A method for imaging a biological tissue sample

Abstract

The present application relates to a kind of biological tissue sample imaging method, the method includes: (1) in embedding container is placed magnetic material sheet, gel precursor solution and biological tissue sample, then form gel, obtain gel body, wherein embedding has biological tissue sample and the porous sheet with ferromagnetic property;(2) the gel body is fixed on the magnetic base of the sample holder of imaging microscope and is imaged.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological tissue sample imaging methods, and particularly relates to a biological tissue sample imaging method comprising a gel embedding step. BACKGROUND

[0002] High-resolution three-dimensional fluorescence imaging of biological tissues is an effective means to obtain three-dimensional structures of biological tissues and to study biological problems such as gene expression, cell morphology, and cell distribution at subcellular, cellular, and tissue scales.

[0003] Biological tissue transparency technology makes biological tissues transparent, thereby overcoming the main obstacle to high-resolution three-dimensional imaging of biological tissues using fluorescence microscopes, and enabling advanced three-dimensional fluorescence microscope imaging technologies such as light sheet microscopy to be used to efficiently obtain three-dimensional structural information at the cellular and subcellular levels of various biological tissues, thereby helping researchers better understand the structure and function of biological tissues and organs. Due to this significant advantage, biological tissue transparency technology has been quickly applied in various fields of life science research.

[0004] Among various biological tissue transparency technologies, hydrophilic transparency methods such as CUBIC have attracted more attention due to their high biological safety, high endogenous fluorescent protein retention rate, compatibility with immunostaining, and suitability for high-resolution imaging.

[0005] After biological tissue samples are treated using a hydrophilic transparency process, most lipid molecules in the tissues are removed, and thus the mechanical strength of the biological tissues is reduced, the biological tissues become soft and are prone to deformation or breakage, making it difficult to maintain the integrity of the biological tissue samples during subsequent imaging processes, and causing great difficulties in using optical microscopes to perform three-dimensional imaging of the biological tissues after transparency treatment. SUMMARY

[0006] The present inventors have found in practice that gel embedding of biological tissue samples treated using a transparency process using agarose and the like can protect the biological tissue samples to some extent and solve the problem of inconvenience in transfer. However, the gel formed by agarose and the like is prone to cracking and breaking during movement. In addition, gel-embedded biological samples are not easy to fix on the sample holder of an imaging microscope, and the sample position is not easy to adjust, thereby making it inconvenient to obtain high-quality imaging.

[0007] To solve the above problems, the present application provides a biological tissue sample imaging method, which comprises:

[0008] 1. placing a magnetic material sheet, a gel precursor solution, and a biological tissue sample in an embedding container, and then forming a gel to obtain a gel body in which the biological tissue sample and the porous sheet having ferromagnetic properties are embedded;

[0009] 2. fixing the gel on a magnetic base of a sample holder of an imaging microscope for imaging.

[0010] The biological tissue can be a biological tissue selected from the group consisting of brain, spinal cord, etc.

[0011] The biological tissue sample can be a whole of the above-mentioned tissue or a part thereof.

[0012] The organism can be one or more selected from the group consisting of biological research model animals. The biological research model animals can be, for example, nematodes, zebra fish, planarians, fruit flies, frogs, newts, mice, rabbits, pigs, monkeys, etc.

[0013] Alternatively, the organism can be a vertebrate, including mammals, reptiles, birds, etc. The mammals can be, for example, humans, mice, rabbits, pigs, monkeys, etc.

[0014] The magnetic material sheet refers to a sheet formed of a 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, etc.

[0015] The sheet can be selected from hard sheets, flexible sheets, porous sheets, and non-porous sheets.

[0016] In some embodiments, the biological tissue sample is a biological tissue sample that has been subjected to a transparentization treatment using a hydrophilic or hydrogel type transparentization method.

[0017] The gel precursor solution is a solution of 1.5% to 3%, preferably 1.8% to 2.5%, in particular about 2% agarose in a refractive index matching solution by mass percentage concentration.

