A sectioning method and imaging method for high-resolution imaging of intact thick biological tissue

Abstract

The application discloses a sectioning method and imaging method for high-resolution imaging of intact thick biological tissues, and the sectioning method comprises the following steps: S1, placing a cover glass on a first object slide and covering both with adhesive tape; S2, digging off the adhesive tape covered in the area of the cover glass, and processing the edges of the dug-off area with a hydrophobic medium; S3, transferring a tissue sample to be observed to the cover glass, absorbing the sample liquid, adding an anti-fluorescence quencher dropwise, absorbing the sample solvent after standing, adding gel dropwise, and fixing the sample on the cover glass; S4, removing the cover glass and placing it on a second object slide, so that the tissue sample is located between the cover glass and the second object slide, and the cover glass is fixedly connected with the second object slide through the adhesive tape, and the sectioning is completed. The application can observe the distribution and quantity of specific cells in thick tissues at high resolution while keeping the integrity of the tissues, and the preparation method is simple, is not limited by light paths, sizes and the like, and is suitable for most upright or inverted microscopes.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biological tissue microscopic observation, in particular to a sectioning method and imaging method for high-resolution imaging of intact thick biological tissue. BACKGROUND

[0002] In life science research and clinical pathology analysis, high-resolution imaging of the whole tissue within a certain thickness range can directly find the number and arrangement of specific cell groups in the tissue and the expression difference of target proteins, thereby assisting in revealing the causes of disease development. However, when studying thick tissue samples, the following methods are usually used:

[0003] (1) The thick tissue is transparentized, and then imaged using a light sheet microscope. However, the light sheet microscope is expensive, and the sample preparation process is complex, which is suitable for imaging larger volume tissues or samples;

[0004] (2) The tissue is sectioned into thin sections of 5-20 μm thickness, and then observed by microscope. However, this method has the following disadvantages: on the one hand, the tissue is lost during sectioning; on the other hand, after the tissue is sectioned and treated, imaging is performed, which is equivalent to observing the local section of the tissue, and the whole tissue cannot be observed, which is not conducive to judging the distribution and expression characteristics of cells or target genes in the tissue sample as a whole;

[0005] (3) When imaging a certain thickness of tissue, the tissue can be pressed in a viewing dish with a glass bottom by artificial method, but there is always a gap between the sample tissue and the glass bottom, which occupies part of the working distance range of the lens, resulting in a decrease in the depth distance available for the sample.

[0006] In summary, how to high-resolution observe the distribution and number of specific cells in thick tissue while maintaining the integrity of the tissue is a problem that needs to be solved in biological and medical research. SUMMARY

[0007] The purpose of the present application is to provide a sectioning method and imaging method for high-resolution imaging of intact thick biological tissue to solve the problems raised in the background.

[0008] To solve the above problems, the present application first provides a sectioning method for high-resolution imaging of intact thick biological tissue, comprising the following steps:

[0009] S1, placing a coverslip on a first object slide, covering the coverslip and the first object slide with adhesive tape;

[0010] S2, dig out the range of the cover glass area covered by the tape, expose most of the cover glass, and use a hydrophobic medium to treat the edges of the dig-out area;

[0011] S3, transfer the tissue sample to be observed to the cover glass, absorb the sample liquid, add anti-fluorescence quencher, stand for 1-3 hours, then absorb the sample solvent again, add gel, and fix the sample on the cover glass;

[0012] S4, remove the cover glass and place it on the second slide, so that the tissue sample is located between the cover glass and the second slide, and the cover glass is fixed and connected with the second slide around by the tape, and the slide is completed.

[0013] Preferably, the thickness of the tissue sample is 280-480 μm.

[0014] Preferably, in step S1, according to the thickness of the tissue sample, cover one to several layers of tape, and the total thickness of the tape does not exceed the thickness of the tissue sample.

[0015] Preferably, in step S2, the cover glass in the dig-out area is also divided into multiple sample chambers using a hydrophobic medium.

[0016] Preferably, in step S3, the gel is low-melting-point agarose.

[0017] Preferably, in step S4, when the tape is fixed, the height between the cover glass and the second slide is kept consistent.

[0018] Preferably, the size of the cover glass is 24 mm x 60 mm, and the thickness is 0.13-0.17 mm; the size of the first and second slides is both 25 mm x 75 mm, and the thickness is 1-1.2 mm.

[0019] Another aspect of the present application also provides a high-resolution imaging method for intact thick biological tissues, comprising the following steps:

[0020] (1) Obtain the sample tissue to be observed, and prepare a slide sample by using any one of the slide preparation methods described above;

[0021] (2) Place the slide sample horizontally on the microscope stage, add the required medium for the lens, soak the lens, and observe the sample tissue.

