JP7116937B2 - Sample Preparation Method Suitable for Imaging Mass Spectrometry and Conductive Adhesive Film - Google Patents
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本発明は、イメージング質量分析装置による生
物組織中に含まれる特定質量の物質の分布を精度良く観察するのに適した分析用試料を作製するためのイメージング質量分析に好適な試料作製法及びその過程で使用する導電性粘着フィルムに関するものである。The present invention provides a sample preparation method suitable for imaging mass spectrometry and its process for preparing an analysis sample suitable for accurately observing the distribution of substances of a specific mass contained in biological tissue using an imaging mass spectrometer. It relates to a conductive adhesive film used in.
生命科学の分野では、生物組織中に含まれる生体成分の質量を測定するとともに、特定質量の組織内分布を明らかにし、画像化する事が求められている。その目的のために生物試料を凍結薄切し、その薄切試料にレーザー照射、あるいはイオンビーム照射して試料中の分子をイオン化させ、そのイオン化した分子をイメージング質量分析装置で連続的に測定することにより特定質量の物質の分布を画像化している。 In the field of life sciences, it is required to measure the mass of biological components contained in biological tissue, clarify the distribution of specific mass in the tissue, and visualize it. For that purpose, a biological sample is sliced frozen, and the sliced sample is irradiated with a laser or an ion beam to ionize the molecules in the sample, and the ionized molecules are continuously measured with an imaging mass spectrometer. By doing so, the distribution of a substance with a specific mass is imaged.
イメージング質量分析用試料作製では、装置の検出感度を高めるためにレーザー照射等で分析試料をイオン化した際に測定に利用されずに試料表面に残ったイオンを除去する事が必要で、分析試料は図１に示す導電処理した光学顕微鏡用スライドガラス上に凍結ミクロトームで作製した生物試料の凍結切片を載せ、次いで凍結切片を解凍することにより切片を導電処理したスライドガラス上に密着固着してイメージング質量分析装置により分析している。従来の方法では、凍結切片は切片支持材を用いないで作製されるために薄切中に組織が壊れ、組織形態が保たれた凍結切片を作製する事が極めて困難であった。また、凍結切片をスライドガラスに固着するために凍結切片をスライドガラス上に密着して解凍を行っており、この解凍過程で生体内成分が酵素等により分解して分子が小さくなる問題がある。また解凍処理により生体内成分が特定部位から拡散する問題もあった。さらに骨や植物試料等から作製した凍結切片は導電処理したスライドガラス上に貼りつかないためにイメージング質量分析用切片を導電処理したスライドガラス上に作製する事ができない問題もあった。 In the preparation of samples for imaging mass spectrometry, it is necessary to remove ions that remain on the sample surface without being used for measurement when the sample is ionized by laser irradiation, etc., in order to increase the detection sensitivity of the instrument. A frozen section of a biological specimen prepared by a freezing microtome is placed on a conductively treated slide glass for an optical microscope shown in FIG. Analyzed by analyzer. In the conventional method, since the frozen section is prepared without using a section supporting material, the tissue is destroyed during sectioning, and it is extremely difficult to prepare a frozen section in which the tissue morphology is maintained. In addition, in order to fix the frozen section to the slide glass, the frozen section is thawed while being brought into close contact with the slide glass. There is also a problem that the thawing treatment causes the components in the body to diffuse from a specific site. Furthermore, since frozen sections prepared from bones, plant samples, etc. do not adhere to conductively treated slide glasses, there is also the problem that sections for imaging mass spectrometry cannot be prepared on conductively treated slide glasses.
凍結試料の薄切中に起こる切片の損壊はプラスチック製粘着テープを凍結切片の支持材として用いる事により解決し、粘着テープに貼りついた状態の切片をイメーシング質量分析装置で分析する事が試みられている。しかしプラスチック製粘着テープは導電性を有していないため分析試料とイメージング質量分析装置間に導電性を確保できず、試料の分析部位が帯電してイメージング質量分析装置の検出感度が低化して精度良く質量分析を行えない問題があった。導電性の問題を解決するために薄い金属箔に粘着剤を塗布した導電性粘着支持材を用いた凍結切片作製が試みられているが、金属箔は柔軟性に乏しいために凍結切片を確実に粘着支持することができず形状の保たれた凍結切片を作製できない問題があった。 Section damage that occurs during sectioning of frozen specimens has been resolved by using plastic adhesive tape as a support material for frozen sections, and attempts have been made to analyze sections stuck to the adhesive tape with an imaging mass spectrometer. there is However, since the plastic adhesive tape is not conductive, it is not possible to ensure conductivity between the sample to be analyzed and the imaging mass spectrometer. There was a problem that mass spectrometry could not be performed well. In order to solve the problem of electrical conductivity, attempts have been made to prepare frozen sections using a conductive adhesive support, which is a thin metal foil coated with an adhesive. There was a problem that it was not possible to prepare a frozen section that maintained its shape because it could not be adhesively supported.