[0018] In some embodiments, in the obtained gel, the sheet of magnetic material is at the lower part, and the biological tissue sample is located above the sheet of magnetic material. DETAILED DESCRIPTION

[0019] In order for those skilled in the art to better understand the technical solutions of the present disclosure, the present disclosure will be described in detail below in combination with specific embodiments. The embodiments of the present disclosure will be further described in detail below in combination with specific examples, but are not intended to limit the present disclosure.

[0020] The terms "first," "second," and similar words used in this disclosure do not indicate any order, quantity, or importance, but are merely used to distinguish different parts. Words such as "including" or "contains" mean that the element preceding the word encompasses the element listed after it, and do not exclude the possibility of encompassing other elements as well. Terms such as "above," "below," "left," and "right" are used only to indicate relative positional relationships; when the absolute position of the described object changes, this relative positional relationship may also change accordingly.

[0021] In this disclosure, when a specific device is described as being located between a first device and a second device, an intermediary device may or may not be present between the specific device and the first or second device. When a specific device is described as being connected to other devices, the specific device may be directly connected to the other devices without an intermediary device, or it may be not directly connected to the other devices but have an intermediary device.

[0022] All terms used in this disclosure (including technical or scientific terms) have the same meaning as understood by one of ordinary skill in the art to which this disclosure pertains, unless otherwise specifically defined. It should also be understood that terms defined in a general dictionary, such as a dictionary, should be interpreted as having a meaning consistent with their meaning in the context of the relevant art, and not as having an idealized or highly formalized meaning, unless expressly defined herein.

[0023] Techniques, methods, and equipment known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and equipment should be considered part of the specification.

[0024] This invention provides a method for imaging biological tissue samples, comprising:

[0025] 1. Place a magnetic material sheet, a gel precursor solution, and a biological tissue sample into an embedding container, and then form a gel to obtain a gel body in which the biological tissue sample and the ferromagnetic sheet are embedded;

[0026] 2. Fix the gel onto the magnetic base of the sample holder of the imaging microscope for imaging.

[0027] In the method according to the invention, the biological tissue is not particularly limited and can be any tissue from an animal, such as the brain, spinal cord, etc.

[0028] The biological tissue sample may be the entire tissue or a part thereof.

[0029] The animal may be one or more selected from biological research model animals. Examples of biological research model animals include nematodes, zebrafish, planarians, fruit flies, Xenopus laevis, salamanders, mice, rabbits, pigs, and monkeys.

[0030] Alternatively, the organism may be a vertebrate, including mammals, reptiles, birds, etc. The mammal may be, for example, a human, a mouse, a rabbit, a pig, a monkey, etc.

[0031] In step 1 above, there are no particular limitations on the embedding container. Those skilled in the art can select suitable existing containers or molds according to the size, shape, etc. of the biological tissue sample to be imaged, or design and manufacture suitable containers or molds.

[0032] The magnetic material sheet refers to a sheet formed of magnetic material. There are no particular limitations on the magnetic material, as long as it is a material that can be attracted by permanent magnetic materials (such as magnets) or electromagnets. For example, it can be a hard magnetic material or a soft magnetic material, such as ferromagnetic materials, ferrite materials, rare earth permanent magnet materials, etc., such as iron, cobalt, nickel, gadolinium, iron-carbon alloys, iron-nickel alloys, iron-cobalt-nickel alloys, iron-cobalt alloys, iron-aluminum alloys, iron-silicon alloys, iron-silicon-aluminum alloys, iron-nickel-manganese alloys, alloys of iron and rare earth elements, or ferrite materials, etc. Furthermore, the sheet can be a sheet formed entirely of magnetic material, such as iron sheets, silicon steel sheets, nickel sheets, etc., or it can be a sheet formed by a combination of non-magnetic and magnetic materials, such as a sheet formed by coating a non-magnetic material sheet with a magnetic material sheet, or a sheet formed by coating a magnetic material sheet with a non-magnetic material sheet, but it is not limited to these. In addition, the sheet can be a rigid sheet or a flexible sheet, a porous sheet or a non-porous sheet, but it is not limited to these.

