High-throughput microporous slide for biological tissue sample detection

Abstract

The utility model relates to the technical field of medical auxiliary equipment, and aims to solve the technical problems that focus drop and data confusion are very easy to occur in laser scanning microscopic imaging detection. In order to solve the technical problem, the utility model provides the high-throughput microporous slide for biological tissue sample detection. The pore plate slide comprises a main body and a plurality of detection units, a detection area is arranged in the middle of the main body; the plurality of detection units are arranged in the detection area in a rectangular manner; the detection unit comprises a plurality of detection holes and an automatic focusing auxiliary hole; the plurality of detection holes are arranged in a centrosymmetric pattern; the automatic focusing auxiliary hole is formed in the symmetric center of the plurality of detection holes, and a standard substance for focusing is arranged in the automatic focusing auxiliary hole; and the cover slide covers the pore plate slide. According to the utility model, the detection flux of different slice samples in tissues processed under the same condition is improved, and detection images of different groups of samples can be automatically distinguished; the problem of focus drop caused by overlong displacement of the electric objective table when the detection unit is switched is solved.

Description

TECHNICAL FIELD

[0001] The utility model relates to medical auxiliary equipment technical field especially is a kind of high-throughput microwell glass for biological tissue sample detection. BACKGROUND

[0002] In biomedical research and clinical field, the detection technology of biological tissue sample has been the hotspot of research. Especially in oncology, neuroscience and cell biology and other disciplines, accurate analysis of biological tissue sample plays a vital role in the diagnosis and treatment of diseases. Traditional biological tissue sample detection method has many limitations, for example, low detection efficiency, large sample consumption, complex operation and low repeatability and accuracy of results.

[0003] With the development of laser scanning microscope (LSM) technology, the detection microwell plate technology is born, but the existing microwell plate still has many challenges in biological tissue sample detection: 1. Detection hole uniform distribution design, no zoning design for the same group of detection hole point unit, resulting in motor moving time is too long and detection data confusion, it is difficult to detect high-throughput laser scanning imaging detection for different slices of tissue sample under the same condition; 2. Laser scanning microscopic imaging between different holes is prone to defocus. SUMMARY

[0004] Therefore, the technical problem to be solved by the utility model is to overcome the above problems existing in the prior art.

[0005] To solve the above technical problems, the utility model provides a kind of high-throughput microwell glass for biological tissue sample detection, comprising:

[0006] Microwell glass, including main body and multiple detection units;The middle of main body is equipped with detection area;Multiple detection units are arranged in detection area in rectangular arrangement;Detection unit includes multiple detection holes and autofocus auxiliary hole;Multiple detection holes are arranged in center symmetry figure;Autofocus auxiliary hole is arranged at the center of symmetry of multiple detection holes, and standard substance for focusing is arranged in autofocus auxiliary hole;

[0007] Cover glass, cover is arranged on the top of microwell glass.

[0008] In an embodiment of the utility model, the standard substance includes multiple microsphere standard substances of microsphere structure;The top end of microsphere standard substance is flush with the orifice of autofocus auxiliary hole.

[0009] In an embodiment of the utility model, the diameter of microsphere standard substance is 20-30 μm.

[0010] In an embodiment of the utility model, the standard object includes a microgrid pattern standard object arranged at the bottom of the autofocusing auxiliary hole; the depth of the grid standard object from the orifice of the autofocusing auxiliary hole is 10-15 mu m.

[0011] In an embodiment of the utility model, the main body includes a substrate and a polylysine coating arranged at the top of the substrate; the detection unit is arranged on the polylysine coating, and the thickness of the polylysine coating is the same as the depth of the detection hole.

[0012] In an embodiment of the utility model, a plurality of row identifiers and a plurality of column identifiers are arranged at the edge of the main body; the row identifiers are arranged in one-to-one correspondence with the rows of the rectangular array of detection units; and the column identifiers are arranged in one-to-one correspondence with the columns of the rectangular array of detection units.

