WO2023010846A1 - 一种生物组织样品成像方法 - Google Patents
一种生物组织样品成像方法 Download PDFInfo
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- WO2023010846A1 WO2023010846A1 PCT/CN2022/079853 CN2022079853W WO2023010846A1 WO 2023010846 A1 WO2023010846 A1 WO 2023010846A1 CN 2022079853 W CN2022079853 W CN 2022079853W WO 2023010846 A1 WO2023010846 A1 WO 2023010846A1
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- biological tissue
- sheet
- tissue sample
- gel
- magnetic material
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Classifications
-
- 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
- G01N1/36—Embedding or analogous mounting of samples
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
Definitions
- the invention relates to the technical field of biological tissue sample imaging methods, in particular to a biological tissue sample imaging method including a gel embedding step.
- High-resolution 3D fluorescence imaging of biological tissue is an effective means to obtain the 3D structure of biological tissue and study biological issues such as gene expression, cell morphology, and cell distribution at the subcellular, cellular, and tissue scales.
- Biological tissue transparency technology makes biological tissue transparent, thus overcoming the main obstacle of high-resolution three-dimensional imaging of biological tissue using fluorescence microscopy, so that cutting-edge three-dimensional fluorescence microscopy imaging technologies such as light sheet microscopy can be used for efficient Obtain cellular and subcellular three-dimensional structural information of various biological tissues, thereby helping researchers better understand the structure and function of biological tissues and organs. Due to this remarkable advantage, biological tissue transparent technology is quickly applied to various fields of life science research.
- the hydrophilic clearing method of CUBIC has the advantages of high biological safety, high retention rate of endogenous fluorescent proteins, compatible with immunostaining, and suitable for high-resolution imaging, etc. Much attention.
- gel embedding the cleared biological tissue samples can protect the biological tissue samples to a certain extent and solve the problem of inconvenient transfer by using materials such as agarose.
- materials such as agarose are prone to cracking and breaking during movement.
- gel-embedded biological samples are not easy to fix on the sample holder of the imaging microscope, and it is not easy to adjust the position of the sample, which makes it difficult to obtain high-quality imaging.
- the present invention provides a biological tissue sample imaging method, which includes:
- the biological tissue may be a biological tissue selected from brain, spinal cord, etc.
- the biological tissue sample may be the whole or a part of the above-mentioned tissue.
- the organism may be one or more selected from biological research model animals.
- the biological research model animals can be, for example, nematodes, zebrafish, planarians, fruit flies, Xenopus laevis, salamanders, mice, rabbits, pigs, monkeys and the like.
- the organism may be a vertebrate, including mammals, reptiles, birds, and the like.
- the mammal can be, for example, human, mouse, rabbit, pig, monkey, etc.
- the magnetic material sheet refers to a sheet formed of magnetic material.
- the magnetic material can be selected from hard magnetic materials or soft magnetic materials, such as ferromagnetic materials, ferrite materials, rare earth permanent magnetic materials and the like.
- the sheet may be selected from rigid sheets, flexible sheets, porous sheets and non-porous sheets.
- the biological tissue sample is a biological tissue sample that has been cleared by a hydrophilic or hydrogel clearing method.
- the gel precursor solution is a solution of 1.5% to 3%, preferably 1.8% to 2.5%, especially about 2% agarose in a refractive index matching solution in terms of mass percent concentration.
- the sheet with the magnetic material is at the bottom, and the biological tissue sample is located above the sheet of magnetic material.
- a specific device when it is described that a specific device is located between a first device and a second device, there may or may not be an intervening device between the specific device and the first device or the second device.
- the specific device When it is described that a specific device is connected to other devices, the specific device may be directly connected to the other device without an intervening device, or may not be directly connected to the other device but has an intervening device.
- the present invention provides a biological tissue sample imaging method, which comprises:
- the biological tissue is not particularly limited, and it may be any tissue from an animal, such as brain, spinal cord and the like.
