as shown in FIG. 6. Samples other than the part adhere. In contrast, as shown in FIG. 7, the stage is tilted or the cutting laser light optical system is driven as shown in FIG. Can be cut with a cutting laser beam to accurately cut out the target portion from the sample.

  As described above, according to the laser microdie sector according to the first embodiment, in the depth direction of the target portion, the target contour is not only a square, but also a circle, a triangle, a polygon, It becomes possible to cut the sample accurately.

  When ultraviolet (UV) light having a wavelength of 355 nm is used as the cutting laser light, the temperature of the portion of the sample irradiated with the cutting laser light rises, and the intermolecular bond in the sample is broken by the high temperature, and the sample is Disconnected. On the other hand, when DUV light having a wavelength of 266 nm or the like is used as the cutting laser light, the intermolecular bond in the sample is directly cut by the DUV light. Therefore, the cutting part by DUV light suppresses heat generation compared with the cutting part by UV light, and the cutting spatial resolution is improved. Further, when the sample is cut with UV light, heat is generated, and thus the sample needs to be fixed with paraffin or the like. However, when the sample is cut with DUV light, the sample does not have to be fixed.

(Second Embodiment)
In the first embodiment, as shown in FIG. 1, an example in which a confocal Raman microspectroscope is used as the microscope 20 has been described. On the other hand, in the laser micro die sector according to the second embodiment, as shown in FIG. 9, an optical microscope that can acquire a three-dimensional image that is not a confocal microscope may be used as the microscope 20.

  The light source 121 is a laser or a halogen lamp that emits illumination light such as laser light or white light. The illumination light is applied to the sample 1 through the objective lens 48, for example. The transmitted illumination light transmitted through the sample 1 passes through the objective lens 127 and is reflected by the reflecting mirror 128 in the direction parallel to the stage 10. The transmitted illumination light reflected by the reflecting mirror 128 is collected by the lens 130 and received by the detector 131. The detector 131 photoelectrically converts the received transmitted illumination light and outputs an electrical signal.

  In the second embodiment, the image generation unit 302 receives an electrical signal from the detector 131 and first generates a two-dimensional image of the sample 1. Furthermore, the image generation unit 302 generates a three-dimensional image of the sample 1 from the two-dimensional image of the sample 1 by a deconvolution method or the like. Here, the deconvolution method is a method of generating a three-dimensional image from a two-dimensional image by a mathematical calculation using a point spread function (PSF) from the diffusion of light that occurs outside the non-focal plane. The PSF can be obtained from, for example, a two-dimensional image of an object whose shape is known, such as a bead, and the degree of light blur in the two-dimensional image.

  Other components of the laser micro-die sector according to the second embodiment are the same as those of the first embodiment. The illumination light or excitation light of a microscope that is not a confocal microscope tends to have lower energy than the illumination light or excitation light of a confocal microscope. Therefore, when a three-dimensional image of the sample 1 is acquired, if a microscope that is not a confocal microscope is used, damage to the sample 1 can be suppressed.

(Third embodiment)
As shown in FIG. 10, the laser microdissector according to the third embodiment further includes a manipulator 60 that collects and collects the target portion isolated by the cutting laser beam. For example, as shown in FIG. 11, the manipulator 60 includes a capillary 61 in which a liquid including a target portion rises due to a capillary phenomenon. The capillary 61 is made of, for example, an acrylic resin and a transparent material such as polyethylene terephthalate (PET).

  The inner wall of the capillary 61 is preferably hydrophilic so that the target portion does not adhere to the inner wall of the capillary 61. For example, as shown in FIG. 12, the inner wall of the capillary 61 can be made hydrophilic by providing fine irregularities on the inner wall of the capillary 61 or roughening the surface. Alternatively, the inner wall of the capillary 61 may be made hydrophilic by plasma treatment, chemical vapor deposition (CVD), or the like. Further, by increasing the degree of hydrophilicity from the inlet of the capillary 61 toward the back, the liquid including the target portion can be raised deep inside the capillary 61 by capillary action.

  As shown in FIG. 13, the manipulator 60 reads, for example, the three-dimensional coordinate information of the contour of the target portion of the sample 1 from the storage device 403 shown in FIG. 10, and introduces the capillary 61 shown in FIG. 13 near the target portion. You may bring your mouth close. Other components of the laser micro-die sector according to the third embodiment are the same as those in the first or second embodiment.