[0013] In an embodiment of the utility model, a hydrophobic groove is arranged on the orifice plate glass sheet; the surface of the hydrophobic groove is coated with a super-hydrophobic coating; and the detection area is surrounded by the hydrophobic groove.

[0014] In an embodiment of the utility model, the width of the hydrophobic groove is 0.6 mm.

[0015] In an embodiment of the utility model, the diameter of the detection hole is 1-1.5 mm; and the depth of the detection hole is 100 mu m.

[0016] In an embodiment of the utility model, the roughness Ra of the bottom wall of the detection hole is less than 0.1 mu m.

[0017] The above technical solution of the utility model has the following advantages compared with the prior art:

[0018] The high-throughput microhole glass sheet for biological tissue sample detection has a plurality of detection units arranged in the detection area, a plurality of detection holes are arranged on each detection unit, thereby unit division is performed on the same group of detection holes, high-density different grouping and loading capacity of the same group of samples can be provided, the detection throughput of different slice samples in the same condition processing tissue is further improved; meanwhile, the automatic control of the scanning software can automatically distinguish the detection images of different groups of samples. In addition, the standard object for focusing is arranged at the center of the plurality of detection holes in the same detection unit, so as to simulate the focusing effect of the uppermost layer of cells of the tumor tissue; the imaging clarity of the standard object is adjusted, automatic focusing and resolution confirmation before scanning of the detection unit are realized, thereby automatic focusing before scanning of different detection units can be realized, the defocusing problem caused by too long displacement of the motorized stage when the detection units are switched is solved; and the detection efficiency is improved. BRIEF DESCRIPTION OF DRAWINGS

[0019] In order to make the content of the utility model more easily be clearly understood, the following is according to the specific embodiment of the utility model and combining with the drawings, the utility model is further explained in detail, wherein,

[0020] Figure 1 It is the structure diagram of the high-throughput microwell glass plate for biological tissue sample detection in the preferred embodiment of the utility model;

[0021] Figure 2 It is Figure 1 The high-throughput microwell glass plate for biological tissue sample detection;

[0022] Figure 3 It is Figure 2 The schematic diagram of the detection unit in the preferred embodiment of the utility model;

[0023] Figure 4 It is Figure 3 The schematic diagram of the detection unit in the preferred embodiment of the utility model;

[0024] Figure 5 It is Figure 3 The schematic diagram of the detection unit in the preferred embodiment of the utility model;

[0025] Figure 6 It is Figure 5 The A-A section view of the preferred embodiment of the utility model;

[0026] Figure 7 It is Figure 1 The structure diagram of the high-throughput microwell glass plate for biological tissue sample detection in the preferred embodiment of the utility model;

[0027] The description of the drawing of the specification: 100, well plate glass;110, main body;111, row mark;112, column mark;120, detection unit;121, detection hole;122, automatic focusing auxiliary hole;123, standard substance;123a, microsphere standard substance;123b, microgrid pattern standard substance;130, hydrophobic groove;

[0028] 200, cover glass. DETAILED DESCRIPTION

[0029] The utility model is further explained in combination with the drawings and specific embodiment, so that the person skilled in the art can better understand the utility model and can be implemented, but the embodiment is not as the limitation of the utility model.

[0030] In some comparative embodiments, the detection holes are uniformly distributed, and the same group of detection holes (a plurality of detection holes into which samples cultured with the same concentration of drugs are put) are not designed with a targeted unit division, which results in that when laser scanning microscopic imaging is performed, the motor needs to move a long distance and takes a long time when the same group or cross-group imaging is performed, and it is difficult to perform high-throughput detection laser scanning imaging detection on different slices of the same condition processing tissue sample, and it is easy to cause confusion of the detection data. In addition, if high-throughput detection is to be realized, the area of the hole plate is relatively large and the number of detection holes is large. Then, there is a slight deviation between the detection holes at different positions and the focal plane of the scanning laser used for imaging. When laser scanning microscopic imaging is performed on different holes in the scanning process, the motor needs to move to the corresponding detection hole, and in this process, it is easy to lose focus, and the laser focal plane needs to be manually adjusted to find it back, which is low in efficiency.

[0031] To solve the above problems, with reference to Figures 1-7 The utility model embodiment provides a high-throughput microhole glass sheet for biological tissue sample detection, which comprises a hole plate glass sheet 100 and a cover glass 200.

[0032] The hole plate glass sheet 100 comprises a main body 110 and a plurality of detection units 120; the middle of the main body 110 is provided with a detection area; the plurality of detection units 120 are arranged in the detection area in a rectangular manner; the detection unit 120 comprises a plurality of detection holes 121 and an automatic focusing auxiliary hole 122; the plurality of detection holes 121 are arranged in a central symmetric pattern; the automatic focusing auxiliary hole 122 is arranged at the center of symmetry of the plurality of detection holes 121, and a standard substance 123 for automatic focusing is arranged in the automatic focusing auxiliary hole 122. In some embodiments, the hole plate glass sheet 100 is made of transparent high borosilicate glass or silicate glass with a thickness of 270 μm, which is beneficial to the transmission of detection optical signals and friendly to biological samples, and does not interfere with the detection of biological samples, thereby ensuring the high accuracy of imaging detection and the standardization of data processing.

[0033] The cover glass 200 is arranged on the top of the hole plate glass sheet 100. In some embodiments, the thickness of the cover glass 200 is 150-200 μm, and the cover glass 200 is made of transparent borosilicate glass. During detection, the cover glass 200 is arranged on the hole plate glass sheet 100, so that the tissue sample to be detected is effectively attached to the cover glass 200 and the hole plate glass sheet 100, and the tissue sample to be detected can be fully coupled with water.

[0034] In the detection of tumor tissue metabolic inhibition stress, different pre-experimentally treated tumor tissues can be added to the detection holes 121. Different tumor samples cultured under the same metabolic inhibitor at the same concentration are considered as the same detection group and are placed in the detection holes 121 of the same detection unit 120 (for example, each row (such as 1 row) of detection holes 121 corresponds to an experimental group under the previous treatment condition. The tumor tissue samples in column 1 are cultured in a conventional culture medium and are used as a blank control group; the tumor tissue samples in column 2 are cultured in a metabolic marker-containing culture medium and are used as a negative control group; the tumor tissue samples in columns 3-10 are cultured in a metabolic inhibitor-containing culture medium with a gradient of increasing concentrations and a metabolic marker-containing culture medium and are used as experimental groups). In this way, the detection throughput of different section samples of the same condition-treated tissue is improved. Each detection hole 121 is used to carry an independent biological tissue sample, so as to maximize the use area of the microwell glass sheet, ensure the independence of each detection hole 121 and avoid cross contamination, realize large-scale parallel detection of tissue samples, and automatically distinguish the detection images of different groups of samples under the automatic control of the scanning software. Different experimental conditions such as pre-treatment metabolic inhibitors and concentrations can cause different metabolic reactions of tumor tissues to different metabolic markers, which are manifested as differences in specific metabolic product chemical signals. The differences in the chemical signals can be detected by the laser scanning microscope matched with the well plate glass sheet 100.

[0035] Specifically, the application sets multiple detection units 120 in the detection area, each detection unit 120 is provided with multiple detection holes 121, so as to divide the same group of detection holes 121 into units, which can provide high-density different grouping and loading capacity of multiple samples in the same group, further improve the detection throughput of different section samples of the same condition-treated tissue, and automatically distinguish the detection images of different groups of samples under the automatic control of the scanning software. In addition, the application sets a standard substance 123 for focusing in the center of the multiple detection holes 121 in the same detection unit 120, so as to simulate the focusing effect on the topmost cells of the tumor tissue. By adjusting the imaging clarity of the standard substance 123, automatic focusing and resolution confirmation before scanning of the detection unit 120 are realized, so that automatic focusing before scanning of different detection units 120 can be realized, the defocusing problem caused by too long displacement of the motorized stage when switching the detection unit 120 is solved, and the detection efficiency is improved.

[0036] Further, referring to Figures 1-3As shown, the standard 123 includes a plurality of microsphere structures of microsphere standard 123a. The top end of the microsphere standard 123a is flush with the orifice of the autofocusing auxiliary hole 122. The diameter of the microsphere standard 123a is 20-30 μm. In some embodiments, the diameter of the autofocusing auxiliary hole 122 is 1 mm; the depth of the autofocusing auxiliary hole 122 is 20-30 μm, i.e., the diameter of the microsphere standard 123a is equal to the depth of the autofocusing auxiliary hole 122. Specifically, the microsphere standard 123a of the present embodiment can simulate the stereoscopic imaging of the topmost layer of cells of the tumor tissue (the diameter of the cells is generally 20-30 μm), thereby assisting the focusing on the longitudinal (the depth direction of the autofocusing auxiliary hole 122, the Z direction) center position of the topmost layer of cells of the tumor tissue and the confirmation of the horizontal (the XY direction) resolution of the cell boundary.

[0037] Further, as shown in Figure 1 , Figure 2 and Figure 4 , the standard 123 includes a micro-lattice pattern standard 123b disposed at the bottom of the autofocusing auxiliary hole 122; the depth of the lattice standard 123 from the orifice of the autofocusing auxiliary hole 122 is 10-15 μm. Specifically, the micro-lattice pattern standard 123b of the present embodiment can simulate the focusing on the longitudinal (the Z direction) center position of the topmost layer of cells of the tumor tissue, which can detect the horizontal (the XY direction) imaging resolution at the vertical depth (the Z direction) focal plane, thereby assisting the autofocusing on the observation depth of the target cells and the resolution confirmation.

[0038] Further, as shown in Figures 1-2 and Figures 5-6As shown, the standard 123 includes a plurality of microspheres of the microsphere standard 123a and a microgrid pattern standard 123b disposed at the bottom of the autofocusing auxiliary hole 122. The top end of the microsphere standard 123a is flush with the aperture of the autofocusing auxiliary hole 122. The diameter of the microsphere standard 123a is 20-30 μm. The depth of the grid standard 123 from the aperture of the autofocusing auxiliary hole 122 is 10-15 μm. That is, the bottom of the autofocusing auxiliary hole 122 is provided with a drop; the depth of the autofocusing auxiliary hole 122 at the position where the microsphere standard 123a is provided is 20-30 μm, and the depth of the autofocusing auxiliary hole 122 at the position where the grid standard 123 is provided is 10-15 μm (i.e., the grid standard 123 is located at the level of the center of the microsphere standard 123a). Specifically, the embodiment provides the grid standard 123 and the microsphere standard 123a in the autofocusing auxiliary hole 122 (for example, half of the autofocusing auxiliary hole 122 is provided with the grid standard 123, and the other half is provided with the microsphere standard 123a). The grid standard 123 can simulate focusing on the vertical (Z direction) center position of the uppermost layer of cells of the tumor tissue (the diameter of the cells is generally 20-30 μm), and the microgrid pattern of the grid standard 123 can detect the horizontal imaging resolution of the vertical depth (Z direction) focal plane, thereby assisting in achieving automatic focusing and resolution confirmation of the observation depth of the target cells. After the vertical depth (Z direction) focal plane is confirmed based on the aforementioned grid standard 123, the microsphere standard 123a is used to simulate stereoscopic imaging of the uppermost layer of cells of the tumor tissue (the diameter of the cells is generally 20-30 μm), thereby assisting in confirming the focusing on the vertical (Z direction) center position of the uppermost layer of cells of the tumor tissue and the horizontal (XY direction) resolution of the cell boundary.

[0039] Further, the main body 110 includes a substrate and a polylysine coating layer disposed on the top of the substrate, and the detection unit 120 is disposed on the polylysine coating layer, and the thickness of the polylysine coating layer is the same as the depth of the detection hole 121. In some embodiments, the thickness of the main body 110 is 270 μm, wherein: the substrate is made of transparent high borosilicate glass or silicate glass with a thickness of 170 μm. The thickness of the polylysine coating layer is 100 μm. Specifically, the embodiment is to open the detection hole 121 on the polylysine coating layer, thereby reducing the process difficulty and thus reducing the cost.

[0040] Further, the thickness of the main body 110 is 270 μm, and the detection hole 121 with a depth of 100 μm is directly constructed on the top of the main body 110 by etching or laser engraving.

[0041] Further, the edge of the main body 110 is provided with a plurality of row identifiers 111 and a plurality of column identifiers 112; the row identifiers 111 are arranged one-to-one corresponding to the rows of the rectangular array of the detection units 120; and the column identifiers 112 are arranged one-to-one corresponding to the columns of the rectangular array of the detection units 120. The row identifier 111 is “number + row”, and the column identifier 112 is “number + row”. Specifically, the coordinates of one detection unit 120 are marked by one row identifier 111 and one column identifier 112, so as to label each detection unit 120. The experimental condition name corresponding to the row and column number can be set in the corresponding software (automated stage software) according to the experimental conditions, so as to realize batch automatic detection. In addition, the automatic experimental result and condition matching can be realized, and the human operation error can be avoided.

[0042] In the laser scanning imaging process, a certain amount of heat is generated, and a certain amount of moisture needs to be maintained in the detection hole 121 to maintain the coupling of the sample and the detection beam. In some embodiments, the detection slide or the well plate cannot inhibit the problem of water evaporation, which makes it difficult to maintain moisture in the case of batch long-time scanning. In order to solve this problem, the hole plate slide 100 of the present embodiment is provided with a hydrophobic groove 130, the surface of the hydrophobic groove 130 is coated with a super-hydrophobic coating, and the detection area is surrounded by the hydrophobic groove 130. The width of the hydrophobic groove 130 is 0.6 mm.

[0043] Specifically, the present embodiment provides a hydrophobic groove 130 surrounding the detection area, the hydrophobic groove 130 has a very high surface tension, can repel water and other liquids, and then seal the detection area to inhibit water evaporation, so as to ensure that long-time batch laser scanning imaging can be performed; at the same time, the sample is prevented from being contaminated during the detection process, and the accuracy of the detection and the reusability of the sample are ensured.

[0044] Further, the diameter of the detection hole 121 is 1-1.5 mm; and the depth of the detection hole 121 is 100 μm. Specifically, in the present embodiment, the detection hole 121 and the depth are matched with the thickness of the biological tissue sample to be detected having a tumor microenvironment, the thickness and the cross-sectional size of the metabolic activity of the biological tissue sample. The depth can ensure that the to-be-detected tissue sample is effectively attached to the hole plate slide 100 and the cover slide 200, and the hole diameter size can accommodate the to-be-detected tissue and also accommodate an appropriate amount of coupling moisture, which is especially suitable for biological tissue single-cell metabolic detection based on a laser scanning microscope.

[0045] In some embodiments, the distance between two adjacent detection units 120 is 4 mm; and in one detection unit 120, the center distance between two adjacent detection holes 121 is 2.5 mm. In some embodiments, four detection holes 121 are arranged in one detection unit 120.

[0046] Further, the roughness Ra of the bottom wall of the detection hole 121 is less than 0.1 μm. Specifically, the embodiment can ensure that cells can be uniformly attached to the surface.

[0047] The present application can realize high-throughput, automation, high resolution and high efficiency detection of biological tissue samples by uniting arrangement of the detection holes 121 and accurate control of the present application and the autofocus of the scanning microscope.

[0048] The present application can be used for tumor tissue metabolic inhibition stress detection. It should be noted that, in combination with the design of the aperture size and the hole spacing of the detection hole 121, the laser scanning microscope can be programmed by the microscope automatic stage to realize automatic alignment and focusing of the biological tissue sample in each detection hole 121, which greatly improves the speed of imaging detection. The design of the hole plate glass 100 should support automatic operation to improve the efficiency and repeatability of the detection.

[0049] The present application is a new type of micro-hole glass suitable for laser scanning microscope, which helps to realize faster, more accurate and more economical analysis of biological tissue samples, and provides strong technical support for biomedical research and early diagnosis and personalized treatment of related diseases.

[0050] Obviously, the above embodiment is only an example for clarity and is not limited to the implementation. For those skilled in the art, other different forms of changes or variations can be made on the basis of the above description. Here, it is not necessary and impossible to enumerate all the embodiments. The obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A high-throughput microporous slide for biological tissue sample detection, characterized by: include: The well plate slide comprises a main body and a plurality of detection units; a detection area is provided in the middle of the main body; and the plurality of detection units are arranged in a rectangular shape in the detection area; The detection unit includes a plurality of detection holes and an autofocus auxiliary hole; the plurality of detection holes are arranged in a centrally symmetrical pattern; the autofocus auxiliary hole is provided at the symmetrical center of the plurality of detection holes, and a standard object for focusing is provided in the autofocus auxiliary hole; A cover slide is placed on top of the well plate slide.

2. The high-throughput microporous glass slide for biological tissue sample detection according to claim 1, characterized in that: The standard comprises a plurality of microsphere standards having a microsphere structure; the top of the microsphere standard is flush with the opening of the autofocus auxiliary hole.

3. The high-throughput microporous slide for biological tissue sample detection according to claim 2, characterized in that: The diameter of the microsphere standard is 20-30 μm.

4. The high-throughput microporous slide for biological tissue sample detection according to claim 1 or 2, characterized in that: The standard comprises a micro-grid pattern standard disposed at the bottom of the auto-focus auxiliary hole; the depth of the micro-grid pattern standard from the opening of the auto-focus auxiliary hole is 10-15 μm.

5. The high-throughput microporous glass slide for biological tissue sample detection according to claim 1, characterized in that: The main body includes a substrate and a polylysine coating arranged on the top of the substrate. The detection unit is arranged on the polylysine coating, and the thickness of the polylysine coating is the same as the depth of the detection hole.

6. The high-throughput microporous glass slide for biological tissue sample detection according to claim 1, characterized in that: A plurality of row identifiers and a plurality of column identifiers are provided at the edge of the main body; the row identifiers are arranged in a one-to-one correspondence with the rows of the rectangular array of the detection units; and the column identifiers are arranged in a one-to-one correspondence with the columns of the rectangular array of the detection units.

7. The high-throughput microporous slide for biological tissue sample detection according to claim 1, characterized in that: A circle of hydrophobic grooves is provided on the well plate glass slide, the surface of the hydrophobic grooves is coated with a super-hydrophobic coating, and the detection area is surrounded by the hydrophobic grooves.

8. The high-throughput microporous glass slide for biological tissue sample detection according to claim 7, characterized in that: The width of the hydrophobic groove is 0.6 mm.

9. The high-throughput microporous glass slide for biological tissue sample detection according to claim 1, characterized in that: The diameter of the detection hole is 1-1.5 mm; the depth of the detection hole is 100 μm.

10. The high-throughput microporous glass slide for biological tissue sample detection according to claim 1, characterized in that: The roughness of the bottom wall of the detection hole Ra is less than 0.1 μm.

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