- the biological tissue sample may be the whole or a part of the above-mentioned tissue.
- the animal may be one or more selected from biological research model animals.
- the biological research model animals can be, for example, nematodes, zebrafish, planarians, fruit flies, Xenopus laevis, salamanders, mice, rabbits, pigs, monkeys and the like.
- the organism may be a vertebrate, including mammals, reptiles, birds, and the like.
- the mammal can be, for example, human, mouse, rabbit, pig, monkey, etc.
- the embedding container is not particularly limited, and those skilled in the art can select a suitable existing container or mold according to the size and shape of the biological tissue sample to be imaged, or design and make a suitable container or mold .
- the magnetic material sheet refers to a sheet formed of magnetic material.
- the magnetic material is not particularly limited, as long as it is a material that can be attracted by a permanent magnetic material (such as a magnet) or an electromagnet.
- a permanent magnetic material such as a magnet
- an electromagnet such as a permanent magnetic material (such as a magnet) or an electromagnet.
- it can be hard magnetic material or soft magnetic material, such as ferromagnetic material, ferrite material, rare earth permanent magnetic material, etc., such as iron, cobalt, nickel, gadolinium, iron carbon alloy, iron nickel alloy, iron cobalt nickel alloy , iron-cobalt alloy, iron-aluminum alloy, iron-silicon alloy, iron-silicon-aluminum alloy, iron-nickel-manganese alloy, alloy material of iron and rare earth elements, or ferrite material, etc.
- the sheet may be a sheet formed entirely of magnetic materials, such as iron sheet, silicon steel sheet, nickel sheet, etc., or a sheet formed of non-magnetic material and magnetic material, such as a non-magnetic material sheet A sheet formed by coating a magnetic material on the surface of a material or a sheet formed by coating a non-magnetic material on the surface of a magnetic material sheet, but not limited thereto.
- the sheet may be a rigid sheet, may also be a flexible sheet, may be a porous sheet or a non-porous sheet, but is not limited thereto.
- the gel precursor solution refers to a solution capable of further gelation to form a gel, which can be prepared by dissolving any suitable gel agent in a solvent.
- the gel reagent may be agarose, polylysine, collagen, gelatin, polyacrylamide, etc., but not limited thereto, preferably agarose.
- gelation can be achieved, for example, by dissolving the gelling agent in a solvent at high temperature and then cooling.
- the solvent may be water, or a solution prepared for a certain purpose, such as a refractive index matching solution used in a hydrophilic or hydrogel type clearing method.
- the biological tissue sample is a biological tissue sample that has been cleared by a hydrophilic or hydrogel clearing method
- the gel precursor solution is, in terms of mass percent concentration, 1.5 % to 3%, preferably 1.8% to 2.5%, especially about 2% agarose in a refractive index matching solution.
- the transparent treatment includes degreasing treatment and refractive index matching.
- the degreasing treatment and refractive index matching can be performed by using any suitable degreasing agent and refractive index matching agent used in any hydrophilic or hydrogel type clearing method in the art.
- the degreasing reagent may be CUBIC, CUBIC-L degreasing reagent, and the refractive index matching reagent may be CUBIC-R refractive index matching reagent, but not limited thereto.
- Specific degreasing reagents and refractive index matching reagents can be obtained by referring to papers or books related to hydrophilic or hydrogel-type transparent methods.
- the magnetic material sheet in the resulting gel, is at the bottom, and the biological tissue sample is located above the magnetic material sheet.
- the magnetic base refers to a magnetic device in the base, which has the magnetism to absorb the above-mentioned magnetic material sheet.
- the device may be permanently magnetic or electromagnetic.
- the magnetic base is usually processed from a chemically stable material, such as stainless steel and Teflon, and is embedded or attached with a magnetic material for absorbing the gel embedded with the magnetic material sheet.
- the shape of the magnetic base can be designed in different ways according to the characteristics and imaging methods of the corresponding microscope. After the embedding gel is adsorbed on the magnetic base, the magnetic base can be mounted on an optical microscope for imaging the embedded sample, thus allowing the cleared and embedded biological tissue to be examined. 3D imaging.
- the sample after gel embedding can be fixed on the magnetic base of the sample holder of the imaging microscope by magnetic force, making it easier to position the sample Adjustment and three-dimensional imaging, thereby solving the problem that the gel-embedded biological sample is not easy to fix on the sample holder of the imaging microscope, and it is not easy to adjust the position of the sample, so that it is not easy to obtain high-quality images.
- the numerical value should be understood as having the precision of the effective digit of the numerical value.
- the number 40.0 should be understood to cover the range from 39.50 to 40.49.
- all numerical values of parameters (for example, amounts or conditions) in this specification (including the appended claims) should in all cases be understood as being understood by the term "about” modifier, regardless of whether "about” actually precedes the numerical value.
- “About” indicates that the stated value allows for some imprecision (some close to exactness in the value; about or reasonably close to the value; approximation).
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- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Sampling And Sample Adjustment (AREA)
Abstract
一种生物组织样品成像方法，包括：(1)在包埋容器中放入磁性材料片材、凝胶前体溶液和生物组织样品，然后形成凝胶，得到凝胶体，其中包埋有生物组织样品和具有铁磁性的多孔片材；(2)将凝胶体固定于成像显微镜的样品架的磁性底座上进行成像。
Description
本发明涉及生物组织样品成像方法技术领域，具体涉及一种包括凝胶包埋步骤的生物组织样品成像方法。
对生物组织进行高分辨率三维荧光成像是获取生物组织的三维结构，以及在亚细胞、细胞、和组织尺度上研究基因表达、细胞形态、和细胞分布等生物问题的有效手段。
生物组织透明化技术使生物组织变得透明，从而克服了使用荧光显微镜对生物组织进行高分辨率三维成像的主要障碍，使光片显微镜技术等前沿的三维荧光显微镜成像技术可以被用于高效的获取各种生物组织的细胞级和亚细胞级三维结构信息，从而帮助科研人员更好的了解生物组织、器官的结构和功能。由于这一显著优点，生物组织透明化技术很快的被应用于生命科学研究的各个领域。