(Fourth embodiment)
In the laser microdissector according to the fourth embodiment, as shown in FIG. 14, a manipulator 60 that collects and collects a target portion isolated by cutting laser light includes a cantilever 62 shown in FIG. As the cantilever 62, a cantilever used in an atomic force microscope (AFM) can be used.

  For example, by modifying the tip of the cantilever 62 with a molecule that specifically binds to the target moiety, it is possible to recover the target moiety using intermolecular affinity. For example, by previously modifying the target portion with biotin, modifying the tip of the cantilever 62 with avidin, and bringing the tip of the cantilever 62 close to the target portion, the target portion can be collected and recovered by biotin-avidin binding. Is possible. Other protein interactions, nucleic acid interactions, and the like may be used for collecting and collecting the target portion. Alternatively, the target portion may be modified with magnetic particles, and the target portion may be collected and collected with the tip of the cantilever 62 provided with magnetism.

  The manipulator 60 shown in FIG. 14 may read, for example, the three-dimensional coordinate information of the contour of the target portion of the sample 1 from the storage device 403 and bring the tip of the cantilever 62 shown in FIG. 15 close to the target portion. Other components of the laser micro-die sector according to the fourth embodiment are the same as those in the first or second embodiment.

(Other embodiments)
Although the present invention has been described by the embodiments as described above, it should not be understood that the description and drawings constituting a part of this disclosure limit the present invention. From this disclosure, various alternative embodiments, examples and operational techniques should be apparent to those skilled in the art. For example, in the first embodiment, an example in which a confocal Raman microspectroscope is used as a microscope is shown. Good. For example, when the target substance is stained with a fluorescent substance or a dye, only the surface of the target part is observed in the three-dimensional image of the sample. For example, it is possible to obtain a three-dimensional coordinate of a target portion by binarizing a three-dimensional image on the basis of a predetermined value regarding brightness and extracting only a bright portion. The sample may be an artifact other than a biological sample. Further, for example, as shown in FIG. 16, the target portion may be modified with magnetic particles, and the target substance may be recovered with an electromagnet 63 as a manipulator. A plurality of target substances isolated from the sample may be collected at once by the wide electromagnet 63. Thus, it should be understood that the present invention includes various embodiments and the like not described herein.

1 Sample 10 Stage 20 Microscope 21 Excitation light source 22 Line filters 23, 24, 28, 43, 44, 53 Reflectors 25, 30, 46, 51 Lens 26 Half mirror 27, 48 Objective lens 29 Band pass filter 41 Cutting laser light source 42 Line filter 45 Aperture 47 Position control mechanism 50 Raman spectrometer 52 Pinholes 54 and 56 Concave reflector 55 Diffraction grating 57 Detector 60 Manipulator 61 Capillary 62 Cantilever 63 Electromagnet 121 Light source 127 Objective lens 128 Reflector 130 Lens 131 Detector 301 Target Partial identification unit 302 Image generation unit 401 Display device 402 Input device 403 Storage device

Claims (34)

A microscope for acquiring a three-dimensional image of the sample;
A target portion specifying unit for specifying the three-dimensional coordinates of the target portion of the sample from the three-dimensional image;
A cutting laser light source that emits a cutting laser beam for cutting the sample;
A position control mechanism that changes an incident position and an incident angle of the cutting laser with respect to the sample such that the periphery of the target portion of the sample is cut based on the three-dimensional coordinates of the target portion;
With the laser micro die sector.   The laser microdissector according to claim 1, wherein an incident angle of the cutting laser beam with respect to the sample is changed based on a cross-sectional shape of the target portion obtained from the three-dimensional image.   The laser microdissector according to claim 1 or 2, wherein an incident angle of the cutting laser beam with respect to the sample is changed along a contour of a cross-sectional shape of the target portion obtained from the three-dimensional image.   4. The laser micro of claim 1, wherein the position control mechanism drives an optical system of the cutting laser light to change an incident position and an incident angle of the cutting laser light with respect to the sample. 5. Die sector.   The laser microdissector according to any one of claims 1 to 4, wherein the position control mechanism changes an incident angle of the cutting laser light with respect to the sample by tilting a stage on which the sample is placed.   The laser microdissector according to any one of claims 1 to 5, wherein an incident angle of the cutting laser beam with respect to the sample is changed in a range of 0 ° to 90 °.   The laser microdie sector according to any one of claims 1 to 6, wherein the cutting laser light is deep ultraviolet laser light.   The laser microdissector of Claim 7 whose wavelength range of the said cutting | disconnection laser beam is 200 nm or more and 300 nm or less.   The laser microdie sector according to claim 7 or 8, wherein a focal spot size of the cutting laser beam is 400 nm or more and 10 µm or less.   The laser microdissector of any one of claims 1 to 9, further comprising a manipulator that recovers the target portion isolated by the cutting laser light.   The laser microdissector of claim 10, wherein the manipulator comprises a capillary in which a liquid containing the target portion rises due to capillary action.   The laser microdissector of claim 10, wherein the manipulator comprises a cantilever that collects the target portion.   The laser microdissector of any one of claims 10 to 12, wherein the manipulator recovers the target portion using magnetic force.   The laser microdissector of any one of claims 10 to 12, wherein the manipulator recovers the target portion using intermolecular affinity.   The microscope according to any one of claims 1 to 14, wherein the microscope is any one selected from the group consisting of a confocal laser microscope, a confocal Raman microspectroscope, an optical microscope, a fluorescence microscope, and an electron microscope. Laser micro die sector.   The laser microdissector of any one of claims 1 to 15, wherein the sample is a biological sample.   The laser microdissector of any one of claims 1 to 16, wherein the sample is a biological tissue. Acquiring a three-dimensional image of the sample;
Identifying the three-dimensional coordinates of the target portion of the sample from the three-dimensional image;
A cutting laser beam for cutting the sample is emitted, and an incident position and an incident angle of the cutting laser with respect to the sample are changed so that the periphery of the target portion of the sample is cut based on the three-dimensional coordinates of the target portion. When,
A laser microdissection method comprising:   19. The laser microdissection method according to claim 18, wherein an incident angle of the cutting laser beam with respect to the sample is changed based on a cross-sectional shape of the target portion obtained from the three-dimensional image.   The laser microdissection method according to claim 18 or 19, wherein an incident angle of the cutting laser beam with respect to the sample is changed along a cross-sectional outline of the target portion obtained from the three-dimensional image.   21. The laser microdissection method according to claim 18, wherein an optical system of the cutting laser beam is driven to change an incident position and an incident angle of the cutting laser beam with respect to the sample.   The laser microdissection method according to any one of claims 18 to 21, wherein an incident angle of the cutting laser beam to the sample is changed by inclining a stage on which the sample is placed.   The laser microdissection method according to any one of claims 18 to 22, wherein an incident angle of the cutting laser beam with respect to the sample varies in a range of 0 ° to 90 °.   The laser microdissection method according to any one of claims 18 to 23, wherein the cutting laser beam is a deep ultraviolet laser beam.   The laser microdissection method according to claim 24, wherein a wavelength range of the cutting laser light is 200 nm or more and 300 nm or less.   The laser microdissection method according to claim 24 or 25, wherein a focal spot size of the cutting laser beam is 400 nm or more and 10 µm or less.   27. The laser microdissection method according to any one of claims 18 to 26, further comprising recovering the target portion isolated with the cutting laser light with a manipulator.   28. The laser microdissection method according to claim 27, wherein the manipulator comprises a capillary in which a liquid containing the target portion rises by capillary action.   28. The laser microdissection method of claim 27, wherein the manipulator comprises a cantilever that collects the target portion.   30. The laser microdissection method according to any one of claims 27 to 29, wherein the manipulator recovers the target portion using magnetic force.   30. A laser microdissection method according to any one of claims 27 to 29, wherein the manipulator recovers the target portion using intermolecular affinity.   The microscope for acquiring an image of the sample is any one selected from the group consisting of a confocal laser microscope, a confocal Raman microspectroscope, an optical microscope, a fluorescence microscope, and an electron microscope. The laser microdissection method according to claim 1.   The laser microdissection method according to any one of claims 18 to 32, wherein the sample is a biological sample.   The laser microdissection method according to any one of claims 18 to 33, wherein the sample is a living tissue.