[0022] Preferably, the microscope includes an upright laser confocal microscope and an inverted laser confocal microscope.

[0023] Preferably, the microscope uses a 40x water lens.

[0024] Preferably, when using an upright laser confocal microscope for observation, before adding medium water, the following operation is further included: using a hydrophobic medium to draw a medium adding area on the cover glass, the medium adding area covering the sample tissue.

[0025] The present application has the following advantages over the prior art:

[0026] (1) The present application provides a sample preparation method and imaging method for high-resolution imaging of intact thick biological tissues, which can observe the distribution and quantity of specific cells in thick tissues while maintaining the integrity of the tissues (280-480 μm), and the preparation method is simple, not limited by light path, size, etc., and can be applied to most upright or inverted microscopes, with good application prospects.

[0027] (2) The present application uses a hydrophobic medium to divide the cover glass into areas, forming a target circle range around the sample, which facilitates the subsequent search for the sample when taking pictures, and solves the problem of difficulty in finding the target when imaging and observing; after drawing a circle on the inverted cover glass with a hydrophobic material, the required medium is dropped into the lens, which can prevent the medium from spreading and evaporating during photography, thereby maintaining the stability of the photography environment.

[0028] (3) The sample placed on the cover glass is subjected to operations such as water absorption and low-melting-point agarose infiltration, which on the one hand tightly fixes the sample on the cover glass, reduces the space loss between the sample and the cover glass, effectively utilizes the working distance of the lens, and images the sample as a whole; on the other hand, after the sample is infiltrated with low-melting-point agarose, it will be lifted to some extent, relieving the degree of shrinkage after the sample is dried, and thus maintaining the original morphology as much as possible during photography. BRIEF DESCRIPTION OF DRAWINGS

[0029] Figure 1 The present application is a schematic diagram of the sample preparation process for high-resolution imaging of intact thick biological tissues.

[0030] Figure 2 The present application is a schematic diagram of the sample preparation process for high-resolution imaging of intact thick biological tissues.

[0031] Figure 3 The present application is a schematic diagram of the sample preparation process for high-resolution imaging of intact thick biological tissues. DETAILED DESCRIPTION

[0032] The technical solution of the present invention will be further described below in conjunction with the accompanying drawings and embodiments. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and retouches can be made, and these improvements and retouches are also regarded as the protection scope of the present invention.

[0033] In the description of the present invention, it should be noted that the terms "first" and "second" are only used for descriptive purposes and cannot be understood as indicating or implying relative importance.

[0034] The numerical aperture (NA) value of a microscope lens is inversely proportional to the working distance (WD). The larger the NA value, the higher the resolution, but the shorter the working distance, and the thinner the thickness of the tissue sample that can be photographed. Therefore, in order to be able to observe the distribution and quantity of specific cells inside thick tissues with high resolution while maintaining tissue integrity, on the one hand, by comparing the objective lens parameters under different magnification multiples and different lens media, the objective lens parameters that balance high resolution and high working distance imaging are screened out. Finally, it is found that selecting a 40× water lens for imaging thick biological tissues (280 - 480 μm) is beneficial for observing the fine structure, cell distribution characteristics, expression range and trend of target proteins in the samples within this thickness range from the whole, so as to assist in explaining the causes or laws of disease occurrence and development; on the other hand, the inventors of the present application have provided a slide preparation method suitable for high-resolution imaging of intact thick biological tissues (280 - 480 μm). The slide samples prepared by this method, on the one hand, can尽可能 shorten the gap between the sample and the cover glass, thus leaving more thickness space for sample photography; on the other hand, it is not restricted by the optical path, size, etc., and can be applied to most upright or inverted microscopes. Specifically, as Figure 1 shown, the slide preparation method includes the following steps:

[0035] S1, place a cover glass on the first slide, and cover the cover glass and the first slide with tape ( Figure 1 a - c);

[0036] S2, dig out the tape covered within the area of the cover glass, "open a window" for the tape to expose most of the cover glass, and treat the edge of the dug area with a hydrophobic medium ( Figure 1 d - e); Preferably, it further includes: dividing the cover glass within the dug area into multiple sample chambers with a hydrophobic medium to observe multiple tissue samples simultaneously ( Figure 1 f);

[0037] S3, transfer the tissue sample to be observed onto the cover glass, suck dry the sample liquid, drop an anti-fluorescence quenching agent, let it stand for 1 - 3 h, then suck dry the sample solvent again, and drop a gel to fix the sample on the cover glass (<g);

[0038] S4, remove the cover glass, flip and place it on the second slide so that the tissue sample is between the cover glass and the second slide Figure 1 h-k) by taping the cover glass to the second slide around the edges, completing the mounting Figure 1 l-m).

[0039] Preferably, in step S1, according to the thickness of the tissue sample, cover one to several layers of tape to maintain the initial state of the sample as much as possible and reduce deformation caused by extrusion, but the total thickness of the tape should not exceed the thickness of the tissue sample.

[0040] Preferably, in step S3, the gel is low-melting agarose.

[0041] Preferably, in step S4, when the tape is fixed, the height between the cover glass and the second slide should be kept consistent.

[0042] It should be noted that:

[0043] (1) The purpose of using hydrophobic medium in step S2 is to form a target circle around the sample for subsequent observation and photography, which is convenient for finding the sample and solving the problem of difficulty in finding the target during imaging observation.

[0044] (2) In step S3, first, the sample liquid and solvent are absorbed, and the tissue sample will naturally stick to the cover glass; second, low-melting agarose or other gels are added to infiltrate the sample, which can further fix the sample on the cover glass, reduce the space loss between the sample and the cover glass, effectively utilize the working distance of the lens, and image the sample as a whole; on the other hand, after the sample is infiltrated with the gel, it will be lifted to some extent, relieving the degree of sagging after the water is absorbed, so as to maintain the original morphology of the sample as much as possible during photography.

[0045] It should be noted that when the gel is added to infiltrate the sample, it should be added gently so that it slowly infiltrates the sample without pushing the sample. If the sample floats in the agarose, even if the sample is pressed by a sharp needle later, there will still be a gap between the sample and the cover glass. Only when the sample sticks to the cover glass can the gap between the sample and the cover glass be shortened as much as possible, thereby leaving more thickness space for the sample to be photographed.

[0046] Another aspect of the present application also provides a high-resolution imaging method for a complete thick biological tissue, comprising the following steps:

[0047] (1) Obtain the sample tissue to be observed, and prepare a slide sample by using any one of the slide preparation methods described above;

[0048] (2) Place the slide sample horizontally on a microscope stage, drop the medium required by the lens, soak the lens, and observe the sample tissue.

[0049] Preferably, the microscope comprises an upright laser confocal microscope or an inverted laser confocal microscope.

[0050] Preferably, the microscope uses a 40x water lens.

[0051] As shown in Figure 2 , when an inverted microscope is used for imaging, the medium required by the lens can be directly dropped on the lens, and then observation can be performed; when an upright microscope is used for imaging, the following operation is further included before dropping the medium water: using a hydrophobic medium to circle a medium adding area (n) on the cover glass, the medium adding area covering the sample tissue. The medium for soaking the lens is gathered in the medium adding area, which can prevent the medium from spreading and evaporating dry during photographing, thereby maintaining the temperature of the photographing environment. Figure 1

[0052] The application will be described in detail below through examples and drawings.

[0053] Unless otherwise specified in the embodiments of the application, the reagents and consumables used are commercially available.

[0054] Example 1 Comparison of objective lens parameters of confocal microscopes

[0055] The inventors of the present application compared the physical characteristics of lenses with different magnification factors on the market, and the results are shown in Table 1: for a sample of a certain thickness of tissue, the NA value and WD value of the 40x water lens are beneficial to high-resolution imaging of thick biological tissues (280-480 μm); the NA value of the 20x and 25x low-power lenses is not enough, resulting in insufficient resolution, and the WD of the 60x high-power lens is too small, resulting in insufficient working distance.

[0056] Table 1 Comparison of confocal microscope lens parameters

[0057]

[0058] In addition, the present application also found through comparison that, when the magnification factor is also 40x, the NA of the lens of the upright microscope with a non-air medium is similar to that of the lens of the inverted microscope, but the WD is longer, which is more beneficial to imaging of sample tissues within the WD range.

[0059] Example 2 Preparation and observation of zebrafish heart

[0060] ​1. Preparation and imaging of zebrafish heart

[0061] (1) Place a coverslip on the first slide, cover the coverslip and the first slide with two layers of adhesive tape according to the thickness of the zebrafish heart (the thickness of the zebrafish heart in this example is about 400 μm);

[0062] (2) Remove the adhesive tape covering the area of the coverslip, expose most of the coverslip, and use an immunohistochemical pen (i.e. a hydrophobic medium) to treat the edges of the removed area, and use the immunohistochemical pen to draw multiple sample chambers in the removed area of the coverslip;

[0063] (3) Under a general fluorescence microscope, use tweezers to remove the zebrafish heart at day 5 after birth, and after immunofluorescence staining, use a micropipette with a range of 20 μl to transfer the heart to the sample chamber;

[0064] (4) Continue to operate under the microscope, use dust-free paper to absorb the sample and surrounding moisture, add anti-fluorescence quencher dropwise, and after standing for 1-3 h, use dust-free paper to absorb the sample solvent again, and the sample tissue will spontaneously adhere to the chamber of the coverslip, at which time the sample becomes shriveled due to water loss;

[0065] (5) Add melted agarose to one side of the sample, and use the micropipette gun head to gently introduce the agarose liquid drop to the sample, so that it slowly infiltrates the sample without pushing the sample;

[0066] (6) After the agarose solidifies, the sample is fixed on the coverslip. Remove the coverslip fixed by the adhesive tape, turn over the coverslip, and place it on the second slide so that the tissue sample is located between the coverslip and the second slide, cut the adhesive tape to an appropriate size, and fix the coverslip around the second slide by the adhesive tape to ensure that the height between the coverslip and the second slide remains consistent.

[0067] (7) Circle the medium addition area outside the sample area on the coverslip using a hydrophobic pen, then place the sample under the Nikon A1 upright laser scanning confocal microscope with a 40x water lens, add water to the medium addition area to soak the lens, and find the target in the chamber where the sample is located for imaging.

[0068] 2. Imaging results

[0069] As shown in Figure 3 , after the preparation and imaging methods of the present application, it can be clearly seen that fibronectin in the zebrafish heart presents different arrangement patterns at different positions: the nuclei in the zebrafish heart are point-like (A) in Figure 3 ; fibronectin is point-like (B) near the lens; Figure 3 ; and actin F-actin in the zebrafish myocardium is arranged in bundles.Figure 3 C) of FIG. 1 1 ; fibronectin presents a similar arrangement to F-actin in the heart (D) of FIG. 1 1. Figure 3

[0070] In summary, the present application provides a sectioning method and imaging method for high-resolution imaging of intact thick biological tissues, which can observe the distribution and quantity of specific cells in thick tissues at high resolution while maintaining the integrity of the tissues (280-480 μm), and the preparation method is simple, not limited by light path, size, etc., suitable for most upright or inverted microscopes, and has good application prospect.

[0071] Although the present application has been described in detail by the above preferred embodiments, it should be appreciated that the above description should not be considered as limiting the present application. Various modifications and alternatives to the present application will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present application should be defined by the appended claims.​

Claims

1. A method for sectioning of intact thick biological tissue for high resolution imaging, characterized in that, The method comprises the following steps: S1, placing a cover glass on a first slide, covering the cover glass and the first slide with adhesive tape; S2, removing the adhesive tape covering the cover glass, exposing most of the cover glass, and treating the edges of the removed area with a hydrophobic medium; S3, transferring the tissue sample to be observed to the cover glass, absorbing the sample liquid, adding anti-fluorescence quencher, standing for 1-3 hours, then absorbing the sample solvent again, adding gel, and fixing the sample on the cover glass; S4, removing the cover glass and placing it on a second slide, so that the tissue sample is between the cover glass and the second slide, and the cover glass is fixedly connected to the second slide around the cover glass by adhesive tape, completing the slide preparation; In step S1, according to the thickness of the tissue sample, cover one to several layers of adhesive tape, and the thickness of the tissue sample is 280-480 μm; The completed slide is observed by a microscope, which includes an upright laser confocal microscope and an inverted laser confocal microscope, and the microscope uses a 40x water lens.

2. The method for sectioning thick biological tissue for high resolution imaging of claim 1, wherein, In step S2, the cover glass in the removed area is divided into multiple sample chambers using a hydrophobic medium.

3. The method for sectioning thick biological tissue for high resolution imaging as claimed in claim 1, wherein, In step S3, the gel is low-melting-point agarose.

4. The method for sectioning thick biological tissue for high resolution imaging of claim 1, wherein, In step S4, when the adhesive tape is fixed, the height between the cover glass and the second slide is kept consistent.

5. A method of high resolution imaging of intact thick biological tissue, characterized by, The method comprises the following steps: (1) obtaining a sample tissue to be observed, and preparing a slide sample by the slide preparation method of claim 1; (2) placing the slide sample horizontally on the microscope stage, adding the required medium for the lens, wetting the lens, and observing the sample tissue.

6. The method of high resolution imaging of an intact thick biological tissue of claim 5, wherein, When an upright laser confocal microscope is used for observation, the following operations are further included before adding the medium water: Using a hydrophobic medium to circle a medium adding area on the cover glass, and the medium adding area covers the sample tissue.

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