[0033] By incorporating magnetic material sheets during the gel formation process, it becomes easier to move the gel and reduces the occurrence of cracking and breakage during movement.

[0034] The gel precursor solution refers to a solution that can be further gelled by gelation, which can be prepared by dissolving any suitable gelling agent in a solvent.

[0035] In some embodiments, the gelling agent may be agarose, polylysine, collagen, gelatin, polyacrylamide, etc., but is not limited to these, with agarose being preferred. In this case, gelation can be achieved, for example, by dissolving the gelling agent in a solvent at high temperature and then cooling.

[0036] The solvent may be water, or a solution formulated for a specific purpose, such as a refractive index matching solution used in hydrophilic or hydrogel-type transparentization methods.

[0037] In some embodiments, the biological tissue sample is a biological tissue sample that has been cleared using a hydrophilic or hydrogel-type clearing method, and the gel precursor solution is a solution of agarose in a refractive index-matched solution, with a mass percentage concentration of 1.5% to 3%, preferably 1.8% to 2.5%, and particularly about 2%.

[0038] The transparency treatment includes degreasing and refractive index matching. The degreasing and refractive index matching can be performed using any suitable degreasing agent and refractive index matching agent used in any hydrophilic or hydrogel-based transparency treatment in the art. For example, the degreasing agent can be CUBIC or CUBIC-L degreasing agent, and the refractive index matching agent can be CUBIC-R refractive index matching agent, but is not limited to these. Specific degreasing agents and refractive index matching agents can be obtained by referring to relevant papers or books on hydrophilic or hydrogel-based transparency treatments.

[0039] In some embodiments, the magnetic material sheet is located at the bottom of the resulting gel, and the biological tissue sample is located above the magnetic material sheet.

[0040] In step 2, the magnetic base refers to a base containing a magnetic device that has the magnetism to attract the aforementioned magnetic material sheet. The device can be permanent magnetic or electromagnetic. The magnetic base is typically made of chemically stable materials, such as stainless steel or Teflon, and incorporates or otherwise attaches magnetic material to attract the gel containing the magnetic material sheet. The shape of the magnetic base can be designed differently depending on the characteristics of the corresponding microscope and imaging method. After the embedded gel is attracted to the magnetic base, the magnetic base can be mounted on an optical microscope for imaging the embedded sample, thereby allowing three-dimensional imaging of the transparentized and embedded biological tissue.

[0041] By embedding ferromagnetic sheets in the gel and using a magnetic base on the sample holder, the gel-embedded sample can be magnetically fixed to the magnetic base of the imaging microscope's sample holder, making it easier to adjust the sample position and perform three-dimensional imaging. This solves the problem that gel-embedded biological samples are not easy to fix on the imaging microscope's sample holder and it is not easy to adjust the sample position, thus making it difficult to obtain high-quality images.

[0042] In this document, all features or conditions defined in the form of numerical ranges or percentage ranges are for the sake of brevity and convenience only. Accordingly, the description of numerical ranges or percentage ranges should be regarded as covering and specifically disclosing all possible sub-ranges and individual values ​​within the ranges.

[0043] In this document, numerical values ​​are to be understood as having a precision with significant digits, provided that the inventive objectives are achieved. For example, the number 40.0 is to be understood as covering a range from 39.50 to 40.49. Except in the detailed description of the working embodiments provided at the end, all numerical values ​​of parameters (e.g., quantities or conditions) in this application (including the appended claims) are to be understood in all cases as being modified by the term “about,” regardless of whether “about” actually precedes the numerical value. “About” indicates that the numerical value allows for slight inaccuracies (some close to precision on that value; approximately or reasonably close to that value; approximate). If the inaccuracy provided by “about” is not understood in this common sense in the art, then “about” as used herein at least indicates a variation that can be produced by common methods of measuring and using these parameters. For example, “about” can include variations of less than or equal to 10%, less than or equal to 5%, less than or equal to 4%, less than or equal to 3%, less than or equal to 2%, less than or equal to 1%, or less than or equal to 0.5%, and in some respects, less than or equal to 0.1%.

