JP6611610B2 - Laser microdissection device, analyzer including the laser microdissection device, and method for manufacturing microchip - Google Patents

Description

  The present invention relates to a laser microdissection apparatus, an analysis apparatus including the laser microdissection apparatus, and a method for manufacturing a microchip. In particular, the position coordinates of a sample and the collected sample when irradiated with laser light are thermoplastic films. The spatial resolution of the analyzer can be improved by associating and storing the position coordinates to be adhered to the sample, and further, the analyzer can display the sample image and the analysis result by the analyzer together It is.

  In recent years, in life science research, it is necessary to accurately analyze where a molecule exists in a biological sample, and what kind of molecule exists in a biological sample of interest. Is required.

  In addition, when analyzing a biological sample, mass spectrometry imaging that displays what kind of molecules are included in the sample being observed with a microscope is desired rather than simply taking out the sample and analyzing it. Has been. As an apparatus that realizes the request, a mass microscope that can observe a biological sample with a microscope and perform mass analysis on the biological sample is commercially available.

  In a general mass spectrometry method, a sample is irradiated with laser light, and the irradiated portion of the sample is ionized for mass analysis (hereinafter referred to as “ionized laser light”). "). Therefore, in order to continuously analyze a sample, after irradiating the sample with ionized laser light, the sample is moved by a length corresponding to the diameter of the ionized laser light, and again irradiated with ionized laser light. It is possible to analyze continuously. However, in mass spectrometry, not only the portion irradiated with the ionized laser light is ionized, but the sample before the movement can be obtained by irradiating the ionized laser light after moving the length corresponding to the diameter of the ionized laser light. There is a problem of ionization at the same time. For this reason, it is difficult to continuously move the portion irradiated with the ionized laser beam, and the spatial resolution is about 200 μm although it depends on the diameter of the ionized laser beam to be used. Is difficult (see Non-Patent Document 1). In addition, the biological sample in the portion irradiated with the ionized laser beam cannot be completely ionized, and there is a problem in the quantitativeness of the analysis result. Furthermore, the conventional mass microscope is simply a combination of a microscope and a mass spectrometer, and there is a problem that the apparatus becomes large.

  In order to solve the above problems, a mass microscope using an atmospheric pressure ionization method is known (see Patent Document 1). In the mass microscope using the atmospheric pressure ionization method, the spatial resolution can be reduced to about 5 μm by narrowing the ionized laser beam to about 1 μm in diameter, and since there is no need to use a vacuum, the apparatus is downsized. There is an advantage that the sample can be easily analyzed. However, since the mass microscope using the atmospheric pressure ionization method is not evacuated when the sample is ionized, the sample that can be analyzed is limited to those that can be ionized by irradiating with ionized laser light under atmospheric pressure. There is a problem that the molecular weight is about 2000, and various samples cannot be analyzed. In addition, in order to reduce the diameter of the ionized laser light, it is necessary to improve the performance of the ionized laser light source, so that there is a problem that the cost is greatly increased.

  Furthermore, the analysis using the atmospheric pressure ionization method is limited to a time-of-flight mass spectrometer (hereinafter sometimes referred to as “MALDI-TOF-MS”) using a matrix-assisted laser desorption ionization method. Others such as liquid chromatograph mass spectrometry (hereinafter sometimes referred to as “LC-MS”), electrospray ionization mass spectrometry (hereinafter sometimes referred to as “ESI-MS”), etc. There is a problem that it is not compatible with mass spectrometry.

  In addition to the above-described method of directly irradiating a sample with ionized laser light and performing mass spectrometry, for example, a method of collecting a sample at a desired position from a sample with a laser microdissection and performing LC-MS analysis is also known. (See Patent Document 2). However, the method described in Patent Document 2 only detects the protein contained in the collected sample, and it is not possible to display the detection-analyzed data superimposed on a biological sample like a mass microscope. There is a problem that you can not.

  In addition to the sample collection method using the laser microdissection device described in Patent Document 2 above, the sample collection method using the laser microdissection device may include a predetermined measurement from the sample in the submillimeter region by cold laser ablation or multiphoton absorption. (Refer to Patent Document 3), a thermoplastic film is placed on a sample, and laser light (hereinafter, laser light for cutting out the sample is sometimes referred to as “dissection laser light”). There is known a method of adhering a sample to a thermoplastic film by irradiating (see Patent Documents 4 and 5) and the like.

  However, the techniques described in Patent Documents 4 and 5 are merely techniques for taking out cells and DNA from tissue specimens. In the first place, a biological sample adhered to a thermoplastic film is irradiated with ionized laser light. When mass spectrometry is performed, it is usually thought that thermoplastic films are also ionized by ionized laser light, resulting in noise in mass analysis. Therefore, it is known to perform mass spectrometry with the sample adhered to the thermoplastic film. It was not done.

  Furthermore, the laser microdissection device described in Patent Documents 4 and 5 simply collects a sample of a desired portion from a biological sample. The laser microdissection device described in Patent Documents 4 and 5 Even if it is used as a sample collection device for analyzing with a normal mass spectrometer, there is a problem that it is difficult to improve the spatial resolution.

JP 2006-170689 A JP 2013-27387 A JP 2009-103701 A Japanese Patent No. 3786711 Japanese Patent No. 4156851

Makoto Sawada, “Development of High Resolution Mass Spectrometry Imaging Sample Preparation Technology for Subcellular Analysis”, [online], [searched on July 17, 2014], Internet <URL: http: // www. jst. go. jp / sentan / hyouka / h24cyukan / 4-08sawada_m. html>