これらの理由により、組織形態を保ち、しかも生体内成分の組織内分布を保持した切片試料を作製し、しかも試料とイメージング質量分析装置間に導電性を確保した状態で感度良く特定質量の物質の分布をイメージング質量分析装置で観察するのに適した試料作製法はなかった。 For these reasons, it is necessary to prepare a section sample that maintains the tissue morphology and the intra-tissue distribution of the in-vivo components, and to extract a substance of a specific mass with high sensitivity while ensuring electrical conductivity between the sample and the imaging mass spectrometer. There was no suitable sample preparation method for observing the distribution with an imaging mass spectrometer.
上記に述べているように、従来の方法では生体内成分と分布が保たれ、しかも組織形態が保たれた切片試料を導電性支持体上に作製する事ができなかったために、イメージング質量分析装置を用いて組織中に分布している特定質量の物質を感度よく検出し、しかも正確に組織に関連させて画像化する事ができなかった。 As described above, it was not possible to prepare a slice sample on a conductive support in which the in vivo components and distribution were maintained and the tissue morphology was maintained by the conventional method. It has not been possible to sensitively detect a substance of a specific mass distributed in a tissue and to image it accurately in relation to the tissue.
本発明は、この問題を解決するためにイメージング質量分析装置と分析用試料間に導電性を確保する事ができる導電性粘着フィルムを作製し、その導電性粘着フィルムを用いて組織内成分と組織形態を保った凍結切片を作製し、切片が貼り付いた導電性粘着フィルムをスライドガラスに両面テープ等で固定し、そのスライドガラスをイメージング質量分析装置試料台に装着して導電性を確保した状態で分析することにより感度良く、しかも分析データを正確に組織に関連させたイメージング質量分析を可能にするイメージング質量分析に好適な試料作製法を提供するものである。 In order to solve this problem, the present invention produces a conductive adhesive film that can ensure conductivity between the imaging mass spectrometer and the sample for analysis, and uses the conductive adhesive film to A state in which a frozen section that maintains its shape is prepared, the conductive adhesive film with the section attached is fixed to a slide glass with double-sided tape, etc., and the conductivity is secured by mounting the slide glass on the imaging mass spectrometer sample stage. To provide a sample preparation method suitable for imaging mass spectrometry, which enables imaging mass spectrometry in which analysis data are accurately related to tissues with high sensitivity by analyzing with .
上記の課題を解決するために、第一発明では生物組織の形態と生体内成分が保たれた試料片を作製するために、まず生物試料を凍結し、その試料を凍結ミクロトームの試料台に固定し、切削面に導電性粘着フィルムを密着して切削を行って作製した導電性粘着フィルム上の凍結切片を用いてイメージング質量分析装置で分析する事により感度良く生体内成分の分布を観察できるイメージング質量分析に好適な試料作製法を提供する。 In order to solve the above problems, in the first invention, in order to prepare a sample piece in which the morphology and in vivo components of a biological tissue are maintained, the biological sample is first frozen, and then the sample is fixed on a sample stage of a freezing microtome. Imaging that can observe the distribution of in vivo components with high sensitivity by analyzing with an imaging mass spectrometer using a frozen section on the conductive adhesive film prepared by cutting while adhering the conductive adhesive film to the cut surface. A sample preparation method suitable for mass spectrometry is provided.
第二発明では、第一発明で導電性粘着フィルム上に作製した切片中の生体成分の分解や拡散を防ぐために導電性粘着フィルムに貼り付いた状態の凍結切片を凍結ミクロトーム内で凍結乾燥する。 In the second invention, the frozen sections attached to the conductive adhesive film are freeze-dried in a freezing microtome in order to prevent the decomposition and diffusion of the biological components in the sections prepared on the conductive adhesive film in the first invention.
次いで、凍結乾燥後に切片を凍結ミクロトームから取り出し、スライドガラス上に導電性粘着フィルムの試料面を上に向け、両面テープで導電性粘着フィルムの基材側をスライドガラスに固定し、その試料をイメージング質量分析装置で分析する事により感度良く生体内成分の分布を観察できるイメージング質量分析に好適な試料作製法を提供する。 Next, after freeze-drying, the section is taken out from the freezing microtome, the sample side of the conductive adhesive film is turned up on the slide glass, the substrate side of the conductive adhesive film is fixed to the slide glass with double-sided tape, and the sample is imaged. Provided is a sample preparation method suitable for imaging mass spectrometry, in which the distribution of in vivo components can be observed with high sensitivity by analyzing with a mass spectrometer.
第三発明では、第一発明および第二発明で使用する形態を保持した凍結切片を作製するのに適した導電性粘着フィルムを凍結薄切を実施する低温下でも強い粘着力を有する粘着剤に導電性微粒子を混和した導電性粘着剤を薄いプラスチックフィルム上に塗布して作製した導電性粘着フィルムを用いて凍結切片を作製し、凍結乾燥後にイメージング質量分析装置で特定質量の物質の分析を行う事を特徴とするイメージング質量分析に好適な試料作製法を提供する。 In the third invention, a conductive adhesive film suitable for preparing frozen sections that retains the shape used in the first and second inventions is used as an adhesive that has strong adhesive strength even at low temperatures for performing frozen sectioning. Frozen sections are prepared using a conductive adhesive film prepared by coating a thin plastic film with a conductive adhesive mixed with conductive fine particles, freeze-dried, and then analyzed for substances with a specific mass using an imaging mass spectrometer. To provide a sample preparation method suitable for imaging mass spectrometry characterized by:
第四発明では、第一発明および第二発明で使用する形態を保持した凍結切片を作製するのに適した導電性粘着フィルムを凍結薄切を実施する低温下でも強い粘着力を有する粘着剤に導電性微粒子を混和した導電性粘着剤を金属蒸着した薄いプラスチックフィルム上に塗布することにより作製した導電性の優れた導電性粘着フィルムを用いて凍結切片を作製し、凍結乾燥後にイメージング質量分析装置で特定質量の物質の分析を行う事を特徴とするイメージング質量分析に好適な試料作製法を提供する。 In the fourth invention, a conductive adhesive film suitable for preparing frozen sections that retains the shape used in the first and second inventions is used as an adhesive that has strong adhesive strength even at low temperatures for performing frozen sectioning. Frozen sections are prepared using a conductive adhesive film with excellent conductivity, which is produced by coating a conductive adhesive mixed with conductive fine particles on a thin plastic film with metal vapor deposition, freeze-dried, and then subjected to an imaging mass spectrometer. Provided is a sample preparation method suitable for imaging mass spectrometry characterized by analyzing a substance with a specific mass at .
第五発明では、第三発明および第四発明で作製した導電性粘着フィルムを凍結試料の切削面に効率良く正確に貼付できるようにするために第三発明および第四発明で作製した導電性粘着フィルムの両端に金属箔あるいはプラスチックフィルムを付着して非粘着域を設け、凍結試料の切削面に効率良く導電性粘着フィルムを正確に貼付することができるようにした導電性粘着フィルムを用いてイメージング質量分析用試料を作製する事を特徴とするイメージング質量分析に好適な試料作製法 In the fifth invention, the conductive adhesive film produced in the third and fourth inventions can be efficiently and accurately attached to the cut surface of the frozen sample. Imaging using a conductive adhesive film that has a non-adhesive area by attaching metal foil or plastic film to both ends of the film so that the conductive adhesive film can be applied efficiently and accurately to the cut surface of the frozen sample. A sample preparation method suitable for imaging mass spectrometry characterized by preparing a sample for mass spectrometry
本発明は次のような効果を奏する。
第一発明によれば、導電性粘着フィルムを用いる事により、導電性支持体上に生物試料の形態が保たれたイメージング質量分析用切片試料を容易に作製でき、しかも分析用試料とイメージング質量分析装置間に導電性保つことができることから、従来の方法で作製した分析用試料よりも特定質量の物質の組織内分布状態を組織形態に正確に関連させて、しかも感度良く分析する事を特徴とするイメージング質量分析に好適な試料作製法を提供することができる。The present invention has the following effects.
According to the first invention, by using a conductive adhesive film, it is possible to easily prepare a section sample for imaging mass spectrometry in which the morphology of the biological sample is maintained on the conductive support, and furthermore, the sample for analysis and imaging mass spectrometry can be easily prepared. Since electrical conductivity can be maintained between the devices, it is characterized in that the tissue distribution state of a substance of a specific mass can be more accurately related to the tissue morphology than in the analysis sample prepared by the conventional method, and the analysis can be performed with high sensitivity. It is possible to provide a sample preparation method suitable for imaging mass spectrometry.
第二発明によれば、導電性粘着フィルムを用いる事により、導電性支持体上に作製された形態が保たれた凍結切片を凍結乾燥することにより生体成分の分解と特定部位からの拡散がない状態のイメージング質量分析用切片試料を容易に作製でき、しかも分析用試料とイメージング質量分析装置間に導電性保つことができることから、従来の方法で作製した分析用試料よりも特定質量の物質の組織内分布を組織形態に関連させて正確に、しかも感度良く分析する事を特徴とするイメージング質量分析に好適な試料作製法を提供することができる。 According to the second invention, by using a conductive pressure-sensitive adhesive film, freeze-drying a frozen section that has been prepared on a conductive support and whose morphology has been preserved prevents decomposition of biological components and diffusion from a specific site. A section sample for imaging mass spectrometry in a state can be easily prepared, and electrical conductivity can be maintained between the analysis sample and the imaging mass spectrometer. It is possible to provide a sample preparation method suitable for imaging mass spectrometry characterized by accurate and sensitive analysis of internal distribution in relation to tissue morphology.
第三発明では、第一発明および第二発明で使用する形態を保持した凍結切片を作製するのに適した導電性粘着フィルムを凍結薄切を実施する低温下でも凍結試料を確実に粘着支持できる強い粘着力を有する粘着剤に導電性微粒子を混和した導電性粘着剤を薄いプラスチックフィルム上に塗布して作製した導電性粘着フィルムを用いて凍結切片を作製するので、生物組織の形態が保たれた凍結切片を容易に作製できる事を特徴とするイメージング質量分析に好適な試料作製法を提供することができる。 In the third invention, a conductive adhesive film suitable for preparing a frozen section that retains the shape used in the first and second inventions can reliably adhere and support a frozen sample even at a low temperature at which cryosectioning is performed. Cryosections are prepared using a conductive adhesive film prepared by coating a thin plastic film with a conductive adhesive obtained by mixing conductive fine particles with an adhesive having strong adhesive strength, so that the morphology of the biological tissue is preserved. It is possible to provide a sample preparation method suitable for imaging mass spectrometry characterized by the ability to easily prepare frozen sections.
第四発明では、第一発明あるいは第二発明で使用する形態を保持した凍結切片作製に適した導電性粘着フィルムを凍結薄切を実施する低温下でも強い粘着力を有する粘着剤に導電性微粒子を混和した導電性粘着剤を金属蒸着した薄いプラスチックフィルム上に塗布して作製した導電性の優れた導電性粘着フィルムを用いて凍結切片を作製するので分析試料とイメージング質量分析装置間の導電性が改善されて感度良く生体成分を分析する事を可能にする事を特徴とするイメージング質量分析に好適な試料作製法を提供することができる。 In the fourth invention, a conductive adhesive film suitable for making frozen sections that retains the shape used in the first or second invention is applied to an adhesive that has strong adhesive strength even at low temperatures for performing frozen sectioning, and conductive fine particles. A conductive adhesive mixed with is coated on a thin plastic film with metal vapor deposition. is improved, and it is possible to provide a sample preparation method suitable for imaging mass spectrometry, which is characterized by enabling the analysis of biological components with high sensitivity.
第五発明では、第三発明あるいは第四発明で使用する導電性粘着フィルムの粘着面の両端に金属箔あるいはプラスチックフィルムを付着して非粘着領域を設けて導電性粘着フィルムを掴むことができるようにしているために凍結試料の切削面に導電性粘着フィルムを正確に効率良く貼付することができ、しかも金属箔を使用している事から導電性粘着フィルムと分析用試料間の導電性が良くなり、感度の良い分析用試料を作製できる事を特徴とするイメージング質量分析に好適な試料作製法を提供することができる。 In the fifth invention, a metal foil or plastic film is attached to both ends of the adhesive surface of the conductive adhesive film used in the third invention or the fourth invention to provide a non-adhesive area so that the conductive adhesive film can be gripped. Because of this, the conductive adhesive film can be applied accurately and efficiently to the cut surface of the frozen sample, and since metal foil is used, the conductivity between the conductive adhesive film and the sample for analysis is good. Therefore, it is possible to provide a sample preparation method suitable for imaging mass spectrometry, which is characterized by the ability to prepare a sample for analysis with good sensitivity.
以下、添付図面を参照して、本発明の一実施の形態について説明する。 An embodiment of the present invention will be described below with reference to the accompanying drawings.
第一発明では、形態と組織成分が保たれた分析用試料片を導電性が確保された状態で作製するために、まず生物試料中の生体成分の分解と拡散を防ぐために凍結し、ついで導電性粘着フィルムを凍結試料の切削面に貼付し、切削して凍結試料片を作製する。 In the first invention, in order to prepare a sample piece for analysis in which the morphology and tissue components are maintained in a state in which conductivity is ensured, the biological sample is first frozen to prevent decomposition and diffusion of the biological components, and then the conductive material is frozen. A flexible adhesive film is attached to the cut surface of the frozen sample and cut to prepare a frozen sample piece.
第二発明では、第一発明で導電性粘着フィルム上に作製した凍結切片をクリオスタットから取り出す際の解凍による組織中成分の酵素による分解を防ぐこと、解凍による物質の拡散を防ぐためにクリオスタット内で凍結切片を凍結乾燥する。 In the second invention, when the frozen section prepared on the conductive adhesive film in the first invention is taken out of the cryostat, it prevents enzymatic decomposition of the components in the tissue due to thawing. Lyophilize the cryosections at .
上記第一発明と第二発明の凍結切片作製で使用する凍結切片支持のための導電性粘着フィルムには、以下に記載している第三発明、第四発明および第五発明の導電性粘着フィルムを使用する。 The conductive adhesive film for supporting frozen sections used in the preparation of frozen sections of the first and second inventions above includes the conductive adhesive films of the third, fourth and fifth inventions described below. to use.
第三発明の導電性粘着フィルムはプラスチックフィルム上の片面に導電性粘着剤を塗布して作製し、図２に示す構成となっている。 The conductive pressure-sensitive adhesive film of the third invention is produced by applying a conductive pressure-sensitive adhesive to one side of a plastic film, and has the structure shown in FIG.
第四発明の導電性粘着フィルムでは導電性粘着フィルムの導電性を高めることによりイメージング質量分析の感度を高くするために、第三発明の導電性粘着フィルムの基材として使用したプラスチックフィルムに代わってプラスチックフィルムの片面の全面にアルミニウムや銅などの金属を蒸着したプラスチックフィルムを用い、金属蒸着した上に導電性粘着剤を塗布して作製し、図３の構成となっている。 In the conductive adhesive film of the fourth invention, in order to increase the sensitivity of imaging mass spectrometry by increasing the conductivity of the conductive adhesive film, instead of the plastic film used as the base material of the conductive adhesive film of the third invention A plastic film in which a metal such as aluminum or copper is vapor-deposited on the entire surface of a plastic film is used, and a conductive adhesive is applied on the metal vapor-deposited plastic film to form the structure shown in FIG.
第五発明の導電性粘着フィルムでは、図４および図５に示すように第三発明および第四発明の導電性粘着フィルムを用いて凍結切片を作製する際に導電性粘着フィルムを掴む領域として導電性粘着フィルムの両端に非粘着域を設け、凍結試料の切削面に導電性粘着フィルムを正確に容易に貼付する事ができるようにしている。非粘着域作製に使用する材料として導電性粘着フィルムとイメージング質量分析装置の導電性を確保するために銅やアルミニウム等の金属箔を使用する。 In the conductive adhesive film of the fifth invention, as shown in FIGS. 4 and 5, a conductive adhesive film is used as a region for gripping the conductive adhesive film when preparing a frozen section using the conductive adhesive film of the third and fourth inventions. Non-adhesive regions are provided at both ends of the conductive adhesive film so that the conductive adhesive film can be accurately and easily attached to the cut surface of the frozen sample. A conductive adhesive film and a metal foil such as copper or aluminum are used to ensure the electrical conductivity of the imaging mass spectrometer as the materials used for forming the non-adhesive area.
第三発明、第四発明および第五発明で使用するプラスチックフィルムとして、凍結切片を作製する－２０℃以下の低温下でも柔軟性を保った材料が適し、具体的には、厚さ３０μｍよりも２０μｍ、２０μｍよりも１５μｍ、さらに１５μｍよりも１２μｍ以下が好適である。 As the plastic film used in the third, fourth and fifth inventions, a material that maintains flexibility even at a low temperature of -20 ° C or less for preparing frozen sections is suitable. Specifically, the thickness is less than 30 μm. 20 μm, 15 μm rather than 20 μm, and 12 μm or less than 15 μm are preferable.
第三発明、第四発明および第五発明で使用する粘着剤としては、凍結切片を作製する－２０℃以下の温度でも凍結切片を確実に粘着できるものを使用する。試料により－３０℃以下の温度での凍結薄切が必要な場合があり、そのような試料薄切には－３０℃以下で凍結切片を粘着支持できる粘着剤を使用する。 The adhesive used in the third, fourth and fifth inventions should be one that can reliably adhere to frozen sections even at a temperature of -20°C or lower at which frozen sections are prepared. Depending on the sample, frozen sectioning at a temperature of −30° C. or lower may be necessary, and for such sample sectioning, an adhesive capable of adhesively supporting the frozen section at −30° C. or lower is used.
第三発明、第四発明および第五発明で粘着剤に混和する導電性微粒子としてカーボン微粒子と金属性微粒子を使用する事ができる。導電性微粒子は小さいほど導電性粘着フィルムの粘着面が平滑になって薄切面と粘着面の接触面積が増加して粘着力が強くなり、良好な凍結切片を作製できる。また分析試料と導電性粘着フィルムとの導電性もよくなって検出感度が良くなることから出来るだけ微粒子が適している。例えば、直径１００ｎｍ以下の微粒子を使用すると平面性の良い粘着面を作製でき、更に直径５０ｎｍ以下の微粒子を使用するとより平滑な平面となり好適である。 In the third, fourth and fifth inventions, carbon fine particles and metallic fine particles can be used as the conductive fine particles mixed with the pressure-sensitive adhesive. The smaller the conductive fine particles, the smoother the adhesive surface of the conductive adhesive film, the greater the contact area between the sliced surface and the adhesive surface, the stronger the adhesive force, and the better the frozen section can be produced. In addition, fine particles are suitable as much as possible because the conductivity between the analysis sample and the conductive adhesive film is improved and the detection sensitivity is improved. For example, fine particles with a diameter of 100 nm or less can be used to form an adhesive surface with good flatness, and fine particles with a diameter of 50 nm or less are preferably used to form a smoother surface.
上記の方法と導電性粘着フィルムで作製した凍結切片は、凍結切片作製後直ちに、あるいは凍結乾燥後クリオスタットから取り出し、図２、図３、図４および図５に示す配置となるように切片が貼り付いた導電性粘着フィルムを両面テープでスライドガラス上に固定する。 The cryosections prepared by the above method and the conductive adhesive film were taken out of the cryostat immediately after preparation of the frozen sections or after freeze-drying, and the sections were arranged as shown in FIGS. The adhered conductive adhesive film is fixed on the slide glass with double-sided tape.
切片中の物質がレーザー光のエネルギーを吸収して生体内物質が蒸発しやすくするためにα－Ｃｙａｎｏ－４－ｈｙｄｒｏｘｙ－ｃｉｎｎａｍｉｃ ａｃｉ、ｔｒａｎｓ－４－ヒドロキシ－３－メトキシケイ皮酸等のマトリクスを切片上に塗布する。 A matrix such as α-Cyano-4-hydroxy-cinnamic acid, trans-4-hydroxy-3-methoxycinnamic acid is added so that the substances in the section absorb the energy of the laser light and the substances in the body can easily evaporate. Apply on the section.
次いで、イメージング質量分析装置の試料台に装着して分析を実施する。 Next, it is mounted on the sample stage of an imaging mass spectrometer and analyzed.
次に具体的な実施例について説明する。 Next, specific examples will be described.
まず、分析用試料を凍結包埋し、凍結ミクロトームに装着して分析対象部位が現れるまで切削する。 First, the sample for analysis is cryo-embedded, mounted on a cryomicrotome, and cut until the site to be analyzed appears.
次に、－３０℃の凍結切片を確実に粘着支持できる低温度でも使用できる粘着剤に直径５０ｎｍ以下のカーボン微粒子を加えて作製した導電性粘着剤を厚さ２０μｍ以下の厚みのプラスチックフィルムに塗布して作製した第三、第四あるいは第五発明の導電性粘着フィルムを凍結ブロックの切削面に密着し、切削を行って凍結切片を作製する。 Next, a conductive adhesive made by adding carbon particles with a diameter of 50 nm or less to an adhesive that can be used even at low temperatures that can reliably adhere to -30 ° C frozen sections is applied to a plastic film with a thickness of 20 μm or less. The electrically conductive adhesive film of the third, fourth or fifth invention produced by the above method is adhered to the cut surface of the frozen block and cut to produce a frozen section.
次いで、凍結切片をクリオスタット内に放置し、完全に凍結乾燥してからクリオスタットから取り出す。解凍切片を使用する場合は、凍結薄切後直ちにクリオスタットから取り出して解凍する。 The cryosections are then left in the cryostat and completely lyophilized before being removed from the cryostat. If using thawed sections, remove from the cryostat immediately after frozen sectioning and thaw.
クリオスタットから取り出した粘着フィルムに貼り付いた切片をスライドガラス上に両面テープで固定する（図２、図３、図５）。この固定作業では切片面を上に向け、粘着フィルムの基材側を両面テープでスライドガラスに固定する。 The section attached to the adhesive film taken out from the cryostat is fixed on the slide glass with double-sided tape (Figs. 2, 3 and 5). In this fixing operation, the cut surface faces upward, and the substrate side of the adhesive film is fixed to the slide glass with double-sided tape.
次いで、切片上にマトリクス（例えば、α－Ｃｙａｎｏ－４－ｈｙｄｒｏｘｙ－ｃｉｎｎａｍｉｃ ａｃｉｄ等）をコートする。Then, the sections are coated with a matrix (eg, α-Cyano-4-hydroxy-cinnamic acid, etc.).
次いで、切片をイメージング質量分析装置に装着し分析する。 Sections are then mounted on an imaging mass spectrometer and analyzed.
上記の手順により、組織形態と生体内物質の組織内分布が保たれ、しかも高い感度で検出する事ができるイメージング質量分析用試料片を容易に、しかも確実に作製できる。 By the above-described procedure, it is possible to easily and reliably prepare a sample piece for imaging mass spectrometry that maintains the tissue morphology and the intra-tissue distribution of substances in the body and that can be detected with high sensitivity.
１…スライドガラス、２…導電膜、３…分析試料、４…レーザー光、５…イメージング質量分析装置と試料片との導電性を確保するための電極、６…分析中に試料中に生じた不要なイオンが流れる方向を示す矢印、７…導電性粘着剤、８…粘着プラスチックフィルムの基材であるプラスチックフィルム、９…両面テープ、１０…金属蒸着膜、１１…金属箔 1
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| US20070278400A1 (en) | 2006-04-27 | 2007-12-06 | Bruker Daltonik Gmbh | Sample preparation for mass spectrometric imaging |
| JP2016511402A (en) | 2013-02-25 | 2016-04-14 | イマビオテクＩｍａｂｉｏｔｅｃｈ | Method for assessing tissue targeting of a molecule of interest |
| JP2018014345A (en) | 2016-07-19 | 2018-01-25 | 和生 渡辺 | Electromagnetic wave shielding material, electromagnetic wave shielding cable, electromagnetic wave shielding tape, and wire harness |
| JP2019109146A (en) | 2017-12-19 | 2019-07-04 | 旭化成株式会社 | Imaging-purpose sample fabrication method for mass spectrometer |
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| US20070278400A1 (en) | 2006-04-27 | 2007-12-06 | Bruker Daltonik Gmbh | Sample preparation for mass spectrometric imaging |
| JP2016511402A (en) | 2013-02-25 | 2016-04-14 | イマビオテクＩｍａｂｉｏｔｅｃｈ | Method for assessing tissue targeting of a molecule of interest |
| JP2018014345A (en) | 2016-07-19 | 2018-01-25 | 和生 渡辺 | Electromagnetic wave shielding material, electromagnetic wave shielding cable, electromagnetic wave shielding tape, and wire harness |
| JP2019109146A (en) | 2017-12-19 | 2019-07-04 | 旭化成株式会社 | Imaging-purpose sample fabrication method for mass spectrometer |
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