[0044] Furthermore, although exemplary embodiments have been described herein, their scope includes any and all embodiments based on this disclosure that have equivalent elements, modifications, omissions, combinations (e.g., schemes involving intersections of various embodiments), adaptations, or alterations. Elements in the claims will be interpreted broadly based on the language used in the claims and are not limited to the examples described in this specification or during the implementation of this application, and such examples will be interpreted as non-exclusive. Therefore, this specification and examples are intended to be considered illustrative only, and the true scope and spirit are indicated by the full scope of the following claims and their equivalents.

[0045] The above description is intended to be illustrative and not restrictive. For example, the above examples (or one or more thereof) can be used in combination with each other. Other embodiments may be used by those skilled in the art upon reading the above description. Furthermore, in the above detailed description, various features may be grouped together to simplify the disclosure. This should not be construed as an intention that a feature of the disclosure that is not claimed is necessary for any claim. Rather, the subject matter of this disclosure may be less than all the features of a particular disclosed embodiment. Thus, the following claims are incorporated herein by reference as examples or embodiments, wherein each claim is an independent, separate embodiment, and these embodiments are contemplated to be combined with each other in various combinations or arrangements. The scope of this disclosure should be determined by reference to the appended claims and the full scope of their equivalents.

[0046] The above embodiments are merely exemplary embodiments of this disclosure and are not intended to limit this disclosure. The scope of protection of this disclosure is defined by the claims. Those skilled in the art can make various modifications or equivalent substitutions to this disclosure within its substance and scope, and such modifications or equivalent substitutions should also be considered to fall within the scope of protection of this disclosure.

Claims

1. A method for imaging biological tissue samples, comprising: (1) Place a magnetic material sheet, a gel precursor solution and a transparent biological tissue sample into an embedding container, and then form a gel to obtain a gel body in which the biological tissue sample and the magnetic material sheet are embedded. (2) The gel is fixed on the magnetic base of the sample holder of the imaging microscope for imaging.

2. The method according to claim 1, wherein, The biological tissue is selected from the brain and spinal cord.

3. The method according to claim 1, wherein, The biological tissue sample is the whole biological tissue or a part thereof.

4. The method according to claim 1, wherein, The organism is selected from one or more of the biological research model animals.

5. The method according to claim 1, wherein, The creature in question is a vertebrate.

6. The method according to claim 5, wherein, The vertebrates include mammals, reptiles, and birds.

7. The method according to claim 6, wherein, The mammals mentioned are humans, mice, rabbits, pigs, and monkeys.

8. The method according to claim 1, wherein, The magnetic material is selected from hard magnetic materials or soft magnetic materials.

9. The method according to claim 1, wherein, The magnetic material is selected from ferromagnetic materials.

10. The method according to claim 1, wherein, The magnetic material is selected from ferrite materials.

11. The method according to claim 1, wherein, The magnetic material is selected from rare earth permanent magnet materials.

12. The method according to claim 1, wherein, The sheet material is selected from rigid sheets, flexible sheets, porous sheets, and non-porous sheets.

13. The method according to claim 1, wherein, The biological tissue sample is a biological tissue sample that has been transparentized using a hydrophilic or hydrogel-type transparentization method.

14. The method according to claim 1, wherein, The gel precursor solution is a solution of 1.5% to 3% agarose in a refractive index-matched solution by mass percentage.

15. The method according to claim 1, wherein, The gel precursor solution is a solution of 1.8% to 2.5% agarose in a refractive index-matched solution by mass percentage.

16. The method according to claim 1, wherein, The gel precursor solution is a solution of 2% agarose in a refractive index-matched solution by mass percentage.

17. The method according to claim 1, wherein, In the resulting gel, the magnetic material sheet is located at the bottom, and the biological tissue sample is located above the magnetic material sheet.