在各种生物组织透明化技术中，例如CUBIC的亲水型透明化方法具有高生物安全性，高内源荧光蛋白保留率，兼容免疫染色，和适合高分辨率成像等优点，因而获得了更多的关注。
生物组织样品在使用亲水型透明化处理后，组织中的大多数脂质分子被去除，因此生物组织的机械强度变低，生物组织变的柔软，易变形或者破碎，使得生物组织样品的在后续的成像过程中很难保持生物组织的完整性，为使用光学显微镜对透明化处理后的生物组织进行三维成像造成极大的困难。
发明内容
本发明人在实践中发现，通过使用琼脂糖等材料对经过透明化处理的生物组织样品进行凝胶包埋可以在一定程度上保护生物组织样品，并解决转移不便的问题。然而，琼脂糖等材料形成的凝胶在移动过程中容易开裂破碎。此外，凝胶包埋的生物样品在成像显微镜的样品架上不易固定，且不容易调整样品位置，从而不便于得到高质量的成像。
为了解决上述问题，本发明提供一种生物组织样品成像方法，其包括：
1.在包埋容器中放入磁性材料片材、凝胶前体溶液和生物组织样品，然 后形成凝胶，得到凝胶体，其中包埋有生物组织样品和所述具有铁磁性的多孔片材；
2.将所述凝胶体固定于成像显微镜的样品架的磁性底座上进行成像。
所述生物组织可以是选自脑、脊髓等的生物组织
所述生物组织样品可以是上述组织的整体或其一部分。
所述生物可以是选自生物研究模式动物中的一种或多种。所述生物研究模式动物例如可以为线虫、斑马鱼、涡虫、果蝇、爪蟾、蝾螈、小鼠、兔、猪、猴等。
或者，所述生物可以是脊椎动物，包括哺乳动物、爬行动物、鸟等。所述哺乳动物例如可以是人、鼠、兔、猪、猴等。
所述磁性材料片材指的是由磁性材料形成的片材。所述磁性材料可以选自硬磁材料或软磁材料，例如铁磁性材料、铁氧体材料、稀土永磁材料等。
所述片材可以选自硬质片材、柔性片材、多孔片材和非多孔片材。
在一些实施方式中，所述生物组织样品为采用亲水型或水凝胶型透明化方法进行透明化处理后的生物组织样品。
所述凝胶前体溶液为以质量百分浓度计，1.5％至3％，优选1.8％至2.5％，特别是约2％的琼脂糖在折射率匹配溶液中的溶液。
在一些实施方式中，所得凝胶体中，所述具有磁性材料片材处于下部，以及所述生物组织样品位于所述磁性材料片材上方。
为使本领域技术人员更好的理解本公开的技术方案，下面结合具体实施方式对本公开作详细说明。下面结合具体实施例对本公开的实施例作进一步详细描述，但不作为对本公开的限定。
本公开中使用的“第一”、“第二”以及类似的词语并不表示任何顺序、数量或者重要性，而只是用来区分不同的部分。“包括”或者“包含”等类似的词语意指在该词前的要素涵盖在该词后列举的要素，并不排除也涵盖其他要素的可能。“上”、“下”、“左”、“右”等仅用于表示相对位置关系，当被描述对象的绝对位置改变后，则该相对位置关系也可能相应地改变。
在本公开中，当描述到特定器件位于第一器件和第二器件之间时，在该特定器件与第一器件或第二器件之间可以存在居间器件，也可以不存在居间器件。 当描述到特定器件连接其它器件时，该特定器件可以与所述其它器件直接连接而不具有居间器件，也可以不与所述其它器件直接连接而具有居间器件。
本公开使用的所有术语(包括技术术语或者科学术语)与本公开所属领域的普通技术人员理解的含义相同，除非另外特别定义。还应当理解，在诸如通用字典中定义的术语应当被解释为具有与它们在相关技术的上下文中的含义相一致的含义，而不应用理想化或极度形式化的意义来解释，除非这里明确地这样定义。
对于相关领域普通技术人员已知的技术、方法和设备可能不作详细讨论，但在适当情况下，所述技术、方法和设备应当被视为说明书的一部分。
本发明提供一种生物组织样品成像方法，其包括：
1.在包埋容器中放入磁性材料片材、凝胶前体溶液和生物组织样品，然后形成凝胶，得到凝胶体，其中包埋有生物组织样品和所述具有铁磁性的片材；
2.将所述凝胶体固定于成像显微镜的样品架的磁性底座上进行成像。
在根据本发明的方法中，所述生物组织没有特别限制，其可以是来自动物的任何组织，例如脑、脊髓等。
所述生物组织样品可以是上述组织的整体或其一部分。
所述动物可以是选自生物研究模式动物中的一种或多种。所述生物研究模式动物例如可以为线虫、斑马鱼、涡虫、果蝇、爪蟾、蝾螈、小鼠、兔、猪、猴等。
或者，所述生物可以是脊椎动物，包括哺乳动物、爬行动物、鸟等。所述哺乳动物例如可以是人、鼠、兔、猪、猴等。
在上述步骤1中，所述包埋容器没有特别限制，本领域技术人员可以根据所要成像的生物组织样品的尺寸、形状等选用适合的现有容器或模具，或者设计并制作适合的容器或模具。
所述磁性材料片材指的是由磁性材料形成的片材。所述磁性材料没有特别限制，只要其是能够被永磁性材料(例如磁铁)或者电磁铁等吸附的材料即可。例如，其可以为硬磁材料或软磁材料，例如铁磁性材料、铁氧体材料、稀土永磁材料等，例如铁、钴、镍、钆、铁碳合金、铁镍合金、铁钴镍合金、铁钴合金、铁铝合金、铁硅合金、铁硅铝合金、铁镍锰合金、铁与稀土元素的合金材料，或铁氧体材料等。此外，所述片材可以是完全由磁性材料形成的片材，例如铁片、硅钢片、镍片等，也可以是由非磁性材料和磁性材料共同形成的片材，例如在非磁性材料片材的表面包覆磁性材料形成的片材或者在磁性材料片材 的表面包覆非磁性材料形成的片材，但是不限于此。此外，所述片材可以是硬质片材，也可以是柔性片材，可以是多孔片材或非多孔片材，但是不限于此。
通过在凝胶体形成过程中加入磁性材料片材，更加便于移动凝胶体并减少凝胶体在移动过程中的开裂破碎的发生。
所述凝胶前体溶液指的是能够进一步通过凝胶化而形成凝胶的溶液，其可通过将任何合适的凝胶试剂溶解在溶剂中而配制。
在一些实施方式中，所述凝胶试剂可以为琼脂糖、多聚赖氨酸、胶原蛋白、明胶、聚丙烯酰胺等，但不限于此，优选为琼脂糖。此时，例如可以通过在高温下将凝胶试剂溶解在溶剂中，然后冷却而实现凝胶化。
所述溶剂可以为水，或者为某种目的而配制的溶液，例如为在亲水型或水凝胶型透明化方法中使用的折射率匹配溶液。
在一些实施方式中，所述生物组织样品为采用亲水型或水凝胶型透明化方法进行透明化处理后的生物组织样品，所述凝胶前体溶液为以质量百分浓度计，1.5％至3％，优选1.8％至2.5％，特别是约2％的琼脂糖在折射率匹配溶液中的溶液。
所述透明化处理包括脱脂处理和折射率匹配。所述脱脂处理和折射率匹配可以采用本领域中任何进行亲水型或水凝胶型透明化方法使用的任何合适的脱脂试剂和折射率匹配试剂进行处理。例如所述脱脂试剂可以为CUBIC、CUBIC-L脱脂试剂，所述折射率匹配试剂可以为CUBIC-R折射率匹配试剂，但是不限于此。具体的脱脂试剂和折射率匹配试剂可以参考亲水型或水凝胶型透明化方法相关的论文或书籍而得到。
在一些实施方式中，所得凝胶体中，所述磁性材料片材处于下部，以及所述生物组织样品位于所述磁性材料片材上方。
在步骤2中，所述磁性底座指的是该底座中具有一种磁性装置，其具有吸附上述磁性材料片材的磁性。所述装置可以是永磁性的或者电磁的。该磁性底座通常由化学性质稳定的材料，例如不锈钢，特氟龙，加工而成，并且嵌入或者以其他方式附加磁性材料用于吸附包埋有磁性材料片材的凝胶体。磁性底座的外形可以根据相应显微镜的特性和成像方式而采用不同的设计。将包埋完成后的凝胶体吸附在磁性底座上以后，所述磁性底座可以安装在用于对包埋样品进行成像的光学显微上，从而允许对完成透明化和包埋的生物组织进行三维成像。
通过在凝胶体中包埋具有铁磁性的片材以及在样品架上采用磁性底座，凝 胶包埋后的样品可以通过磁力被固定于成像显微镜的样品架的磁性底座上，更加易于样品位置的调整和三维成像，由此解决了凝胶包埋的生物样品在成像显微镜的样品架上不易固定，且不容易调整样品位置，从而不便于得到高质量的图像的问题。
在本文中，所有以数值范围或百分比范围形式界定的特征或条件仅是为了简洁及方便。据此，数值范围或百分比范围的描述应视为已涵盖且具体公开所有可能的次级范围及范围内的个别数值。
在本文中，在可实现发明目的的前提下，数值应理解成具有该数值有效位数的精确度。举例来说，数字40.0则应理解成涵盖从39.50至40.49的范围。除了在详细描述最后提供的工作实施例之外，本申请文件(包括所附权利要求)中的参数(例如，数量或条件)的所有数值在所有情况下都应被理解为被术语“大约”修饰，不管“大约”是否实际出现在该数值之前。“大约”表示所述的数值允许稍微不精确(在该值上有一些接近精确；大约或合理地接近该值；近似)。如果“大约”提供的不精确性在本领域中没有以这个普通含义来理解，则本文所用的“大约”至少表示可以通过测量和使用这些参数的普通方法产生的变化。例如，“大约”可以包括小于或等于10％，小于或等于5％，小于或等于4％，小于或等于3％，小于或等于2％，小于或等于1％或者小于或等于0.5％的变化，并且在某些方面，小于或等于0.1％的变化。
此外，尽管已经在本文中描述了示例性实施例，其范围包括任何和所有基于本公开的具有等同元件、修改、省略、组合(例如，各种实施例交叉的方案)、改编或改变的实施例。权利要求书中的元件将被基于权利要求中采用的语言宽泛地解释，并不限于在本说明书中或本申请的实施期间所描述的示例，其示例将被解释为非排他性的。因此，本说明书和示例旨在仅被认为是示例，真正的范围和精神由以下权利要求以及其等同物的全部范围所指示。
以上描述旨在是说明性的而不是限制性的。例如，上述示例(或其一个或更多方案)可以彼此组合使用。例如本领域普通技术人员在阅读上述描述时可以使用其它实施例。另外，在上述具体实施方式中，各种特征可以被分组在一起以简单化本公开。这不应解释为一种不要求保护的公开的特征对于任一权利要求是必要的意图。相反，本公开的主题可以少于特定的公开的实施例的全部特征。从而，以下权利要求书作为示例或实施例在此并入具体实施方式中，其中每个权利要求独立地作为单独的实施例，并且考虑这些实施例可以以各种组合或排列彼此组合。本公开的范围应参照所附权利要求以及这些权利要求赋权 的等同形式的全部范围来确定。
以上实施例仅为本公开的示例性实施例，不用于限制本公开，本公开的保护范围由权利要求书限定。本领域技术人员可以在本公开的实质和保护范围内，对本公开做出各种修改或等同替换，这种修改或等同替换也应视为落在本公开的保护范围内。
Claims (10)
- 一种生物组织样品成像方法，其包括：(1)在包埋容器中放入磁性材料片材、凝胶前体溶液和生物组织样品，然后形成凝胶，得到凝胶体，其中包埋有生物组织样品和所述具有铁磁性的多孔片材；(2)将所述凝胶体固定于成像显微镜的样品架的磁性底座上进行成像。
- 根据权利要求1所述的方法，其中，所述生物组织是选自脑、脊髓的生物组织。
- 根据权利要求1所述的方法，其中，所述生物组织样品是生物组织的整体或其一部分。
- 根据权利要求1所述的方法，其中，所述生物是选自生物研究模式动物中的一种或多种。
- 根据权利要求1所述的方法，其中，所述生物是脊椎动物，包括哺乳动物、爬行动物、鸟；特别地，所述哺乳动物是人、鼠、兔、猪、猴。
- 根据权利要求1所述的方法，其中，所述磁性材料选自硬磁材料或软磁材料，例如铁磁性材料、铁氧体材料、稀土永磁材料。
- 根据权利要求1所述的方法，其中，所述片材选自硬质片材、柔性片材、多孔片材和非多孔片材。
- 根据权利要求1所述的方法，其中，所述生物组织样品为采用亲水型或水凝胶型透明化方法进行透明化处理后的生物组织样品。
- 根据权利要求1所述的方法，其中，所述凝胶前体溶液为以质量百分浓度计，1.5％至3％，优选1.8％至2.5％，特别是2％的琼脂糖在折射率匹配溶液中的溶液。
- 根据权利要求1所述的方法，其中，所得凝胶体中，所述磁性材料片材处于下部，以及所述生物组织样品位于所述磁性材料片材上方。
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