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WO2015053039A1 - Dispositif de microdissection laser, dispositif d'analyse contenant un dispositif de microdissection laser, et procédé pour produire une micropuce - Google Patents

Dispositif de microdissection laser, dispositif d'analyse contenant un dispositif de microdissection laser, et procédé pour produire une micropuce Download PDF

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Publication number
WO2015053039A1
WO2015053039A1 PCT/JP2014/074064 JP2014074064W WO2015053039A1 WO 2015053039 A1 WO2015053039 A1 WO 2015053039A1 JP 2014074064 W JP2014074064 W JP 2014074064W WO 2015053039 A1 WO2015053039 A1 WO 2015053039A1
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WO
WIPO (PCT)
Prior art keywords
sample
thermoplastic film
collected
laser
dissection
Prior art date
Application number
PCT/JP2014/074064
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English (en)
Japanese (ja)
Inventor
誠 澤田
Original Assignee
国立大学法人名古屋大学
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Application filed by 国立大学法人名古屋大学 filed Critical 国立大学法人名古屋大学
Priority to JP2015541497A priority Critical patent/JP6611610B2/ja
Publication of WO2015053039A1 publication Critical patent/WO2015053039A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • G01N2001/2833—Collecting samples on a sticky, tacky, adhesive surface
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
    • G01N2001/2833—Collecting samples on a sticky, tacky, adhesive surface
    • G01N2001/284—Collecting samples on a sticky, tacky, adhesive surface using local activation of adhesive, i.e. Laser Capture Microdissection
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/286—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q involving mechanical work, e.g. chopping, disintegrating, compacting, homogenising
    • G01N2001/2873—Cutting or cleaving
    • G01N2001/2886—Laser cutting, e.g. tissue catapult

Definitions

  • the present invention relates to a laser microdissection apparatus, an analysis apparatus including the laser microdissection apparatus, and a method for manufacturing a microchip.
  • 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.
  • 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.
  • a mass microscope that can observe a biological sample with a microscope and perform mass analysis on the biological sample is commercially available.
  • 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.
  • 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.
  • 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.
  • 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.
  • MALDI-TOF-MS time-of-flight mass spectrometer
  • 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.
  • LC-MS liquid chromatograph mass spectrometry
  • ESI-MS electrospray ionization mass spectrometry
  • Patent Document 2 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.
  • 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.
  • 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”).
  • laser light for cutting out the sample is sometimes referred to as “dissection laser light”.
  • a method of adhering a sample to a thermoplastic film by irradiating see Patent Documents 4 and 5 and the like.
  • Patent Documents 4 and 5 are merely techniques for taking out cells and DNA from tissue specimens.
  • a biological sample adhered to a thermoplastic film is irradiated with ionized laser light.
  • mass spectrometry 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.
  • 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.
  • 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.
  • thermoplastic film is applied to the thermoplastic film from the interval between samples collected by irradiating the dissection laser light.
  • the distance between the collected samples to be adhered can be increased, and (3) the distance between the collected samples to be adhered to the thermoplastic film is a distance at which the adjacent sample is not affected by the ionized laser light when irradiated with the ionized laser light. Therefore, 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 analyzer can be improved, and (4) the position of the sample at the location where the disection laser beam is irradiated.
  • thermoplastic film where the collected sample adheres are stored in the storage means in association with each other, and the position coordinates of the sample adhered to the thermoplastic film and the analysis result of the sample by the analyzer are linked.
  • 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.
  • the sample is contaminated as a solution by applying a liquid to the collected sample.
  • the present invention has been completed by newly finding out 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.
  • an object of the present invention is to provide a laser microdissection apparatus, an analysis apparatus including the laser microdissection apparatus, and a method for manufacturing a microchip.
  • 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.
  • 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
  • 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 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.
  • 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.
  • 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.
  • 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. 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
  • 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
  • FIG. 9 (2) is an enlarged view of the collection range displayed on the display unit.
  • 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 of the collection range, a result of Amyloid beta staining, and a HeatMap display of a mass analysis 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.
  • the laser microdissection apparatus of the present invention the analysis apparatus including the laser microdissection apparatus, and the manufacturing method of the microchip will be described 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.
  • 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.
  • a driving force transmission mechanism for rotating and moving the arm 32 is included.
  • 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.
  • a thermoplastic resin having a melting point of about 50 to 70 ° C. as a raw material.
  • EVA ethyl vinyl acetate
  • Examples include polyolefin, polyamide, acrylic, and polyurethane.
  • 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 Co., Ltd.), a thermoplastic EVA film (manufactured by Sigma-Aldrich Japan Co., Ltd.), and the like.
  • 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). .
  • FIG. 2 is a diagram showing an example of the principle of laser microdissection.
  • 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).
  • 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.
  • the sample 23 cut out by irradiation with the dissection laser beam 43 can be adhered to the thermoplastic film 31.
  • 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 of the hollow ring 34, which is composed of a hollow base 35 and a hollow convex portion 36 for holding a support with a thermoplastic film 31 attached to 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.
  • 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.
  • thermoplastic film 31 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.
  • the thermoplastic film 31 may be directly attached to a slide glass subjected to a conductive treatment.
  • 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.
  • 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.
  • 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.
  • 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. .
  • the section a of the sample 23 is irradiated with a dissection laser beam by the sample moving means 2.
  • 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.
  • thermoplastic film 31 and the sample 23 are brought into contact with each other by being lowered in the direction.
  • 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.
  • the samples 23b, c,... are bonded to 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 ⁇ m to 5 ⁇ m for subcellular structure analysis or high spatial resolution, 15 ⁇ m to 30 ⁇ m for collecting single cells, and 50 ⁇ m to 100 ⁇ m for 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.
  • 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.
  • the sample collected from the sample 23 can be adhered to the thermoplastic film 31 at an arbitrary interval larger than the interval of the sample 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.
  • the diameter should 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 ⁇ m.
  • 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 ⁇ m + 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 ⁇ m + 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.
  • 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 ⁇ m + 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.
  • 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.
  • 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 disposed below the sample mounting table 22 of the laser microdissection apparatus 1.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the sample 23 is continuously cut out.
  • 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.
  • 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 at which the sample collected by the irradiation is bonded to the thermoplastic film 31 is associated and stored in the storage means (hereinafter, the stored information may be referred to as “collected information”).
  • 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.
  • 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.
  • 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”.
  • 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.
  • 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.
  • the laser controller 63 controls to irradiate the dissection laser light from the dissection laser light source 41.
  • 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.
  • an analytical device 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.
  • 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
  • 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.
  • analysis information 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.
  • 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.
  • an image is constructed based on collection information and analysis information, and a sample image and analysis results may be displayed together on the display unit.
  • 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.
  • 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.
  • three-dimensional imaging can also be performed using the laser microdissection apparatus 1 of the present invention or an analysis apparatus incorporating the apparatus.
  • 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.
  • 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.
  • the thickness of each section is very thin, the consecutive sample sections cut out from the tissue are almost the same size.
  • 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.
  • dissolved thermoplastic film 31 penetrates the recessed part from which the sample was already extract
  • the sample mounting table 22 may be moved in the vertical direction in addition to the movement in the horizontal direction.
  • the depth of the concave portion was measured by moving the sample mounting table 22 in the vertical direction and focusing the optical system on the concave 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.
  • Example 1 Based on an inverted microscope (Olympus IX series), stepping motor (Bio Precision; made by Rudol) as the driving source, 3D-A-LCS software (made by Lucille) as the moving means drive control unit, disection laser light source By attaching Z-808-200-SM (manufactured by Lucille Co.) as a 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.
  • 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.
  • OCT compound embedding agent
  • a sample section was prepared by the following procedure. 1. Sections were prepared from the frozen block with a thickness of 10 ⁇ m. 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
  • Example 1 [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
  • 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.
  • the position coordinates were set so that the collected sample was adhered to the EVA film at a sample center distance of 233 ⁇ m. 4).
  • Live Cell Imaging System V7 manufactured by Lucille
  • the sample fixed to the slide glass is irradiated with a dissection laser beam (output: 300 mA, irradiation time: 5 msec, irradiation diameter: 30 ⁇ m), 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 ⁇ m.
  • FIG. 9 (1) shows the whole image of the section when the collection range is set
  • 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.
  • 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.
  • Example 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.
  • FIG. 12 is a mass spectrum of the peptide analysis result obtained from the sample. Peptides with molecular weights of 14 and 173 could be detected.
  • FIG. 13 shows an image of a section, a sample collection range, a sample collection position of the collection range, a result of Amyloid beta staining, and a HeatMap display of the results of mass spectrometry of Amyloid beta 1-40.
  • Amyloid beta staining as apparent from the photograph, only a few stainings were confirmed.
  • mass imaging result of Amyloid beta 1-40 Amyloid beta 1-40 was detected in a shape similar to that of the hippocampus (the dark portion of mass imaging).
  • 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 Three-dimensional imaging was performed using a mass spectrometer including the laser microdissection apparatus of the present invention.
  • samples APP / PS1 mice and normal litter mice (Alzheimer's disease model mice; Jackson Laboratories) were prepared, and colored nylon floss was inserted as a position marker at the appropriate place of the embedding agent when embedding the specimen. 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.
  • mass analysis was performed on 10 sections (sample thickness is 100 ⁇ m 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
  • FIG. 14 (2) is a three-dimensional image of the hippocampus of an APP / PS1 mouse.
  • 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.
  • the laser microdissection apparatus 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.
  • 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.

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  • General Health & Medical Sciences (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • Immunology (AREA)
  • Physics & Mathematics (AREA)
  • Health & Medical Sciences (AREA)
  • Pathology (AREA)
  • General Physics & Mathematics (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
  • Microscoopes, Condenser (AREA)

Abstract

L'invention concerne un dispositif de collecte d'échantillon pouvant améliorer une résolution spatiale tout en utilisant un dispositif d'analyse classique tel quel. Le dispositif de microdissection laser selon l'invention est caractérisé par le fait qu'il contient : un moyen de déplacement d'échantillon sur lequel peut être monté un échantillon, et pouvant déplacer l'échantillon dans la direction horizontale ; un moyen de déplacement de film thermoplastique sur lequel peut être monté un film thermoplastique, et pouvant déplacer le film thermoplastique dans les directions verticale et horizontale ; une unité de rayonnement laser pour exposer l'échantillon à un faisceau laser de dissection, séparer la section d'échantillon exposée au faisceau laser de dissection, et coller ladite section en tant qu'échantillon collecté au film thermoplastique ; un moyen de stockage pour associer et stocker les coordonnées de position de la section d'échantillon à être exposée au faisceau laser de dissection, et les coordonnées de position de la section du film thermoplastique sur lequel l'échantillon collecté doit être collé ; et une unité de commande d'entraînement de moyen de déplacement pour entraîner et commander le moyen de déplacement d'échantillon et le moyen de déplacement du film thermoplastique, sur la base des coordonnées de position de l'échantillon et des coordonnées de position du film thermoplastique qui sont stockées dans le moyen de stockage.
PCT/JP2014/074064 2013-10-07 2014-09-11 Dispositif de microdissection laser, dispositif d'analyse contenant un dispositif de microdissection laser, et procédé pour produire une micropuce WO2015053039A1 (fr)

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JP2018091634A (ja) * 2016-11-30 2018-06-14 国立大学法人名古屋大学 試料の処理方法、および試料処理用キット
JP2019132831A (ja) * 2018-01-29 2019-08-08 東ソー株式会社 質量分析計を用いた粒子測定法
WO2020045291A1 (fr) * 2018-08-28 2020-03-05 国立大学法人名古屋大学 Dispositif de microdissection au laser, appareil d'analyse contenant un dispositif de microdissection au laser, et procédé de collecte d'échantillon
CN111108375A (zh) * 2017-09-21 2020-05-05 浜松光子学株式会社 激光解吸电离法和质量分析方法
WO2021186577A1 (fr) * 2020-03-17 2021-09-23 国立大学法人東海国立大学機構 Dispositif de microdissection laser, procédé de microdissection laser et système d'analyse quantitative
JP2021148679A (ja) * 2020-03-23 2021-09-27 株式会社島津製作所 イメージング質量分析システム、及び、イメージング質量分析を利用した分析方法
WO2022003890A1 (fr) * 2020-07-02 2022-01-06 株式会社島津製作所 Spectromètre de masse d'imagerie et procédé de traitement de données de spectrométrie de masse d'imagerie
US11380532B2 (en) 2020-06-17 2022-07-05 Shimadzu Corporation Method for imaging mass spectrometry and imaging mass spectrometer
US11545348B2 (en) 2020-06-15 2023-01-03 Shimadzu Corporation Imaging mass spectrometer and method for imaging mass spectrometry

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EP3147659A4 (fr) * 2014-05-19 2017-06-07 National University Corporation Nagoya University Procédé d'analyse de constituants d'un échantillon, procédé de séparation spécifique de constituants dans un échantillon et échantillon destiné à être utilisé dans la spectrométrie de masse
JP2018091634A (ja) * 2016-11-30 2018-06-14 国立大学法人名古屋大学 試料の処理方法、および試料処理用キット
CN111108375A (zh) * 2017-09-21 2020-05-05 浜松光子学株式会社 激光解吸电离法和质量分析方法
JP2019132831A (ja) * 2018-01-29 2019-08-08 東ソー株式会社 質量分析計を用いた粒子測定法
JP7262220B2 (ja) 2018-01-29 2023-04-21 東ソー株式会社 質量分析計を用いた粒子測定法
JP7136490B2 (ja) 2018-08-28 2022-09-13 国立大学法人東海国立大学機構 レーザーマイクロダイセクション装置、レーザーマイクロダイセクション装置を含む分析装置、および、試料の採取方法
WO2020045291A1 (fr) * 2018-08-28 2020-03-05 国立大学法人名古屋大学 Dispositif de microdissection au laser, appareil d'analyse contenant un dispositif de microdissection au laser, et procédé de collecte d'échantillon
JPWO2020045291A1 (ja) * 2018-08-28 2021-08-12 国立大学法人東海国立大学機構 レーザーマイクロダイセクション装置、レーザーマイクロダイセクション装置を含む分析装置、および、試料の採取方法
WO2021186577A1 (fr) * 2020-03-17 2021-09-23 国立大学法人東海国立大学機構 Dispositif de microdissection laser, procédé de microdissection laser et système d'analyse quantitative
JP2021148679A (ja) * 2020-03-23 2021-09-27 株式会社島津製作所 イメージング質量分析システム、及び、イメージング質量分析を利用した分析方法
US11469087B2 (en) 2020-03-23 2022-10-11 Shimadzu Corporation Imaging mass spectrometry system and analytical method using imaging mass spectrometry
JP7375640B2 (ja) 2020-03-23 2023-11-08 株式会社島津製作所 イメージング質量分析システム、及び、イメージング質量分析を利用した分析方法
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US11380532B2 (en) 2020-06-17 2022-07-05 Shimadzu Corporation Method for imaging mass spectrometry and imaging mass spectrometer
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