CN112113937A - Tissue and organ three-dimensional imaging and analyzing method based on continuous section, multicolor fluorescence and three-dimensional reconstruction - Google Patents
Tissue and organ three-dimensional imaging and analyzing method based on continuous section, multicolor fluorescence and three-dimensional reconstruction Download PDFInfo
- Publication number
- CN112113937A CN112113937A CN202010698695.0A CN202010698695A CN112113937A CN 112113937 A CN112113937 A CN 112113937A CN 202010698695 A CN202010698695 A CN 202010698695A CN 112113937 A CN112113937 A CN 112113937A
- Authority
- CN
- China
- Prior art keywords
- fluorescence
- dimensional
- tissue
- dimensional reconstruction
- tissue organ
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 238000000034 method Methods 0.000 title claims abstract description 32
- 210000000056 organ Anatomy 0.000 title claims abstract description 32
- 238000003384 imaging method Methods 0.000 title claims abstract description 21
- 238000004458 analytical method Methods 0.000 claims abstract description 28
- 238000010186 staining Methods 0.000 claims abstract description 20
- 238000003125 immunofluorescent labeling Methods 0.000 claims abstract description 15
- 230000008569 process Effects 0.000 claims abstract description 9
- 238000004445 quantitative analysis Methods 0.000 claims abstract description 8
- 210000004027 cell Anatomy 0.000 claims description 19
- 239000012188 paraffin wax Substances 0.000 claims description 8
- 210000003850 cellular structure Anatomy 0.000 claims description 6
- 230000006870 function Effects 0.000 claims description 6
- 238000010276 construction Methods 0.000 claims description 5
- 230000003915 cell function Effects 0.000 claims description 4
- 238000007710 freezing Methods 0.000 claims description 4
- 230000008014 freezing Effects 0.000 claims description 4
- 239000003550 marker Substances 0.000 claims description 4
- 238000004043 dyeing Methods 0.000 claims description 3
- 238000010219 correlation analysis Methods 0.000 abstract description 3
- 238000011002 quantification Methods 0.000 abstract description 3
- 108091006146 Channels Proteins 0.000 description 22
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 21
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 12
- 239000000427 antigen Substances 0.000 description 10
- 102000036639 antigens Human genes 0.000 description 10
- 108091007433 antigens Proteins 0.000 description 10
- 235000019441 ethanol Nutrition 0.000 description 9
- 238000011160 research Methods 0.000 description 8
- 239000001993 wax Substances 0.000 description 8
- 238000011532 immunohistochemical staining Methods 0.000 description 5
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 4
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 4
- 238000001816 cooling Methods 0.000 description 4
- 230000007547 defect Effects 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 239000008096 xylene Substances 0.000 description 4
- 230000006835 compression Effects 0.000 description 3
- 238000007906 compression Methods 0.000 description 3
- 230000018044 dehydration Effects 0.000 description 3
- 238000006297 dehydration reaction Methods 0.000 description 3
- 238000003745 diagnosis Methods 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000009826 distribution Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 238000012545 processing Methods 0.000 description 3
- 238000007789 sealing Methods 0.000 description 3
- 229910001220 stainless steel Inorganic materials 0.000 description 3
- 239000010935 stainless steel Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 239000000945 filler Substances 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 239000003517 fume Substances 0.000 description 2
- 230000004927 fusion Effects 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 230000001575 pathological effect Effects 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 238000012800 visualization Methods 0.000 description 2
- 238000005406 washing Methods 0.000 description 2
- 102000017420 CD3 protein, epsilon/gamma/delta subunit Human genes 0.000 description 1
- 238000000116 DAPI staining Methods 0.000 description 1
- 102000018651 Epithelial Cell Adhesion Molecule Human genes 0.000 description 1
- 108010066687 Epithelial Cell Adhesion Molecule Proteins 0.000 description 1
- 101000934372 Homo sapiens Macrosialin Proteins 0.000 description 1
- 102100025136 Macrosialin Human genes 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 102100024616 Platelet endothelial cell adhesion molecule Human genes 0.000 description 1
- 239000007984 Tris EDTA buffer Substances 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- 238000013475 authorization Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000005282 brightening Methods 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000005138 cryopreservation Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000000975 dye Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 230000007310 pathophysiology Effects 0.000 description 1
- 238000007781 pre-processing Methods 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- 230000011218 segmentation Effects 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000013334 tissue model Methods 0.000 description 1
- 238000009966 trimming Methods 0.000 description 1
Images
Classifications
-
- 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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
-
- 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
-
- 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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1468—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5302—Apparatus specially adapted for immunological test procedures
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
- G01N33/532—Production of labelled immunochemicals
- G01N33/533—Production of labelled immunochemicals with fluorescent label
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
-
- G—PHYSICS
- G06—COMPUTING; CALCULATING OR COUNTING
- G06T—IMAGE DATA PROCESSING OR GENERATION, IN GENERAL
- G06T17/00—Three dimensional [3D] modelling, e.g. data description of 3D objects
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1434—Optical arrangements
- G01N2015/144—Imaging characterised by its optical setup
- G01N2015/1445—Three-dimensional imaging, imaging in different image planes, e.g. under different angles or at different depths, e.g. by a relative motion of sample and detector, for instance by tomography
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Optical investigation techniques, e.g. flow cytometry
- G01N15/1468—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle
- G01N2015/1472—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle with colour
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Physics & Mathematics (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- General Health & Medical Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Medicinal Chemistry (AREA)
- Food Science & Technology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Dispersion Chemistry (AREA)
- Computer Graphics (AREA)
- Geometry (AREA)
- Software Systems (AREA)
- Theoretical Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a tissue organ stereo imaging and analyzing method based on continuous slicing, multicolor fluorescence and three-dimensional reconstruction, which mainly comprises the steps of taking a reconstruction object to carry out strict continuous slicing; performing multicolor immunofluorescence staining alternately in various combinations according to a standard process line; performing multi-channel fluorescence scanning on all immunofluorescent staining slices to obtain digital slice images; aligning the continuous sections according to the fluorescence channel signals, and performing three-dimensional reconstruction by inserting a plurality of staining combinations to obtain a tissue organ fluorescence three-dimensional model with cell level resolution; the local region of interest is STL modeled and subjected to various analyses. The invention can construct and obtain a high-resolution tissue organ fluorescence three-dimensional model which can be subjected to quantitative analysis, and can faithfully restore the tissue organ three-dimensional shape to the maximum extent; one or more fluorescence channels can be viewed individually or in superposition; STL modeling can be carried out on the region of interest to complete the space quantification and correlation analysis of various cells and functional markers.
Description
Technical Field
The invention relates to the field of tissue and organ stereo imaging analysis, in particular to a tissue and organ stereo imaging and analyzing method based on continuous slicing, multicolor fluorescence and three-dimensional reconstruction.
Background
Tissue and organ specimens are important research materials in the biomedical research field and are also key specimens for disease diagnosis, and tissue imaging is an important means for diagnosing and researching tissue specimens. The tissue imaging mainly comprises two parts of staining and detecting.
Staining methods include dye staining (e.g., HE staining) and antibody-based staining, which is further classified into immunohistochemical staining (IHC) and immunofluorescence staining (IF). Among them, IF can detect multiple antigens simultaneously, as compared to IHC, contributing to more accurate cell identity confirmation and functional characterization.
The detection part currently mainly takes observation and reading of a single tissue section. The traditional tissue slice has the limitation that the traditional tissue slice can only reflect the condition of one section of the tissue, is far away from the stereo form in a living body, has huge information loss and is difficult to identify the complete picture. In order to observe and study tissues on a stereo model, three-dimensional imaging technologies have been developed. The current mature anatomical reconstruction based on CT or MRI can only observe the anatomical level of organ tissues, mainly serving clinical work and not effectively reacting to the condition of the cellular level inside the tissues. The method of acquiring multi-layer images in the slice thickness range and fusing the images in 2009 by Zheng and Crowdown enables the acquired area to have higher definition (authorization notice number: CN 101615289B), which essentially belongs to the two-dimensional image acquisition and presentation category. In 2017, the cheerful complexion and the like adopt a continuous section IHC staining and layer-by-layer calibration mode to carry out tissue three-dimensional reconstruction (application publication number: CN 106918484A), but the defects mainly comprise single IHC detection index, relatively limited information which can be reflected by a formed three-dimensional model, and large workload of layer-by-layer calibration on a scanned image, so that the method has high labor cost and relatively low clinical or scientific research practical value. In 2018, Wangjingxiang and the like develop a three-dimensional reconstruction algorithm (application publication number: CN 109191510A) which can carry out grid alignment and region segmentation on continuous pathological sections, and the three-dimensional reconstruction algorithm is used for clinical application of pathological sections. In addition, the stereoscopic scanning imaging technology based on the tissue transparentizing staining microscope has the advantage of good tissue integrity, but has the defects of low model resolution, single staining mark, high hardware requirement, high cost, difficulty in popularization and the like.
With the gradual progress of basic research, exploring the distribution relationship of various tissue cells on the spatial structure and the spatial expression characteristics of various functional markers is the key to further deepen the development of tissues and organs and the change of pathophysiology, so how to establish a multi-index high-resolution tissue three-dimensional model becomes a bottleneck to be broken through urgently. The invention provides a three-dimensional reconstruction three-dimensional imaging method based on continuous section multicolor immunofluorescence staining aiming at two requirements of multiple indexes and high resolution.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a tissue and organ three-dimensional imaging and analyzing method based on continuous slicing, multicolor fluorescence and three-dimensional reconstruction.
The purpose of the invention is achieved by the following technical scheme: the tissue and organ stereo imaging and analyzing method based on continuous section, multicolor fluorescence and three-dimensional reconstruction mainly comprises the following steps:
1) strictly and continuously slicing the reconstructed object;
2) performing multicolor immunofluorescence staining alternately in various combinations according to a standard process line;
3) performing multi-channel fluorescence scanning on all immunofluorescent staining slices to obtain digital slice images;
4) aligning the continuous sections according to the fluorescence channel signals, and performing three-dimensional reconstruction by inserting a plurality of staining combinations to obtain a tissue organ fluorescence three-dimensional model with cell level resolution;
5) the local region of interest is STL modeled and subjected to various analyses.
The reconstruction objects are isolated tissues and organs, and are subjected to freezing and paraffin embedding and then strictly continuous slicing.
The thickness of each slice of the strict continuous slices is 2-10 micrometers, the slice of each layer is marked with a serial number, and the section lost in the slicing process is also calculated in a marked sequence.
The multicolor immunofluorescent staining employs a plurality of staining combinations to participate in model construction together, and fluorescent markers of less-marked schemes must be included in fluorescent markers of most-marked schemes.
And performing multi-channel fluorescence scanning on each slice according to the fluorescence channel corresponding to the scheme of marking the maximum number.
The three-dimensional reconstruction has the following properties: zooming at will, observing under different scales; different dyeing combinations are integrated into the same model to form a multicolor fluorescence model which simultaneously reflects a plurality of even dozens of marks; the presentation form of the system is switched randomly, and the system comprises a single channel and superposition of 2 channels and more than 2 channels; the observation angle of the three-dimensional space can be freely rotated to realize any angle.
And performing multiple analyses after the STL modeling, including model qualitative observation and analysis, cell component quantitative analysis, cell function marker quantitative analysis and analysis of spatial relationship between cells and function markers. .
The invention has the beneficial effects that:
1. the technology can construct and obtain a high-resolution tissue organ fluorescence three-dimensional model capable of carrying out quantitative analysis, and is characterized in that the tissue organ three-dimensional shape including but not limited to internal cell and matrix components, distribution characteristics and adjacent relation can be faithfully reduced to the maximum extent; one or more fluorescence channels can be viewed individually or in superposition; STL modeling can be carried out on the region of interest to complete space quantification and correlation analysis of various cells and functional markers, and the interaction relation between the multi-cell functional quantification and the components can be reflected more accurately; the method is helpful for people to know the real form of local tissues and organs of a human body or an animal and plant model more intuitively, and particularly, the method can clearly observe the internal details of the tissue and organ cell level, so that the internal fine structure characteristics which are difficult to be found by the traditional two-dimensional slice and the common three-dimensional model can be found, and meanwhile, the method can carry out various quantitative calculations and analyses on the spatial relationship between cells and non-cell components, thereby better serving the fields of scientific research, disease diagnosis and the like.
2. The invention has cell-level tissue resolution, can simultaneously detect a plurality of markers for a single tissue, can help people to better know the spatial distribution characteristics and the functional states of cells and non-cell components in the tissue, and can help scientific research and disease diagnosis; the three-dimensional reconstruction model based on continuous tissue slice multicolor immunofluorescence is characterized in that an original picture does not need to be compressed, all two-dimensional image information can be completely reserved, a single-cell resolution video and an image can be obtained through high-precision rendering, the model can simultaneously display cell identities and functional markers of a plurality of or even dozens of pseudo-color markers, huge information quantity is presented, and the requirement of high-throughput space detection of clinical and scientific researches can be met; the three-dimensional visualization tissue model analysis method meets the traditional requirement on three-dimensional visualization of the tissue, further meets the requirement on model refinement analysis, and better serves clinical diagnosis for scientific research.
Drawings
FIG. 1 is a schematic view of sequential slice scanning and image alignment according to the present invention.
FIG. 2 is a schematic front view of a multicolor fluorescent three-dimensional model of the present invention.
FIG. 3 is a schematic side view of a multicolor fluorescent three-dimensional model of the present invention.
FIG. 4 is a schematic view of a single channel of the present invention.
FIG. 5 is a schematic view of a combination of multiple channels according to the present invention.
Fig. 6 is a schematic view of the present invention when viewed at different scales.
Fig. 7 is a partially enlarged view of a selected area in fig. 6.
Detailed Description
The invention will be described in detail below with reference to the following drawings:
example 1: as shown in the attached drawings, the tissue and organ stereo imaging and analyzing method based on continuous section, multicolor fluorescence and three-dimensional reconstruction mainly comprises the following steps:
1) strictly and continuously slicing the reconstructed object; the reconstruction objects are isolated tissues and organs, and are subjected to freezing and paraffin embedding and then strictly continuous slicing. Strictly continuous sections each having a thickness of 2-10 microns, each layer of sections being strictly numbered, e.g. the first layer is numbered 1, the second layer is numbered 2, and so on, the sections lost during the slicing process are also counted in the sequence of marks.
2) Performing multicolor immunofluorescence staining alternately in various combinations according to a standard process line; the multicolor immunofluorescence staining adopts a plurality of staining combinations to participate in model construction together. If 2 staining combinations a and B were designed simultaneously, wherein a comprises four markers DAPI, CD3, CD31, CD68, and B comprises three markers DAPI, EpCAM, α -SMA, then in the strict serial sections, section 1 row a staining, section 2 row B staining, section 3 repeat a staining, and so on. And the fluorescent label (DAPI, 488nm, 555nm) of the label-less protocol (such as protocol B) must be included in the fluorescent label (DAPI, 488nm, 555nm, 647nm) of the label-most protocol (such as protocol A).
3) Performing multi-channel fluorescence scanning on all immunofluorescent staining slices to obtain digital slice images; and performing multi-channel fluorescence scanning on each slice according to the fluorescence channel corresponding to the labeling maximum scheme.
4) Aligning the continuous sections according to the fluorescence channel signals, and performing three-dimensional reconstruction by inserting a plurality of staining combinations to obtain a tissue organ fluorescence three-dimensional model with cell level resolution; the three-dimensional reconstruction has the following properties: zooming at will, observing under different scales; different dyeing combinations are integrated into the same model to form a multicolor fluorescence model which simultaneously reflects a plurality of even dozens of marks; the presentation form of the system is switched randomly, and the system comprises a single channel and superposition of 2 channels and more than 2 channels; the observation angle of the three-dimensional space can be freely rotated to realize any angle.
5) The local region of interest is STL modeled and subjected to various analyses. Various analyses after STL modeling include qualitative model observation analysis (e.g., macroscopic signal amount, substructure size, mutual position relationship, etc.), quantitative cell component analysis (e.g., quantitative analysis of cell types defined by one or more markers, correlation analysis, etc.), quantitative cell function marker analysis (e.g., quantitative analysis of expression of one or more markers reflecting cell functions in different cell types or different spatial positions), and spatial relationship analysis between cells and function markers (e.g., average spatial distance analysis between cell components, or spatial distance analysis of function markers between one or more cells, etc.).
Example 2: the tissue and organ stereo imaging and analyzing method based on continuous section, multicolor fluorescence and three-dimensional reconstruction mainly comprises the following steps:
1) tissue acquisition and preservation: taking excised tissue 1cm x 0.5cm, and performing OCT cryopreservation or 10% formalin fixation for 48 hours;
2) tissue treatment: dehydration was performed if the tissue was 10% formalin fixed: taking the tissue out of the fixing liquid, flattening the tissue of the target part in a fume hood by using a scalpel, and putting the trimmed tissue into a dehydration box written with a corresponding number. The dewatering box is put into a hanging basket to be dewatered by gradient alcohol in the dewatering machine in sequence: 2h of 75% alcohol, 2h of 85% alcohol, 2h of 90% alcohol, 1h of 95% alcohol, 1h of absolute ethyl alcohol I1h, 1h of absolute ethyl alcohol II, 45min of xylene I, 45min of xylene II, 1h of wax I-1 h of wax II and 1h of wax III. Paraffin embedding was subsequently performed: embedding the wax-soaked tissue in an embedding machine. Firstly, molten wax is put into a stainless steel embedding mold, and before the wax is solidified, tissues are taken out from a dehydration box and put into the stainless steel embedding mold according to the requirements of an embedding surface. And cooling in a freezing table, taking the wax block out of the stainless steel embedding mould after the wax is solidified, and trimming the wax block.
3) Tissue section: 200-300 serial sections with the thickness of 2-10 μm were taken from the frozen paraffin tissue. The thickness of the slices at one time is kept consistent. In order to maintain the quality of the section, 10-15 pieces of paraffin tissue are cut and then placed on ice for cooling, the section floats on 45 ℃ warm water of a sheet spreading machine to flatten the tissue, and a glass slide is used for taking the tissue out. When fishing out the slices, the serial numbers of the continuous slices need to be closely noticed, and unqualified slices should be brought into the sequence. Taking out and putting into a 65 ℃ oven to bake the slices for at least 30-60 minutes.
4) And (3) slicing treatment: (paraffin section) dewaxing: placing the slices in xylene I for 10min, xylene II for 10min, anhydrous ethanol I for 5min, anhydrous ethanol II for 5min, 95% ethanol for 5min, 90% ethanol for 5min, 80% ethanol for 5min, 70% ethanol for 5min, and washing with distilled water. Antigen retrieval: placing the tissue slices in a repairing box filled with boiling Tris-EDTA buffer solution (PH9.0), continuously heating to 110 deg.C in an antigen repairing pot for 10min, slowly cooling to 95 deg.C, uncovering, cooling at room temperature, taking out the slide, placing in PBS (PH7.4), shaking and washing on a decolorizing shaking table for 3 times, 3min each time. And (3) sealing: after the sections were spin-dried slightly, the tissue was circled with a organizing pen (to prevent antibody from running off), 1% BSA was added dropwise to the circle, and the cells were incubated at room temperature for 20 min. Primary antibody incubation: after the section is slightly dried, primary antibody covering tissues diluted by 1% BSA according to a certain proportion are dripped into the ring, and the section is flatly placed in a wet box and incubated overnight at 4 ℃. And (3) secondary antibody incubation: the slides were washed 3 times in PBS (pH7.4) on a destaining shaker for 3min each; dripping primary antibody host specificity fluorescent secondary antibody in the ring to cover the tissue after the section is slightly dried, and incubating for 30min at room temperature; the primary antibody and secondary antibody incubation process was repeated for the remaining marker staining. (frozen tissue) air drying: and (5) placing the frozen slices in a fume hood to dry at room temperature for 10-30min to remove water vapor. Fixing: circle the tissue with a pen and fix with 4% paraformaldehyde for 15 min. Subsequent steps were incubated with paraffin sections from antigen retrieval to secondary antibody.
5) And (3) sealing sheet scanning: DAPI staining for 5min, sealing with anti-quencher, keeping in dark overnight, drying, and storing at 4 deg.C or scanning with digital slide (instrument: 3DHISTECH Panoramic MIDI) to obtain two-dimensional digital multicolor immunofluorescence continuous image.
6) The three-dimensional reconstruction method mainly comprises the following steps:
image data preparation: the digitized slices corresponding to a series of master images from the slide scanner were manually examined using the viewing software CaseViewer. By using the output image function in the CaseViewer software, the original image (i.e. the main digital image) is output as a TIFF or JPEG file, each channel of each slice resulting in a TIFF file of about 2GB volume;
image preprocessing:
a) TIFF is compressed, and since the original image pixel value is too low, the compression is achieved by neighborhood sum filtering the original TIFF. Assuming that the original image has a width w, a height h, a compression ratio k, and an image pixel value matrix M1, the pixel value M2 of the new image is expressed as:
by MATLAB programming, the original TIFF matrix M1 is processed by the formula to obtain M2, and the TIFF is derived. When k is 3, the size of each TIFF after compression is about 200-300 MB.
b) TIFF is brightened and the summed image brightness is still too low, so the contrast enhancement process is performed. An ultra-lightweight open source image processing framework based on Python is realized through an enhance function, and the algorithm is Contrast construction. Assuming that the lowest value of the current image is c, the highest value is d, the current pixel value is Pin, and a and b are the minimum value and the maximum value of the bit depth, respectively, the pixel value after the brightening is:
image fusion and registration:
a) and (3) utilizing MATLAB software, taking the coordinate mean value of all the image positioning rods as a reference coordinate, obtaining the projective transformation parameters of each picture, performing projective transformation on the corresponding picture, and finally correcting the picture of the specimen positioning rod through the projective transformation.
b) The successive slices must be image registered in order to eliminate slice progressive translation registration errors. Because the reconstructed structure is very tiny, the segmented binary image is registered without adopting the traditional fixed point registration method. The registration between images is realized by adopting the principle of maximizing the gray level similarity between the images, mainly the translation and the rotation of a rigid body. Since multicolor staining requires that the sections be stained in batches, different staining protocols are used, respectively. The only common channel in the different batch protocols was the DAPI channel, so first the registration was performed with the DAPI channel in focus, for the next two sets of stains to perform virtual tissue fusion. The TIFF images of all sections of the DAPI channel (e.g., a in staining protocol 1 and b in protocol 2) are first collected in a folder, and the DAPI images in the folder are registered using a Python-generated registration plug-in. And the registration is based on feature matching, and a rigid body mode capable of freely moving and rotating is adopted for registration to obtain a Z-Stack.
c) For each picture mixed by the two sets of dyed DAPI, an image with the same size and the pixel value of pure 0 is manufactured and is reserved as a matched image filler source which is relatively lacked in the two sets of dyed images.
d) Next, TIFFs corresponding to each antigen are collected separately, all missing position slices are found compared with the most complete DAPI channel, pure black images of the same position are copied from the filler source, and virtual renaming is performed by referring to the image name under the antigen to supplement the defect. Thus, a + b pictures were taken for each antigen.
e) And applying the registration parameters of the DAPI to all the antigen channels for rapid equal processing, wherein each antigen channel obtains a Z-stack.
f) Integrating Z-stacks of all antigen channels and Z-stacks of DAPI, renaming to C0 and C1 …, then opening in MATLAB, and carrying out overall registration according to actual pixel size to obtain a registered multi-channel slice three-dimensional image library.
And (3) registration effect detection: after pictures are transparentized by Photoshop 5.0 software, fine adjustment is carried out on every two continuous pictures, the pictures are stored after the pictures are adjusted to be completely overlapped. And after the image registration processing is finished, all the images are sequentially input into an MATLAB program to draw a three-dimensional graph. The three-dimensional reconstruction result is made into a rotation animation; and finally, carrying out three-dimensional position observation, skin construction and analysis.
It should be understood that equivalent substitutions and changes to the technical solution and the inventive concept of the present invention should be made by those skilled in the art to the protection scope of the appended claims.
Claims (7)
1. A tissue organ stereo imaging and analyzing method based on continuous section, multicolor fluorescence and three-dimensional reconstruction is characterized in that: the method mainly comprises the following steps:
1) strictly and continuously slicing the reconstructed object;
2) performing multicolor immunofluorescence staining alternately in various combinations according to a standard process line;
3) performing multi-channel fluorescence scanning on all immunofluorescent staining slices to obtain digital slice images;
4) aligning the continuous sections according to the fluorescence channel signals, and performing three-dimensional reconstruction by inserting a plurality of staining combinations to obtain a tissue organ fluorescence three-dimensional model with cell level resolution;
5) the local region of interest is STL modeled and subjected to various quantitative analyses.
2. The method for tissue organ stereoscopic imaging and analysis based on serial sections, polychromatic fluorescence and three-dimensional reconstruction according to claim 1, characterized in that: the reconstruction objects are isolated tissues and organs, and are subjected to freezing and paraffin embedding and then strictly continuous slicing.
3. The method for tissue organ stereoscopic imaging and analysis based on serial sections, polychromatic fluorescence and three-dimensional reconstruction according to claim 1, characterized in that: the thickness of each slice of the strict continuous slices is 2-10 micrometers, the slice of each layer is marked with a serial number, and the section lost in the slicing process is also calculated in a marked sequence.
4. The method for tissue organ stereoscopic imaging and analysis based on serial sections, polychromatic fluorescence and three-dimensional reconstruction according to claim 1, characterized in that: the multicolor immunofluorescent staining employs a plurality of staining combinations to participate in model construction together, and fluorescent markers of less-marked schemes must be included in fluorescent markers of most-marked schemes.
5. The method for tissue organ stereoscopic imaging and analysis based on serial sections, polychromatic fluorescence and three-dimensional reconstruction according to claim 1, characterized in that: and performing multi-channel fluorescence scanning on each slice according to the fluorescence channel corresponding to the scheme of marking the maximum number.
6. The method for tissue organ stereoscopic imaging and analysis based on serial sections, polychromatic fluorescence and three-dimensional reconstruction according to claim 1, characterized in that: the three-dimensional reconstruction has the following properties: zooming at will, observing under different scales; different dyeing combinations are integrated into the same model to form a multicolor fluorescence model which simultaneously reflects a plurality of even dozens of marks; the presentation form of the system is switched randomly, and the system comprises a single channel and superposition of 2 channels and more than 2 channels; the observation angle of the three-dimensional space can be freely rotated to realize any angle.
7. The method for tissue organ stereoscopic imaging and analysis based on serial sections, polychromatic fluorescence and three-dimensional reconstruction according to claim 1, characterized in that: and performing multiple analyses after the STL modeling, including model qualitative observation and analysis, cell component quantitative analysis, cell function marker quantitative analysis and analysis of spatial relationship between cells and function markers.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010698695.0A CN112113937A (en) | 2020-07-20 | 2020-07-20 | Tissue and organ three-dimensional imaging and analyzing method based on continuous section, multicolor fluorescence and three-dimensional reconstruction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202010698695.0A CN112113937A (en) | 2020-07-20 | 2020-07-20 | Tissue and organ three-dimensional imaging and analyzing method based on continuous section, multicolor fluorescence and three-dimensional reconstruction |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112113937A true CN112113937A (en) | 2020-12-22 |
Family
ID=73798804
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202010698695.0A Pending CN112113937A (en) | 2020-07-20 | 2020-07-20 | Tissue and organ three-dimensional imaging and analyzing method based on continuous section, multicolor fluorescence and three-dimensional reconstruction |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112113937A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115082529A (en) * | 2022-06-30 | 2022-09-20 | 华东师范大学 | System and method for collecting and analyzing multi-dimensional information of gross tissue |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106408648A (en) * | 2015-08-03 | 2017-02-15 | 青岛海信医疗设备股份有限公司 | Medical-tissue slice-image three-dimensional reconstruction method and equipment thereof |
| CN106918484A (en) * | 2017-03-28 | 2017-07-04 | 武汉瑞福宁科技有限公司 | A kind of construction method of the threedimensional model based on histotomy |
| CN108074243A (en) * | 2018-02-05 | 2018-05-25 | 志诺维思（北京）基因科技有限公司 | A kind of cellular localization method and cell segmentation method |
-
2020
- 2020-07-20 CN CN202010698695.0A patent/CN112113937A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106408648A (en) * | 2015-08-03 | 2017-02-15 | 青岛海信医疗设备股份有限公司 | Medical-tissue slice-image three-dimensional reconstruction method and equipment thereof |
| CN106918484A (en) * | 2017-03-28 | 2017-07-04 | 武汉瑞福宁科技有限公司 | A kind of construction method of the threedimensional model based on histotomy |
| CN108074243A (en) * | 2018-02-05 | 2018-05-25 | 志诺维思（北京）基因科技有限公司 | A kind of cellular localization method and cell segmentation method |
Non-Patent Citations (5)
| Title |
|---|
| 唐军民: "《组织学与胚胎学》", 31 January 2009, 北京大学医学出版社 * |
| 彭瑞云等: "《形态计量与图像分析学》", 31 August 2012, 军事医学科学出版社 * |
| 范玉明等: "《毒理学安全性评价标准操作规程指南 上》", 31 July 2009, 电子科技大学出版社 * |
| 赵亮: "基于云计算的医学教育3D打印模型管理系统设计", 《中国教育信息化》 * |
| 银平章等: "《临床病理学实验技术》", 31 July 1997, 中原农民出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115082529A (en) * | 2022-06-30 | 2022-09-20 | 华东师范大学 | System and method for collecting and analyzing multi-dimensional information of gross tissue |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4376058B2 (en) | Quantitative video microscopy and related systems and computer software program products | |
| BRPI0611381A2 (en) | automated image analysis | |
| US20050123181A1 (en) | Automated microscope slide tissue sample mapping and image acquisition | |
| JP6120675B2 (en) | Microscope system, image generation method and program | |
| Quintana et al. | Optical projection tomography of vertebrate embryo development | |
| US11521317B2 (en) | Method for analyzing tissue specimens | |
| Armer et al. | Imaging transient blood vessel fusion events in zebrafish by correlative volume electron microscopy | |
| CN106023291A (en) | Imaging device and method for quickly acquiring 3D structure information and molecular phenotype information of large sample | |
| TW201709150A (en) | Spatial multiplexing of histological stains | |
| CN111795879A (en) | Application of pathological specimen manufactured by dehydration set in digital evaluation system | |
| Maco et al. | Correlative in vivo 2-photon imaging and focused ion beam scanning electron microscopy: 3D analysis of neuronal ultrastructure | |
| WO2020210679A1 (en) | High resolution alignment of 3d imaging with 2d imaging | |
| TW202111304A (en) | Three dimensional histopathology imaging method and system thereof | |
| Čapek et al. | Volume reconstruction of large tissue specimens from serial physical sections using confocal microscopy and correction of cutting deformations by elastic registration | |
| CN106706643A (en) | Liver cancer comparison section detection device and detection method | |
| CN112113937A (en) | Tissue and organ three-dimensional imaging and analyzing method based on continuous section, multicolor fluorescence and three-dimensional reconstruction | |
| Clarke et al. | Increasing specimen coverage using digital whole-mount breast pathology: implementation, clinical feasibility and application in research | |
| CN109087390A (en) | Micro- nerve digitlization and three-dimensional nerve network establishing method around the big organ of pelvic cavity | |
| Mourik et al. | Correlative light microscopy and electron tomography to study von Willebrand factor exocytosis from vascular endothelial cells | |
| Tian et al. | Three-dimensional reconstruction of laryngeal cancer with whole organ serial immunohistochemical sections | |
| CN109859304B (en) | Three-dimensional printing technology for establishing three-dimensional structure digital model outside cornea limbal tissue | |
| CN115943305A (en) | Information processing apparatus, information processing method, program, and information processing system | |
| CN113138110A (en) | Three-dimensional reconstruction method for pathological picture | |
| Kurien et al. | Three dimensional reconstruction of a human breast carcinoma using routine laboratory equipment and immunohistochemistry | |
| Malloci et al. | Label-free imaging of large samples: 3D rendering and morphological analysis within histological workflows using serial block face imaging |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |