CN114199658B - Method for manufacturing core by organizing chip - Google Patents
Method for manufacturing core by organizing chip Download PDFInfo
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- CN114199658B CN114199658B CN202010986636.3A CN202010986636A CN114199658B CN 114199658 B CN114199658 B CN 114199658B CN 202010986636 A CN202010986636 A CN 202010986636A CN 114199658 B CN114199658 B CN 114199658B
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- 238000000034 method Methods 0.000 title claims abstract description 36
- 238000004519 manufacturing process Methods 0.000 title abstract description 13
- 239000001993 wax Substances 0.000 claims abstract description 14
- 239000012188 paraffin wax Substances 0.000 claims abstract description 13
- 239000011159 matrix material Substances 0.000 claims description 8
- 230000000740 bleeding effect Effects 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 206010061218 Inflammation Diseases 0.000 claims description 6
- 230000007850 degeneration Effects 0.000 claims description 6
- 230000004054 inflammatory process Effects 0.000 claims description 6
- 238000012216 screening Methods 0.000 claims description 6
- 230000017074 necrotic cell death Effects 0.000 claims description 4
- 102000008186 Collagen Human genes 0.000 claims description 3
- 108010035532 Collagen Proteins 0.000 claims description 3
- 229920001436 collagen Polymers 0.000 claims description 3
- 239000000835 fiber Substances 0.000 claims description 3
- 238000002372 labelling Methods 0.000 claims description 3
- 230000001338 necrotic effect Effects 0.000 claims description 3
- 238000005070 sampling Methods 0.000 claims description 3
- 238000005553 drilling Methods 0.000 claims description 2
- 230000002792 vascular Effects 0.000 claims description 2
- 239000000758 substrate Substances 0.000 claims 4
- 108090000623 proteins and genes Proteins 0.000 abstract description 7
- 238000011160 research Methods 0.000 abstract description 6
- 238000003364 immunohistochemistry Methods 0.000 abstract description 4
- 239000000463 material Substances 0.000 abstract description 4
- 201000011510 cancer Diseases 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 description 12
- 230000001575 pathological effect Effects 0.000 description 10
- 230000009286 beneficial effect Effects 0.000 description 3
- 238000007901 in situ hybridization Methods 0.000 description 3
- 238000002493 microarray Methods 0.000 description 3
- 239000003086 colorant Substances 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 230000000877 morphologic effect Effects 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000000018 DNA microarray Methods 0.000 description 1
- 229960000074 biopharmaceutical Drugs 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 230000002055 immunohistochemical effect Effects 0.000 description 1
- 238000007850 in situ PCR Methods 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
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- 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
- G01N21/84—Systems specially adapted for particular applications
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- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The application relates to a method for manufacturing a core by using a tissue chip, which belongs to the technical field of tissue chips and comprises the following steps: scanning paraffin sections by using a digital scanner to obtain scanning images amplified by scanning; and carrying out microscopic marking on the basis of the scanning image, and obtaining effective target tissues from wax blocks corresponding to the sections according to marking results to serve as tissue chip column cores. According to the application, accurate target tissue marking is performed based on the digital intelligent scanning slice, so that invalid points of a constructed medium-high density tissue chip are greatly reduced, the core quality and accuracy of the tissue chip are obviously improved, the heterogeneity difference of each point of the malignant tumor tissue chip is reduced, the freshness of cells in a target tissue is effectively ensured, and the accuracy of immunohistochemistry and gene research is further improved. And the marking work intensity of a pathologist can be reduced, the tissue chip manufacturing time is shortened, and the rejection rate is reduced, so that the manpower, material resources and financial resources are saved.
Description
Technical Field
The application belongs to the technical field of tissue chips, and particularly relates to a method for manufacturing a core by using a tissue chip.
Background
Tissue Chip (TC), also known as tissue microarray (tissue microarray, TMA), is a component of biochip technology, like gene chip, protein chip, and cell chip, and belongs to biological high-tech technology. The tissue chip is a novel high-throughput multi-sample research tool for arranging tens or hundreds of tissue samples of different individuals on a carrier according to a preset sequence and carrying out morphological or molecular biology experiments. The tissue chip technology has the advantages of large information quantity, small experiment error, strong comparability, time saving, labor saving, great saving of expenditure and the like, is combined with the traditional pathology technology, histochemistry technology, immunohistochemical technology (IHC), in situ hybridization technology (ISH), fluorescent nucleic acid in situ hybridization technology (FISH), in situ PCR technology and the like, can realize morphological observation of normal or pathological tissue specimens and research on different molecular levels of specific genes and expressed proteins thereof, and is gradually applied to the fields of scientific research, clinic, teaching, biological reagent test, quality monitoring, standardization and the like in the fields of medicine, biology, animal and plant.
One of the cores of microarray tissue chip technology is the accuracy of information in each site and the amount of target tissue, and in recent years, as biopharmaceuticals continue to develop, demands for tissue chips are increasing, especially medium-high density large sample tissue chips are increasing. The tissue chip with higher density and more information content is manufactured, the diameter of each site must be reduced, for example, the sample application diameter is from 1.5mm diameter of the tissue chip with 80 points to 1.0mm-0.6mm diameter of the tissue chip with more than 100 points and even more than 240 points. The diameter of each site is reduced along with the increase of the number of points, and the spotting diameter is reduced, so that the sufficient target cell quantity and the target tissue effectiveness are ensured, and the difficulty of the core quality of each site tissue chip is increased.
In the TMA construction process, pathological histology marks are always penetrated through, and at present, the pathological marks are mainly positioned under a manual light microscope, so that the marking range is large, the positioning accuracy is low, the freshness of target tissues and the target tissue quantity are inaccurate to grasp, the experimental results of immunohistochemistry and the like are unstable, and false negative and false positive occur; meanwhile, the existing pathological markers have the problems of uneven level, too little target tissue quantity, necrosis, bleeding and fibrous tissue caused by tissue layering, and even more invalid points caused by falling and losing slices, so that the quality of the core-making chip is low, and the experimental effect is not ideal, and the manpower, material resources and financial resources are wasted.
The foregoing is provided merely for the purpose of facilitating understanding of the technical solutions of the present invention and is not intended to represent an admission that the foregoing is prior art.
Disclosure of Invention
In order to overcome the problems in the related art to at least a certain extent, the application provides a method for manufacturing the tissue chip, which is used for precisely marking the target tissue based on the digital intelligent scanning slice and is beneficial to better realizing the manufacturing marking of the high-density tissue chip.
In order to achieve the above purpose, the application adopts the following technical scheme:
the application provides a method for manufacturing a core by organizing a chip, which comprises the following steps:
scanning paraffin sections by using a digital scanner to obtain scanning images amplified by scanning;
and carrying out microscopic marking on the basis of the scanning image, and obtaining effective target tissues from wax blocks corresponding to the sections according to marking results to serve as tissue chip column cores.
Optionally, the performing the under-lens marking based on the scanned image includes:
the slice is observed and evaluated based on the scanned image,
Aiming at the situation that the effective target tissues are less and the ineffective tissues are more in the slice, a pathological tissue local marking method is adopted for marking under the mirror image,
And aiming at the situation that the number of effective target tissues in the slice is large and the number of ineffective tissues is small, the precise graffiti method is adopted for marking under the mirror image.
Optionally, the marking under the mirror image is performed by adopting a pathological tissue local marking method, which is specifically as follows:
And determining an invalid tissue area on the scanning image, marking by adopting a first mark, comparing the section under a microscope, avoiding the invalid tissue in the section and the invalid area of the target tissue matrix, and marking the effective target tissue concentrated area by adopting a second mark.
Alternatively, the process may be carried out in a single-stage,
Ineffective tissue within the slice includes tissue that interferes with inflammatory reactions, tissue that is necrotic by bleeding, and tissue that is degenerated around both types of tissue;
the ineffective area of the target tissue matrix comprises an area corresponding to collagen fibers in the target tissue matrix.
Alternatively, the process may be carried out in a single-stage,
The marking by adopting the first mark is specifically to mark by adopting a picture fork;
The marking by the second mark is specifically to marking by an oily stroke circle.
Optionally, the marking under the mirror image is performed by adopting an accurate graffiti method, which specifically comprises the following steps:
And determining an invalid region on the scanning image, marking by adopting a third mark, comparing the section under a microscope, marking the color of the preliminary screening invalid region in the section, and marking the color of the tiny invalid region under a high-magnification view field by matching the scanning image with the non-color-coated region of the section.
Optionally, the preliminary screening ineffective area in the slice comprises a bleeding necrosis area, a thick-wall vascular area and a fibrous tissue area in the slice;
the minor ineffective areas include a tissue degeneration area, a cell degeneration area and an inflammation interference area.
Optionally, the marking with the third mark is specifically marking with an oily stroke fork; the color mark is specifically a black mark made by an oil pen.
Optionally, the scanning paraffin section by using a digital scanner, and acquiring a scanned and amplified scanned image specifically includes:
Wiping paraffin sections clean by absolute alcohol and putting the paraffin sections into a carrying tray of a scanner in sequence;
the digital scanner is configured to distinguish the color of the slice target tissue, and focusing scanning is performed in a precise mode so as to obtain a scanned and amplified scanning image.
Optionally, the effective target tissue is obtained from the wax block corresponding to the slice according to the marking result as a tissue chip column core, specifically:
and (3) based on the label and the shape of the slice, confirming a wax block corresponding to the slice, and vertically drilling an effective target tissue at a position corresponding to the wax block according to a marking area of a marking result by using an array instrument sampling needle to serve as a tissue chip column core.
The application adopts the technical proposal and has at least the following beneficial effects:
The method has the advantages that accurate target tissue marking is performed based on the digital intelligent scanning section, invalid points of a constructed medium-high density tissue chip are greatly reduced, the core quality and accuracy of the tissue chip are obviously improved, the heterogeneity difference of each point of the malignant tumor tissue chip is reduced, the freshness of cells in a target tissue is effectively ensured, and the accuracy of immunohistochemistry and gene research is further improved. And the marking work intensity of a pathologist can be reduced, the tissue chip manufacturing time is shortened, and the rejection rate is reduced, so that the manpower, material resources and financial resources are saved.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention. The objectives and other advantages of the invention will be realized and attained by the structure particularly pointed out in the written description and claims hereof as well as the appended drawings.
Drawings
The accompanying drawings are included to provide a further understanding of the technical aspects or prior art of the present application, and are incorporated in and constitute a part of this specification. The drawings, which are used to illustrate the technical scheme of the present application, are not limited to the technical scheme of the present application.
Fig. 1 is a flow chart of a method for organizing chips into cores according to an embodiment of the present application.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the technical solutions of the present application will be described in detail below. It will be apparent that the described embodiments are only some, but not all, embodiments of the application. All other embodiments, based on the examples herein, which are within the scope of the application as defined by the claims, will be within the scope of the application as defined by the claims.
Based on the background technology, the invention aims to provide a method for manufacturing a tissue chip, which adopts a precise marking method in manufacturing a high-density paraffin tissue chip core, so that the content and the accuracy of effective target tissues in the paraffin tissue chip core are greatly improved.
As shown in fig. 1, in an embodiment, the method for manufacturing a core of a tissue chip according to the present application includes:
Step S110, a digital scanner is used for scanning paraffin sections, and scanning amplified scanning images are obtained;
Specifically, in the step, paraffin sections are wiped clean by absolute alcohol and are sequentially placed into a carrying tray of a scanner; the digitized scanner is configured to distinguish the color of the slice target tissue (based on the HE staining technology of the slice), and focusing scanning is performed in a precise mode so as to obtain a scanned image after scanning and amplifying. For example, the model number of the digital scanner is PRECICE B, and the scanning magnification is used for automatically focusing and scanning pathological section target tissues according to a 40-time objective lens so as to obtain scanned and enlarged scanning images.
Then, step S120 is performed, in which the obtained scanned image is subjected to microscopic marking, and the effective target tissue is obtained as a tissue chip column core from the wax block corresponding to the slice according to the marking result, which is easily understood by a professional having pathological knowledge.
In the step, firstly, a slice is observed and evaluated according to a scanned image, the content comparison condition of effective target tissues and ineffective tissues in the slice is evaluated and judged, and different methods are adopted for processing based on different conditions;
In the application, for the situation that the effective target tissues in the slice are less and the ineffective tissues are more, a pathological tissue local marking method is adopted for mirror image lower marking, and for the situation that the effective target tissues in the slice are more and the ineffective tissues are less, an accurate graffiti method is adopted for mirror image lower marking, and the two marking methods are respectively introduced as follows:
A. the process of marking under mirror image by adopting a pathological tissue local marking method comprises the following steps:
The method comprises the steps of determining an invalid tissue area on a scanned image and marking the invalid tissue area by using a first mark, such as a fork, comparing a slice under a microscope, avoiding invalid tissues in the slice (such as tissues interfering with inflammatory reaction, bleeding necrotic tissues, degenerated tissues around the two tissues and the like) and avoiding an invalid area of a target tissue matrix (such as an invalid area comprising an area corresponding to collagen fibers in the target tissue matrix), and marking an effective target tissue concentrated area by using a second mark, such as an oily Marker pen precise circle.
B. The process of marking under the mirror image by adopting the accurate graffiti method comprises the following steps:
The invalid region is determined on the scanned image and marked with a third mark, for example, here marked with an oily stroke fork, then the section is compared under a microscope, the preliminary screening invalid region in the section is marked with colors (for example, the preliminary screening invalid region in the section comprises a bleeding necrosis region, a thick wall blood vessel region, a fibrous tissue region and the like in the section), and then the section is marked with colors for the fine invalid region (for example, the fine invalid region comprises a tissue degeneration region, a cell degeneration region, an inflammation interference region and the like) under a high magnification view in cooperation with the scanned image. The two-time coloring marking can be black marking by an oil pen.
In step S120, after the labeling process of a or B is completed, the effective target tissue is continuously obtained from the wax block corresponding to the slice according to the labeling result as the tissue chip column core, and the tissue chip core making is completed based on the obtained column core.
Specifically, based on the label and shape of the slice, the wax block corresponding to the slice is confirmed (namely, the marked target tissue slice and the same wax block are required to be compared with each other in number and shape), then the effective target tissue is vertically drilled at the corresponding position of the wax block according to the marked area of the slice marking result by using an array instrument sampling needle to serve as a tissue chip column core, and the tissue chip core making is completed based on the column core.
The application adopts the technical proposal and has at least the following beneficial effects:
The method has the advantages that accurate target tissue marking is performed based on the digital intelligent scanning section, invalid points of a constructed medium-high density tissue chip are greatly reduced, the core quality and accuracy of the tissue chip are obviously improved, the heterogeneity difference of each point of the malignant tumor tissue chip is reduced, the freshness of cells in a target tissue is effectively ensured, and the accuracy of immunohistochemistry and gene research is further improved. And the marking work intensity of a pathologist can be reduced, the tissue chip manufacturing time is shortened, and the rejection rate is reduced, so that the manpower, material resources and financial resources are saved.
The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the scope of the present invention are intended to be included in the scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.
Claims (7)
1. A method of organizing a chip core, comprising:
scanning paraffin sections by using a digital scanner to obtain scanning images amplified by scanning;
Performing microscopic marking based on the scanning image, and acquiring an effective target tissue from a wax block corresponding to the slice according to a marking result to serve as a tissue chip column core; wherein the performing the under-mirror marking based on the scanned image includes:
the slice is observed and evaluated based on the scanned image,
Aiming at the condition that effective target tissues are few and ineffective tissues are many in a slice, determining an ineffective tissue area on the scanning image, marking by adopting a first mark, comparing the slice under a microscope, avoiding the ineffective tissues in the slice and the ineffective areas of the target tissue matrix, and marking the effective target tissue concentrated area by adopting a second mark;
And aiming at the situation that the number of effective target tissues in the slice is large and the number of ineffective tissues is small, determining an ineffective area on the scanning image, marking by adopting a third mark, comparing the slice with the slice under a microscope, marking the color of the preliminary screening ineffective area in the slice, and marking the color of the tiny ineffective area under a high-magnification view field by matching the scanning image with the uncolored area of the slice.
2. The method of claim 1, wherein the step of determining the position of the substrate comprises,
Ineffective tissue within the slice includes tissue that interferes with inflammatory reactions, tissue that is necrotic by bleeding, and tissue that is degenerated around both types of tissue;
the ineffective area of the target tissue matrix comprises an area corresponding to collagen fibers in the target tissue matrix.
3. The method of claim 1, wherein the step of determining the position of the substrate comprises,
The marking by adopting the first mark is specifically to mark by adopting a picture fork;
The marking by the second mark is specifically to marking by an oily stroke circle.
4. The method of claim 1, wherein the step of determining the position of the substrate comprises,
The preliminary screening ineffective area in the slice comprises a bleeding necrosis area, a thick-wall vascular area and a fibrous tissue area in the slice;
the minor ineffective areas include a tissue degeneration area, a cell degeneration area and an inflammation interference area.
5. The method of claim 1, wherein the step of determining the position of the substrate comprises,
The marking by adopting the third mark is specifically to mark by adopting an oily stroke fork;
The color mark is specifically a black mark made by an oil pen.
6. The method according to claim 1, wherein the paraffin section is scanned by a digital scanner to obtain a scanned and magnified scanned image, specifically:
Wiping paraffin sections clean by absolute alcohol and putting the paraffin sections into a carrying tray of a scanner in sequence;
the digital scanner is configured to distinguish the color of the slice target tissue, and focusing scanning is performed in a precise mode so as to obtain a scanned and amplified scanning image.
7. The method according to claim 1, wherein the obtaining the effective target tissue from the wax block corresponding to the slice as the tissue chip column core according to the labeling result comprises:
and (3) based on the label and the shape of the slice, confirming a wax block corresponding to the slice, and vertically drilling an effective target tissue at a position corresponding to the wax block according to a marking area of a marking result by using an array instrument sampling needle to serve as a tissue chip column core.
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| CN202010986636.3A CN114199658B (en) | 2020-09-18 | 2020-09-18 | Method for manufacturing core by organizing chip |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002048680A1 (en) * | 2000-12-13 | 2002-06-20 | THE GOVERNMENT OF THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SEVICES. The National Institutes of Health | Method and system for processing regions of interest for objects comprising biological material |
| CN112534237A (en) * | 2018-06-21 | 2021-03-19 | 基美健有限公司 | System and method for analyzing pre-substrate processing |
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| WO2011150316A1 (en) * | 2010-05-28 | 2011-12-01 | Sridharan Rajagopalan | Obtaining analytes from a tissue specimen |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2002048680A1 (en) * | 2000-12-13 | 2002-06-20 | THE GOVERNMENT OF THE UNITED STATES OF AMERICA AS REPRESENTED BY THE SECRETARY OF THE DEPARTMENT OF HEALTH AND HUMAN SEVICES. The National Institutes of Health | Method and system for processing regions of interest for objects comprising biological material |
| CN112534237A (en) * | 2018-06-21 | 2021-03-19 | 基美健有限公司 | System and method for analyzing pre-substrate processing |
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