  The present invention has been made in order to solve the above-mentioned conventional problems, and as a result of intensive research, (1) a laser section of a laser microdissection device is cut out by irradiating the sample with a section laser beam. Mass spectrometry is performed by directly irradiating the sample adhering to the sample (hereinafter referred to as “collected sample”, which is cut out with a dissection laser beam and adhered to a thermoplastic film) by ionizing the laser beam. Even if it is performed, there is no noise derived from the thermoplastic film, and the collected sample can be mass-analyzed with good quantitativeness. (2) The position coordinates of the sample where the dissection laser beam is irradiated and the heat where the collected sample adheres The position coordinates of the plastic film are associated and stored in the storage means, and the position coordinates of the sample and the thermoplasticity stored in the storage means are stored. Based on the position coordinates of the film, a moving means drive control unit that drives and controls the sample moving means and the thermoplastic film moving means adheres to the thermoplastic film based on the distance between the samples collected by irradiating the dissection laser light. (3) The distance between the collected samples to be adhered to the thermoplastic film is the distance at which the adjacent sample is not affected by the ionized laser light when irradiated with the ionized laser light. By doing so, it is possible to analyze a small sample continuously collected by a laser microdissection device without the influence of the adjacent sample without changing the ionization laser light source of the conventional mass spectrometer. The spatial resolution of the device can be improved, and (4) the position of the sample at the location where the dissection laser beam is irradiated The position coordinates of the thermoplastic film at the location where the mark and the collected sample are bonded are associated and stored in the storage means, and the position coordinates of the sample bonded to the thermoplastic film and the analysis result of the sample by the analyzer are linked. By doing so, mass imaging can be performed easily, and (5) the interval between the collected samples to be adhered to the thermoplastic film can be arbitrarily set. For example, the sample is contaminated as a solution by applying a liquid to the collected sample. Thus, the present invention has been completed by newly finding that it can be used for sample preparation of various analyzers such as LC-MS by setting it within a range that can be recovered without any problems.

  That is, an object of the present invention is to provide a laser microdissection apparatus, an analysis apparatus including the laser microdissection apparatus, and a microchip manufacturing method.

  The present invention relates to a laser microdissection apparatus, an analysis apparatus including the laser microdissection apparatus, and a method for manufacturing a microchip, which will be described below.

(1) Sample moving means capable of placing a sample and moving the sample in the horizontal direction;
A thermoplastic film moving means capable of placing a thermoplastic film and moving the thermoplastic film in a horizontal direction and a vertical direction;
A laser irradiation part for irradiating a sample with a dissection laser beam, cutting out a sample of the portion irradiated with the dissection laser beam, and bonding the sample to a thermoplastic film as a sample,
Memory means for associating and memorizing the position coordinates of the sample where the dissection laser beam is irradiated and the position coordinates of the thermoplastic film where the sample is adhered, and the position coordinates and thermoplasticity of the sample stored in the memory means A moving means driving control unit for driving and controlling the sample moving means and the thermoplastic film moving means based on the position coordinates of the film;
A laser microdissection apparatus comprising:
(2) At least two or more collected samples can be adhered to the thermoplastic film, and the distance between centers of any collected sample and another collected sample adjacent to the collected sample is a dissection laser beam. The laser microdissection apparatus according to (1) above, wherein the laser microdissection apparatus is larger than the distance between the centers of the samples when irradiated.
(3) The above (2) is characterized in that the center-to-center distance between the arbitrary collected sample and the collected sample adjacent to the collected sample is larger than (diameter of ionized laser light + diameter of collected sample) / 2. The laser microdissection device described.
(4) The laser microdissection apparatus according to any one of (1) to (3) above, wherein the sample moving means can also move in the vertical direction.
(5) An analyzer including the laser microdissection device according to any one of (1) to (4) above.
(6) The analysis apparatus according to (5), wherein the analysis apparatus is one selected from a mass analysis apparatus, an analysis apparatus including chromatography, an elemental analysis apparatus, a nucleic acid sequence analysis apparatus, and a microchip analysis apparatus. Analysis equipment.
(7) The analyzer according to (5) or (6) above, wherein the analyzer includes a display unit that can display a sample image and an analysis result by the analyzer together.
(8) A method for producing a microchip in which a sample is adhered to a thermoplastic film, using the laser microdissection device according to any one of (1) to (4).

The laser microdissection apparatus of the present invention includes a storage means for storing the position coordinates of the sample at the location where the section laser beam is irradiated and the position coordinates of the thermoplastic film at the location where the collected sample adheres, and stores the relationship. The interval between the collected samples to be adhered to can be set at an interval arbitrarily larger than the interval between the samples collected by irradiating the dissection laser beam.
Therefore, spatial resolution can be improved by combining the laser microdissection apparatus of the present invention with, for example, a conventional mass spectrometer. Furthermore, since the position coordinates of the sample and the position coordinates of the thermoplastic film are stored, it is possible to easily perform two-dimensional and three-dimensional mass imaging by associating the analyzed position coordinates with the analysis results. Therefore, mass imaging can be performed at low cost using a conventional mass spectrometer.
In addition, when a sample is prepared using the laser microdissection apparatus of the present invention, the sample ionization method is not limited to the atmospheric pressure ionization method, and thus a sample having a molecular weight of about 15,000 can be analyzed.
Furthermore, the laser microdissection apparatus of the present invention can set the interval between the collected samples adhered to the thermoplastic film to an arbitrary size. Therefore, for example, by applying a liquid to the collected sample so that the sample can be collected without contamination as a solution, the sample can be used as a sample preparation device for various analyzers such as LC-MS.

FIG. 1 is a diagram showing an outline of a laser microdissection apparatus 1 of the present invention. FIG. 2 is a diagram showing the principle of laser microdissection. FIG. 3 is a photograph substituted for a drawing and a photograph of the hollow ring 34. FIG. 4 is a diagram showing an example in which the thermoplastic film 31 is irradiated with the disection laser light through the sample 23. FIG. 5 is a diagram showing the relationship between the position coordinates of the sample where the die section laser light is irradiated and the position coordinates of the thermoplastic film 31 to which the collected sample adheres, and shows an example in which the sample is continuously cut out. . FIG. 6 is a diagram showing the relationship between the position coordinates of the sample where the dissection laser beam is irradiated and the position coordinates of the thermoplastic film 31 to which the collected sample adheres, and an example in which the position to cut out the sample is arbitrarily set Show. FIG. 7 is a cross-sectional view of an example of the laser microdissection apparatus 1 of the present invention and a diagram showing an outline of the system. FIG. 8 is a photograph substituted for a drawing and showing the appearance of the produced laser microdissection apparatus. FIG. 9 is a drawing-substituting photograph, FIG. 9 (1) is an overall image of the section when the collection range is set, and FIG. 9 (2) is an enlarged view of the collection range displayed on the display unit. In FIG. 9 (3), numerals and broken circles are overwritten on the collected sections after irradiation with the dissection laser. FIG. 10 is a drawing-substituting photograph, which is a photograph after the matrix is applied to the EVA film to which the collected sample is adhered. FIG. 11 is mass imaging of the sample obtained in Example 2. FIG. 12 is a mass spectrum of the analysis result of the peptide obtained in Example 2. FIG. 13 is a drawing-substituting photograph showing a section map, a sample collection range, a sample collection position in the collection range, a result of Amyloid beta staining, and a HeatMap display of the mass spectrometry result of Amyloid beta 1-40. . FIG. 14 is a drawing-substituting photograph, FIG. 14 (1) is a three-dimensional image of the hippocampus of a normal mouse, and FIG. 14 (2) is a three-dimensional image of the hippocampus of an Alzheimer's disease model mouse.

  Below, the laser microdissection apparatus of this invention, the analyzer including this laser microdissection apparatus, and the manufacturing method of a microchip are demonstrated in detail.

  FIG. 1 schematically shows a laser microdissection apparatus 1 according to the present invention, including a sample moving means 2, a thermoplastic film moving means 3, a laser irradiation section 4, a storage means (not shown), and a moving means drive control section. Yes.

  The sample moving means 2 shown in FIG. 1 has a sample mounting table 22 on which a slide glass 21 or the like on which a sample is mounted can be mounted, and the sample mounting table 22 is moved in the horizontal direction (X and Y axis directions). And a driving force transmission mechanism that transmits the driving force of the driving source to the sample mounting table 22. As a driving source, a pulse motor, an ultrasonic motor, or the like may be used. The driving force transmission mechanism may be a known one such as a driving force transmission mechanism for driving a sample mounting table used in an inverted microscope or the like in the horizontal direction.

  The thermoplastic film moving means 3 shown in FIG. 1 has an arm 32 on which the thermoplastic film 31 can be placed at one end and the other end can be attached to an arm column 33, and the arm 32 is placed in the horizontal direction (X, Y axes). Arm strut 33 that can rotate and move in the vertical direction (Z-axis direction), a driving source (not shown) for rotating the arm 32 in the horizontal direction and moving in the vertical direction, and the driving force of the driving source are transmitted. Thus, a driving force transmission mechanism for rotating and moving the arm 32 is included. As a driving source, a pulse motor, an ultrasonic motor, or the like may be used. The driving force transmission mechanism may be a known arm mechanism that can rotate in the horizontal direction and move in the vertical direction, such as an arm mechanism for moving the sample of the automatic analyzer. The thermoplastic film moving means 3 is not limited to the embodiment illustrated in FIG. 1, and is not particularly limited as long as the thermoplastic film 31 for adhering the collected sample can be moved in the horizontal direction and the vertical direction.

  The thermoplastic film 31 is not particularly limited as long as it can be dissolved by a dissection laser beam irradiated from a laser irradiation unit 4 to be described later and can be bonded without modifying the collected sample. As the thermoplastic film, it is preferable to use a thermoplastic resin having a melting point of up to about 50-70 ° C. as a raw material since the lower melting point can prevent thermal denaturation of the sample. For example, ethyl vinyl acetate (EVA), Examples include polyolefin, polyamide, acrylic, and polyurethane. In addition, an organic dye such as naphthalene cyanine dye may be added to the thermoplastic film in order to selectively absorb the spectrum of the wavelength section of the dissection laser light source, depending on the wavelength section of the used dissection laser light source. A suitable organic dye may be selected. The thermoplastic film 31 may be prepared by appropriately blending the above-described thermoplastic resin and organic dye, or a commercially available thermoplastic film may be used. Examples of the commercially available thermoplastic film include a thermoplastic transfer film (manufactured by Electro Seal), a thermoplastic EVA film (manufactured by Sigma-Aldrich Japan), and the like.

  As the dissection laser light source included in the laser irradiation unit 4, it is preferable to use a laser beam with a single mode fiber output in order to minimize the irradiation spot, and a near-infrared high NA long focus objective for focusing. It is preferable to use a lens. The pulse width is 0.1 to 100 milliseconds, preferably 5 milliseconds, the wavelength is 785 to 900 nanometers, preferably 808 nanometers, and the output is 0.2 to 0.3 watts. The laser power is preferably from 0.1% to 100%, preferably from 80% to 100%, which can generate pulsed laser light. Specific examples include Z-808-200-SM (manufactured by Lucille Co.). .

  FIG. 2 is a diagram illustrating an example of the principle of laser microdissection. As shown in FIG. 2 (1), a sample 23 fixed to the slide glass 21, and a resin-made support such as a light-transmitting acrylic resin or polycarbonate resin with a thermoplastic film 31 mounted thereon are shown in FIG. 2 (2). As shown in the figure, the thermoplastic film 31 is brought into contact with the sample 23, the sample 23 is irradiated with the disection laser beam 43 through the support and the thermoplastic film 31, and then the thermoplastic film 31 is applied as shown in FIG. By pulling away from the sample 23, the sample 23 cut out by irradiation with the dissection laser beam 43 can be adhered to the thermoplastic film 31.

  If there is a gap between the thermoplastic film 31 and the sample 23, it is difficult for the collected sample to adhere to the thermoplastic film 31. Therefore, for example, the support can be held in the hollow portion of the hollow ring 34 made of metal such as stainless steel or titanium, and the thermoplastic film 31 can be pressed against the sample 23 by the weight of the hollow ring 34. . The contact condition may be adjusted as appropriate by adjusting the thickness of the hollow ring and changing the weight.

  FIG. 3 (1) is a side view photograph of the hollow ring 34, which is composed of a hollow base portion 35 and a hollow convex portion 36 that holds a support body on which a thermoplastic film 31 is mounted at the tip. FIG. 3B is a photograph taken from the tip direction of the convex portion 36, and a support body on which the thermoplastic film 31 is mounted is held in the hollow portion 37 of the base portion 35 and the convex portion 36. Further, in order to prevent displacement when the hollow ring 34 is brought into contact with the slide glass 21, the convex portion 36 may be provided with an O-ring 38 made of silicon rubber, synthetic rubber or the like. FIG. 3 (3) is a photograph in which a hollow ring 34 is attached to the tip of the arm 32. A hole larger than the convex portion 36 and smaller than the base 35 is formed at the tip of the arm 32, and the convex portion 36 may be inserted.

  When the thermoplastic film 31 is attached to the support, it is necessary to peel the thermoplastic film 31 from the support in order to analyze the collected sample. In order to improve efficiency by allowing the collected sample to be directly analyzed by a mass spectrometer or the like, for example, the thermoplastic film 31 may be directly attached to a slide glass subjected to a conductive treatment. In that case, for example, a fixing means for fixing the slide glass may be provided at the tip of the arm 32, and the arm 32 may be attached so as to hold the slide glass.

  Further, since the laser microdissection apparatus shown in FIG. 2 is based on an inverted microscope, the dissection laser light 43 is irradiated onto the sample 23 through the thermoplastic film 31, but the dissection laser light 43 is The thermoplastic film 31 may be irradiated through the sample 23. FIG. 4 is a view showing an example in which the thermoplastic film 31 is irradiated with the dissection laser beam 43 through the sample 23. The slide is made of a material that can transmit the dissection laser beam 43, for example, glass or light transmissive resin. The thermoplastic film 31 attached to the base material 39 may be brought into contact with the sample 23 fixed to 24 and the dissection laser light 43 may be irradiated from the slide 24 side. In this case, since the base material 39 may or may not transmit light, in addition to a light-transmitting material such as glass, it may be made of a light-impermeable material that can be used in various analyzers as it is. Moreover, when using a mass spectrometer as an analyzer, the base material 39 may be subjected to a conductive treatment, for example, a conductive material such as metal, a slide glass that has been subjected to a conductive process, or an optional adhesive tape that has been subjected to a conductive process. What is necessary is just to produce with the material of this.

  FIG. 5 is a diagram showing the relationship between the position coordinates of the sample where the die section laser light is irradiated and the position coordinates of the thermoplastic film 31 to which the collected sample adheres, and shows an example in which the sample is continuously cut out. . For example, when the sample 23 in FIG. 5A is continuously cut out as a, b, c... (I) The section a of the sample 23 is irradiated with a dissection laser beam by the sample moving means 2. Move to the position where (Ii) Next, the thermoplastic film moving means 3 moves the location a ′ where the collected sample a of the thermoplastic film 31 shown in FIG. 5 (2) adheres to a position overlapping the sample 23a, and vertically moves the arm 32. The thermoplastic film 31 and the sample 23 are brought into contact with each other by being lowered in the direction. (Iii) By irradiating the dissection laser beam, the sample collected from the location of the sample 23a is bonded to the position a 'of the thermoplastic film 31, and then the arm 32 is raised in the vertical direction to raise the thermoplastic film 31. Is separated from the sample 23, and the sample 23a is adhered to a predetermined portion of the thermoplastic film 31. For the samples 23b, c,..., The above steps (i) to (iii) are repeated to adhere the samples 23b, c,... To the b ', c',. be able to.

  The size A of the sample collected from the sample 23 may be changed according to the target tissue section or the purpose. For example, 1 μm to 5 μm when analyzing subcellular structures and obtaining high spatial resolution, 15 μm to 30 μm when collecting single cells, and 50 μm to 100 μm when collecting cancer or degenerated sites The sample may be cut out and collected from the sample 23 by irradiating a dissection laser beam. The size of the sample to be cut can be adjusted by adjusting the diameter and intensity of the dissection laser light to be radiated, and a sample with the same size as the diameter of the dissection laser light can be cut out. The sample larger than the diameter of the dissection laser beam can also be cut out by increasing the irradiation time. What is necessary is just to adjust the diameter and intensity | strength of a dissection laser beam suitably according to the sample to extract | collect. The diameter of the dissection laser beam may be reduced by using an optical aperture, a condenser lens, or the like. The intensity of the dissection laser beam may be changed by changing the voltage of the laser oscillator using a variable resistor or the like.

  In the present invention, the sample collected from the sample 23 can be adhered to the thermoplastic film 31 at an arbitrary interval larger than the interval between the samples before collection. Therefore, the spatial resolution of the analysis can be improved by adjusting the interval for adhering the collected samples according to the spatial resolution of the analyzer for analyzing the collected samples, the pretreatment of the samples, and the like. For example, in the case of analyzing with a mass spectrometer, the diameter may be larger than (diameter of ionized laser light + diameter of collected sample) / 2. In the case of general-purpose MALDI-TOF-MS, the diameter of ionized laser light is about 200 μm. Therefore, the distance between the centers of the collected sample adhered to the thermoplastic film 31 and the adjacent collected sample may be larger than (200 μm + the diameter of the collected sample) / 2. If the shape of the sample to be collected does not become circular depending on the type of sample, the longest line connecting any outer peripheral point and the outer peripheral point of the sample to be collected may be used as the diameter, and the diameter of the dissection laser beam and / or The diameter of the sample to be cut out set by adjusting the intensity of the dissection laser beam may be used as the diameter of the collected sample. Note that (200 μm + sampled sample diameter) / 2 is a case where irradiation is performed so that the center of the sampled sample and the center of the ionized laser beam are the same. For example, the diameter of the sampled sample is larger than the diameter of the ionized laser beam. If the irradiation control is performed so that the end portion of the ionized laser beam can be irradiated to the collected sample, the distance between the center of the collected sample and the adjacent collected sample is smaller than (200 μm + the diameter of the collected sample) / 2. Also good. If the sample is liquefied and analyzed by liquid chromatography, etc., when liquefying and collecting the sample, consider the amount of liquid required for liquefaction so that it will not be contaminated with the adjacent sample, What is necessary is just to adjust suitably.

  FIG. 6 is a diagram showing the relationship between the position coordinates of the sample where the dissection laser beam is irradiated and the position coordinates of the thermoplastic film 31 to which the collected sample adheres, and an example of arbitrarily setting the position where the sample is collected. Is shown. In the example shown in FIG. 6, it is sufficient to set in advance in which order the samples of the sample 23 are collected, and to determine in advance in which location of the thermoplastic film 31 the sample is to be adhered. In the example shown in FIG. 6, a sample at an arbitrary position in the sample can be cut out, which is effective for, for example, analysis of a sub-cellular or the like, and differential collection of a plurality of objects scattered on the sample.

  FIG. 7 is a cross-sectional view of an example of the laser microdissection apparatus 1 of the present invention and a diagram showing an outline of the system. The laser microdissection apparatus 1 shown in FIG. 7 shows an example manufactured based on an inverted optical microscope, and an illumination unit 50 is provided above the sample mounting table 22. In addition to the laser irradiation unit 4, an illumination light source 51, an optical lens 52, a dichroic mirror 53, and a condenser lens 54 are provided inside the illumination unit 50, and illumination light radiated from the illumination light source 51 is optical lens 52. The sample 23 is irradiated from above the sample mounting table 22 through the dichroic mirror 53 and the condenser lens 54. The sample 23 is fixed to the center of the slide glass 21 without being covered with a cover glass. The sample 23 may be fixed by providing the sample mounting table 22 with a known fixing means for holding the slide glass 21 by spring force and fixing the slide glass 21 to the sample mounting table 22.

  The laser irradiation unit 4 is provided with a dissection laser light source 41 and a collimator lens 42 disposed on the optical axis of the dissection laser light output from the dissection laser light source 41, and is output from the dissection laser light source 41. The dissection laser light is reflected by the dichroic mirror 53 through the collimator lens 42, travels on the same axis as the optical axis of the illumination light, and irradiates the sample 23 through the thermoplastic film 31 at the center of the observation field. Yes.

  Below the sample mounting table 22 of the laser microdissection apparatus 1, an observation optical system 61 and a CCD camera 62 including an objective lens 57, a half mirror 58, a mirror 59, and an eyepiece lens 60 are arranged. The observation optical system 61 reflects the observation light irradiated with the illumination light from the illumination light source 51 and transmitted through the sample 23, reflected by the mirror 59 through the objective lens 57 and the half mirror 58, and can be visually observed by the eyepiece 60. The CCD camera 62 is arranged so that the imaging surface is positioned at the imaging position of the observation light on the spectral optical axis of the half mirror 58 so that the same observation image as that visually observed by the eyepiece 60 can be captured. It has become. Note that FIG. 7 shows an example manufactured based on an inverted microscope. However, it is not necessary to observe the sample 23 with the eyepiece 60, and when only the display on the display unit 65 is performed, the half mirror 58 and the mirror 59 are used. There is no need to provide the eyepiece 60, and the CCD camera 62 may be disposed at a position where the observation light that has passed through the objective lens 57 forms an image directly or via a mirror.

  A laser controller 63 is connected to the laser irradiation unit 4, and a control computer 64 is connected to the laser controller 63. The control computer 64 is connected to a display unit 65, for example, a pointing device 66 such as a mouse, and moving means drive control for controlling the driving of the CCD camera 62, the sample moving means 2 and the thermoplastic film moving means 3. The part 67 is connected.

  The control computer 64 is a personal computer or the like incorporating software dedicated to the laser microdissection apparatus 1 of the present invention, and is output from the dissection laser light source 41 by the laser controller 63 based on the command of the control computer 64. The output state of the disection laser light to be controlled is controlled, and the moving means drive control unit 67 controls the power supply and pulse signals to the driving sources of the sample moving means 2 and the thermoplastic film moving means 3. Yes.

  A joystick controller 68 is connected to the moving means drive control unit 67. A joystick (not shown) is incorporated in the joystick controller 68, and a pulse signal corresponding to the operation of the joystick is given to the sample moving means 2. Here, for example, the selection of the driving source of the sample moving means 2 for sending the pulse signal and the rotation direction of the driving source are determined in the tilt direction of the joystick, and the pulse source is oscillated at a frequency corresponding to the tilt angle. It rotates so that the sample moving means 2 moves. The joystick controller 68 is not indispensable. For example, an image photographed by the CCD camera 62 is displayed on the display unit 65, and the position where the pointing device 66 is inserted becomes the center of the display unit 65. Is selected on the screen having a touch function and is controlled so that the touched position becomes the center of the display unit 65, so that the driving source of the sample moving means 2 can be selected and the driving source can be rotated without using the joystick. The direction may be controlled.

  Next, a procedure for cutting out a sample by the laser microdissection apparatus 1 of the present invention and an operation by dedicated software incorporated in the control computer 64 will be described.

  First, the laser microdissection apparatus 1 is turned on to start the dedicated software of the control computer 64, and the sample mounting table 22 is brought into a predetermined position for initialization in accordance with an automatically executed initialization operation. Moving. Then, the origin position is detected by an origin detector (not shown), moved to the irradiation area of the dissection laser beam separated from the origin by a predetermined distance, and waits. The position of the sample mounting table 22 during standby is a position where the sample on the slide glass 21 fixed on the sample mounting table 22 enters the irradiation area of the disection laser light and the irradiation area of the illumination light. In this state, the slide glass 21 to which the sample 23 is fixed is attached to the sample mounting table 22.

  Next, the illumination light from the illumination light source 51 is irradiated onto the sample 23 on the slide glass 21 from above the sample mounting table 22 through the optical lens 52, the dichroic mirror 53, and the condenser lens 54, and the observation light transmitted through the sample 23 is objective. The image is captured by the CCD camera 62 through the lens 57, the half mirror 58, and the mirror 59 and is displayed on the display unit 65. At that time, the position of the sample mounting table 22 is finely adjusted by the touch function of the joystick controller 68 or the display unit 65 so that the position where the sample 23 is desired to be collected can be displayed on the display unit 65.

  Next, as shown in FIG. 5, in the case where the sample is continuously cut out from the sample 23, a range in which the sample 23 displayed on the display unit 65 is desired to be collected is set by the pointing device 66 or the like. Based on the diameter of the dissection laser light to be irradiated, the control computer 64 calculates the position coordinates of the sample irradiated with the dissection laser light so that the sample 23 in the set range can be continuously cut out. In the above example, the sample 23 is continuously cut out. For example, the position coordinates of the sample irradiated with the dissection laser beam are set so that the sample in the set range can be cut out at an arbitrary interval. It may be set.

  Next, the position coordinates of the sample when the collected sample is bonded to the thermoplastic film 31 are set. The position coordinates are set by, for example, a list stored in the control computer 64 in advance such as bonding the sample 23 in the set range to the thermoplastic film 31 with a spot of 5 × 5, 25 × 25, 50 × 50 or the like. The coordinate position may be calculated by the control computer 64 by inputting a desired interval value to the control computer 64. The control computer 64 stores the selected or calculated position coordinates in the storage means (not shown) of the control computer 64, the sample image captured by the CCD camera, the position coordinates of the sample irradiated with the dissection laser light, and the dissection laser light. The position coordinates for bonding the sample collected by the irradiation to the thermoplastic film 31 are associated and stored in the storage means (hereinafter, the stored information may be referred to as “collection information”).

  In addition, as shown in FIG. 6, when arbitrarily setting the position where the sample 23 is to be collected, the pointing device 66 or the like is used to irradiate the dissection laser light of the sample 23 displayed on the display unit 65 to be collected. The storage means of the control computer 64 is configured to automatically identify and set each position coordinate using color information, staining intensity, the area of the object, perimeter, major axis, shape, etc. by setting or image analysis software The position coordinates of the location set in is stored. At that time, at which part of the sample 23 displayed on the display unit 65 is set by the pointing device 66, for example, which part of the sample is to be cut out, such as displaying a mark or displaying a number for each set order. It is preferable to display.

  Next, in the same manner as described above, the position coordinates for adhering the collected sample to the thermoplastic film 31 are calculated, and the sample imaged by the CCD camera, preferably the sample at any location, is cut out in the storage means (not shown) of the control computer 64. The image of the displayed sample, the position coordinates of the sample irradiated with the dissection laser light, and the position coordinates where the collected sample is adhered to the thermoplastic film 31 by irradiation of the dissection laser light are stored in the storage means in association with each other (hereinafter referred to as the storage section). The stored information may be referred to as “collection information”.)

  After storing the collection information according to the above procedure, the moving means drive controller 67 determines that the location of the sample 23 to be irradiated with the first section of the dissection laser beam is on the optical axis of the dissection laser light based on the position coordinate information of the collection information. The sample moving means 2 is driven and controlled so as to be positioned at the position. Next, based on the position coordinate information stored in the storage unit, the moving unit drive control unit 67 determines the location of the thermoplastic film 31 to which the first sampled portion of the sample 23 is bonded on the optical axis of the dissection laser beam. Then, the thermoplastic film moving means 3 is driven and controlled so that the thermoplastic film 31 comes into contact with the sample 23.

  Next, in accordance with an instruction from the control computer 64, the laser controller 63 controls to irradiate the dissection laser light from the dissection laser light source 41. After the die section laser light irradiation, the movement control means 67 drives and controls the thermoplastic film moving means 3 so as to move the thermoplastic film 31 upward, so that the sample 23 collected first is bonded to the thermoplastic film 31. Peel from the sample section in a state of being allowed to stand. Thereafter, the above procedure is repeated until all of the samples to be collected that have been set in advance are adhesively peeled from the thermoplastic film 31.

  The sample 23 adhered to the thermoplastic film 31 by the above procedure may be analyzed by a known method. For example, necessary pretreatment is performed while the sample 23 is adhered to the thermoplastic film, and the sample 23 is set in a vacuum chamber of a known mass spectrometer, and the adhered sample 23 is irradiated with ionized laser light in order. What is necessary is just to perform ionization and mass spectrometry. The analysis using the conventional atmospheric pressure ionization method was limited to MALDI-TOF-MS, but in the present invention, analysis can be performed using all mass spectrometers such as LC-MS and ESI-MS. it can. In addition to mass spectrometry, for example, by liquefying a sample adhered to a thermoplastic film, an analyzer including chromatography such as HPLC-fluorescence spectrometer, HPLC-electrochemical detector, electron beam microanalyzer, Elemental analyzers such as X-ray photoelectron spectrometers, nucleic acid sequence analyzers that amplify genes by PCR or LCR and analyze the DNA sequence contained in the sample using a sequencer, and hybridize DNA using the nucleic acid contained in the sample as a template Analysis can be performed using a microchip analyzer such as a DNA chip to be reacted or an antibody chip for reacting an antibody with a protein.

  Further, the laser microdissection apparatus of the present invention is incorporated in the analysis apparatus as a sample collection apparatus of the above-described mass analysis apparatus, analysis apparatus including chromatography, elemental analysis apparatus, nucleic acid sequence analysis apparatus, and microchip analysis apparatus. You can also. Further, when the laser microdissection apparatus of the present invention is used, the collected sample can be adhered and arranged on the thermoplastic film at a desired interval. For example, as a manufacturing apparatus for manufacturing a DNA chip or an antibody chip It can also be used.

  When the analysis result of the collected sample 23 obtained by the analysis is stored in the storage means, the position coordinate of each collected sample 23 and the analysis result are associated and stored in the storage means (hereinafter, the stored information is referred to as “analysis information”). "). And by performing image composition based on the collection information and analysis information stored in the storage means, the image of the sample is displayed and the analysis result of the sample contained in the location where the sample was collected is also displayed Can do.

  When the laser microdissection device 1 and the analysis device are separate from each other, the sample information and the analysis result are displayed by, for example, reading the analysis information by the control computer 64 of the laser microdissection device 1 and the laser microdissection device 1. It is only necessary to construct an image based on the collection information stored in the storage means and the read analysis information, and display the sample image and the analysis result together on the display unit 65. If you want to display on the display unit of the analyzer, the collected information is read by the control computer of the analyzer, and the image is constructed based on the analysis information stored in the storage means of the analyzer and the read collected information. What is necessary is just to display a sample image and an analysis result collectively on the display part of an apparatus.

  In the case of an analyzer incorporating the laser microdissection device 1, an image is constructed based on the collection information and the analysis information, and the sample image and the analysis result may be displayed together on the display unit. Further, the collection information and the analysis information may be read by a personal computer or the like separate from the laser microdissection device 1 and the analysis device, and the sample image and the analysis result may be displayed together on the display unit of the personal computer. Moreover, since this apparatus can also be automated, it can also be remotely operated and analyzed, and can also perform analysis and inspection from a remote location.

  Although the above example shows an example in which a two-dimensional analysis is performed from a collected sample, three-dimensional imaging can also be performed using the laser microdissection apparatus 1 of the present invention or an analysis apparatus incorporating the apparatus. . For example, when a plurality of sample sections are prepared from the same tissue and the collection information and analysis information are obtained for each sample section in the same procedure as described above, the sample section of the sample section prepared from the tissue By associating and storing whether or not there is, three-dimensional imaging can be easily performed. At this time, as means for preventing the positional deviation of the image of each section, (1) inserting a colored nylon floss or the like as a position marker at an appropriate place of the embedding agent at the time of embedding the specimen, (2) a plurality of specimens A position marker may be created by marking with a black ink mark or a female mark by micro-cutting at a specific location, and an image corrected by the image processing function incorporated in the control computer 64 may be displayed. In addition, when the thickness of each section is very thin, the consecutive sample sections cut out from the tissue are almost the same size. When the image captured by the CCD camera of the sample is displayed on the display unit 65, it is displayed so as to be superimposed on the first image so that it is the same as the sample collection range set by the first image. The sample collection range may be set by the second image.

  The thermoplastic film 31 dissolves by absorbing the dissection laser light, solidifies again by stopping the irradiation of the dissection laser light, and adheres to the sample cut out by the dissection laser light when solidifying. . Therefore, the position coordinates of the sample 23 irradiated with the dissection laser light are made the same, and the sample collected by the first irradiation of the dissection laser light is bonded to the preset position of the thermoplastic film 31, and then the sample 23 is not moved, but only the thermoplastic film 31 is moved to the bonding position of the sample to be collected next, and is irradiated with a dissection laser beam. Then, although the melt | dissolved thermoplastic film 31 penetrates the recessed part from which the sample was already extract | collected, since the thermoplastic film 31 is viscous, the melt | dissolved thermoplastic film 31 is not melt | dissolved when it solidifies. Since it is solidified so as to be pulled back to the film 31, it can be collected from the sample 23 that is bonded to the slide glass 21 by adhering the sample cut out by the dissection laser beam.

  When a sample is collected by the above method, the amount of sample that can be collected may vary depending on the depth of the recess. Therefore, the sample mounting table 22 may be moved in the vertical direction in addition to the movement in the horizontal direction. The sample mounting table 22 was moved in the vertical direction, and the depth of the recess was measured and collected by focusing the optical system on the recess surface of the sample fixed to the slide glass 21 after the sample was collected. Since the amount of the sample can be obtained, the quantitativeness when performing three-dimensional imaging can be improved.

  The present invention will be described in detail with reference to the following examples, which are provided merely for the purpose of illustrating the present invention and for reference to specific embodiments thereof. These exemplifications are for explaining specific specific embodiments of the present invention, but are not intended to limit or limit the scope of the invention disclosed in the present application.

<Example 1>
Based on an inverted microscope (IX series manufactured by Olympus), a stepping motor (Bio Precision; manufactured by Rudol) as a driving source, 3D-A-LCS software (manufactured by Lucille) as a moving means drive control unit, and a dissection laser light source A Z-808-200-SM (manufactured by Lucille Co., Ltd.) was attached to produce the laser microdissection device of the present invention. FIG. 8 is a photograph of the appearance of the manufactured laser microdissection apparatus.

<Example 2>
Using the laser microdissection device prepared in Example 1 above, a sample fixed to a slide glass was collected by the following procedure and subjected to an imaging process.

[Acquisition of analysis organization]
As an analysis tissue, the brain of an APP / PS1 mouse (10 months old, about 25 g) obtained by the following procedure was used.
1. The mice were anesthetized with diethyl ether, placed in the supine position, and the limbs were fixed.
2. After laparotomy, the diaphragm was incised, and the left and right ribs were incised toward the head.
3. The xiphoid process was pinched and inverted toward the head, and fixed with forceps to expose the heart.
4). The left ventricle was pierced with a winged needle, and 1 × PBS solution (physiological saline) was injected.
5. The right atrial appendage was incised with a scissors, and blood was removed and perfused with about 70 ml of physiological saline.
6). After perfusion, the head was cut and the brain was removed after craniotomy.
7). The excised brain was half-cut with a sagittal cut, and the cut surface was placed on the lower surface (cut surface) and then frozen in an embedding agent (OCT compound) to prepare a frozen block.

[Preparation of sample section]
From the frozen block obtained by the above procedure, a sample section was prepared by the following procedure.
1. Sections were prepared from the frozen block with a thickness of 10 μm. A slide glass without a coat was used.
2. The frozen section was dried according to the following procedure.
(1) 100% acetone 10 minutes (2) PBS 1 minute (3) 70% ethanol 1 minute (4) 100% ethanol 1 minute (5) 100% ethanol 1 minute (6) 100% xylene 2 minutes (7) 100 % Xylene 2 minutes

[Extraction of specimen from section and adhesion to thermoplastic film]
The sample of the location set from the frozen section obtained by said procedure was extract | collected with the following procedures, and was adhere | attached on the thermoplastic film.
1. The laser microdissection apparatus manufactured in Example 1 was turned on to initialize the sample mounting table, and the obtained frozen section was set on the sample mounting table of the laser microdissection apparatus. Further, a hollow ring with an EVA film (manufactured by Sigma-Aldrich Japan) attached to the tip was inserted into the hole at the tip of the arm of the thermoplastic film moving means.
2. The image of the frozen section was displayed on the display unit, and the position coordinates for irradiating the sample with the dissection laser beam were set so that a square part with a side of 800 μm could be collected at 15 × 15 = 225 points.
3. The position coordinates were set so that the collected sample was adhered to the EVA film at a sample center distance of 233 μm.
4). According to the program of Live Cell Imaging System V7 (manufactured by Lucille), 2.3. In accordance with the position coordinates, the sample fixed to the slide glass is irradiated with a dissection laser beam (output: 300 mA, irradiation time: 5 msec, irradiation diameter: 30 μm), and the cut sample is adhered to a predetermined location on the EVA film. It was collected. The laser intensity was adjusted so that the diameter of the sample collected in this example was 60 μm.

FIG. 9 (1) shows the whole image of the section when the collection range is set, and FIG. 9 (2) is an enlarged view of the collection range displayed on the display unit. FIG. 9 (3) shows the section obtained after the irradiation with the disection laser overwritten with a numeral and a circle with a broken line.

[Mass analysis of collected sample]
1. Take out the resin support attached from the hollow ring, peel off the EVA film so that the collected sample is on top, and mark it with black oily magic so that you can see which sample is the first sample collected An EVA film was attached to the conductive double-sided tape.
2. A matrix for use in MALDI-TOF-MS was applied to the collected sample using a chemical printer. CHCA (50% acetonitrile, 0.1% TFA) was used for the matrix, and an amount of 10,000 pl (100 pl × 5 drops / 1 spot × 20 times) was applied. FIG. 10 is a photograph after the matrix is applied to the EVA film to which the collected sample is adhered.
3. The carrier brand position information was set using Angiotensin 2 (MW 1046.3) and Insulin (MW 5804.6).
4). A CSV file (spot position coordinates) was created.
5. The EVA film was transferred to a desiccator, dried with a vacuum pump for 20 minutes, and then subjected to mass spectrometry with AXIMA Performance (Shimadzu Corporation). The measurement conditions of mass spectrometry were Laser Power 65, Profile 1, and Shots 200, and each parameter was set in ChIP Imaging Experiment.

[Sample and mass imaging display]
The analysis result by AXIMA Performance was imaged using the HeatMap display of BioMap software or Image Pro Plus software, the image was constructed in association with the position coordinates of the sample, and the result was displayed on the display unit. FIG. 11 shows an image displayed on the display unit. From the above results, by using the laser microdissection apparatus of the present invention, mass imaging can be efficiently performed in association with the position coordinates of the sample.

  FIG. 12 is a mass spectrum of the analysis result of the peptide obtained from the sample. Peptides with molecular weights of 14 and 173 could be detected.

<Example 3>
Next, mass imaging of the hippocampus of the left brain of APP / PS1 mice (Jackson Laboratories) was performed. The sample was adhered to the thermoplastic film in the same manner as in Example 2, except that a slice was prepared from the mouse in the same procedure as in Example 2 and the hippocampus portion of the left brain was collected from the slice at 30 × 30 = 900 points. -The collected and collected samples were analyzed. Further, the hippocampus portion of the left brain was stained with Amyloid beta using FSB solution (Doujin Chemical Co., Ltd.) in another section adjacent to the section. FIG. 13 shows an image of a section, a sample collection range, a sample collection position in the collection range, a result of Amyloid beta staining, and a HeatMap display of the results of mass spectrometry of Amyloid beta 1-40. In the Amyloid beta staining, as apparent from the photograph, only a few stainings were confirmed. On the other hand, in the mass imaging result of Amyloid beta 1-40, Amyloid beta 1-40 was detected in a shape similar to the shape of the hippocampus (the dark part of mass imaging). From the above results, the use of the laser microdissection apparatus of the present invention has a higher sensitivity in vivo using a general mass spectrometer than the specific fluorescence staining of peptides, which is said to be the most sensitive at present. The peptide could be analyzed.

<Example 4>
Next, three-dimensional imaging was performed using a mass spectrometer including the laser microdissection apparatus of the present invention. As samples, APP / PS1 mice and normal litter mice (Alzheimer's disease model mice; Jackson Laboratories) were prepared, and colored nylon floss was inserted into the appropriate place of the embedding agent as a position marker when the specimen was embedded. Except for the above, a section was prepared in the same procedure as in Example 2, and mass spectrometry was performed in the same procedure. In Example 4, mass analysis was performed on 10 sections (sample thickness is 100 μm in total) to obtain a three-dimensional image, and the analysis result was stored in association with the section number when performing analysis. The images imaged in the same procedure as 2 were superimposed. FIG. 14 (1) is a three-dimensional image of the hippocampus of a normal mouse, and FIG. 14 (2) is a three-dimensional image of the hippocampus of an APP / PS1 mouse. As is clear from FIGS. 14 (1) and (2), amyloid beta 1-40 was not observed in normal mice, but in APP / PS1 mice, as shown in FIG. 14 (2), amyloid beta 1-40 Was confirmed in three dimensions.

By using the laser microdissection apparatus according to the present invention, the spatial resolution of a conventional mass spectrometer can be improved, and mass imaging can be easily performed. By using the laser microdissection apparatus according to the present invention, it is possible to analyze not only the mass spectrometer but also the collected sample with various analyzers, and display the result of imaging. Therefore, it can be used as a device for tissue analysis in medical institutions, research institutions such as university medical departments, general hospitals, and the like.

Claims (13)

A sample moving means capable of placing a sample and moving the sample in a horizontal direction;
A thermoplastic film on which a support, a slide glass or a substrate on which a thermoplastic film is mounted can be attached, and the thermoplastic film mounted on the support, the slide glass or the substrate can be moved in the horizontal and vertical directions. transportation,
A laser irradiation part for irradiating a sample with a dissection laser beam, cutting out a sample of the portion irradiated with the dissection laser beam, and bonding the sample to a thermoplastic film as a sample,
Memory means for associating and memorizing the position coordinates of the sample where the dissection laser beam is irradiated and the position coordinates of the thermoplastic film where the sample is adhered, and the position coordinates and thermoplasticity of the sample stored in the memory means A moving means driving control unit for driving and controlling the sample moving means and the thermoplastic film moving means based on the position coordinates of the film;
Including
At least two or more collected samples can be adhered to the thermoplastic film,
The position coordinates of the thermoplastic film at the location where the collected sample adheres is larger than the distance between the samples before the sample is collected, and the center-to-center distance between any collected sample to be adhered and the other collected sample adjacent to the collected sample; The center-to-center distance between an arbitrary collected sample to be bonded and another collected sample adjacent to the collected sample is set to an arbitrary interval.
Laser microdissection device characterized by that.   The laser microdissection apparatus according to claim 1, further comprising a pointing device for arbitrarily setting a position coordinate of a sample at a position where the dissection laser light is irradiated.   3. The laser micro of claim 2, wherein a distance between centers of the arbitrary collected sample and a collected sample adjacent to the collected sample is larger than (diameter of ionized laser light + diameter of collected sample) / 2. Dissection device.   The laser microdissection apparatus according to any one of claims 1 to 3, wherein the sample moving means can also move in a vertical direction.   4. The laser microdissection apparatus according to claim 3, wherein the slide glass and the base material are subjected to a conductive treatment.   An analyzer including the laser microdissection device according to claim 1.   The analyzer according to claim 6, wherein the analyzer is one selected from a mass spectrometer, an analyzer including chromatography, an element analyzer, a nucleic acid sequence analyzer, and a microchip analyzer.   A mass spectrometer comprising the laser microdissection device according to claim 5.   The analyzer according to any one of claims 6 to 8, wherein the analyzer includes a display unit capable of displaying a sample image and an analysis result by the analyzer together.   The manufacturing method of the microchip which the sample adhered to the thermoplastic film using the laser microdissection apparatus as described in any one of Claims 1-5. Set the position coordinates of the sample where the dissection laser light is irradiated, set the position coordinates of the thermoplastic film where the collected sample adheres, and associate the position coordinates of the sample and the position coordinates of the thermoplastic film Memorizing process,
A step of moving the sample and the thermoplastic film to a position to be irradiated with the dissection laser beam based on the position coordinate of the sample and the position coordinate of the thermoplastic film stored in association with each other;
A process of adhering the collected sample to a thermoplastic film by irradiating a dissection laser beam;
Including
In the step of associating and storing the position coordinates of the sample and the position coordinates of the thermoplastic film, at least two or more position coordinates of the sample and the position coordinates of the thermoplastic film are stored in association with each other;
The position coordinates of the thermoplastic film at the location where the collected sample adheres is larger than the distance between the samples before the sample is collected, and the center-to-center distance between any collected sample to be adhered and the other collected sample adjacent to the collected sample; The center-to-center distance between an arbitrary collected sample to be bonded and another collected sample adjacent to the collected sample is set to an arbitrary interval.
A method of collecting a sample by a laser microdissection characterized by the above. 12. The method of collecting a sample by a laser microdissection according to claim 11, wherein the position coordinates of the sample at the place where the dissection laser light is irradiated is arbitrarily set by a pointing device. A step of irradiating a sample collected by the laser microdissection method according to claim 11 or 12 with an ionized laser beam from a mass spectrometer onto a sample collected on a thermoplastic film,
A mass spectrometric method comprising: