CN109632420A - A kind of processing method and its application of the rapid tissue transparence based on water-soluble reagent - Google Patents
A kind of processing method and its application of the rapid tissue transparence based on water-soluble reagent Download PDFInfo
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- 238000003672 processing method Methods 0.000 title claims abstract description 30
- 239000003153 chemical reaction reagent Substances 0.000 title claims abstract description 26
- 239000003795 chemical substances by application Substances 0.000 claims abstract description 59
- 238000003384 imaging method Methods 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 23
- 230000000694 effects Effects 0.000 claims abstract description 9
- 229930040373 Paraformaldehyde Natural products 0.000 claims abstract description 7
- 229920002866 paraformaldehyde Polymers 0.000 claims abstract description 7
- 230000008961 swelling Effects 0.000 claims abstract description 7
- 150000002632 lipids Chemical class 0.000 claims abstract description 6
- 210000001519 tissue Anatomy 0.000 claims description 122
- 241000699666 Mus <mouse, genus> Species 0.000 claims description 49
- 210000004556 brain Anatomy 0.000 claims description 43
- FDLQZKYLHJJBHD-UHFFFAOYSA-N [3-(aminomethyl)phenyl]methanamine Chemical compound NCC1=CC=CC(CN)=C1 FDLQZKYLHJJBHD-UHFFFAOYSA-N 0.000 claims description 29
- 238000012545 processing Methods 0.000 claims description 27
- 210000004185 liver Anatomy 0.000 claims description 19
- 230000008520 organization Effects 0.000 claims description 15
- 210000002216 heart Anatomy 0.000 claims description 11
- 210000003734 kidney Anatomy 0.000 claims description 11
- 210000004072 lung Anatomy 0.000 claims description 11
- 230000013011 mating Effects 0.000 claims description 11
- 210000000952 spleen Anatomy 0.000 claims description 11
- 210000002784 stomach Anatomy 0.000 claims description 11
- 239000008363 phosphate buffer Substances 0.000 claims description 9
- 210000000936 intestine Anatomy 0.000 claims description 8
- 241000699670 Mus sp. Species 0.000 claims description 6
- 238000007654 immersion Methods 0.000 claims description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 2
- 210000001161 mammalian embryo Anatomy 0.000 claims 2
- 210000000056 organ Anatomy 0.000 abstract description 13
- 210000004204 blood vessel Anatomy 0.000 abstract description 7
- 210000003792 cranial nerve Anatomy 0.000 abstract description 3
- 238000012986 modification Methods 0.000 description 5
- 230000004048 modification Effects 0.000 description 5
- 230000008901 benefit Effects 0.000 description 4
- 238000000799 fluorescence microscopy Methods 0.000 description 4
- 239000004615 ingredient Substances 0.000 description 4
- 230000000007 visual effect Effects 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 210000005013 brain tissue Anatomy 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 210000004218 nerve net Anatomy 0.000 description 3
- 230000003287 optical effect Effects 0.000 description 3
- 238000009738 saturating Methods 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 241000220324 Pyrus Species 0.000 description 2
- 238000012632 fluorescent imaging Methods 0.000 description 2
- JEIPFZHSYJVQDO-UHFFFAOYSA-N iron(III) oxide Inorganic materials O=[Fe]O[Fe]=O JEIPFZHSYJVQDO-UHFFFAOYSA-N 0.000 description 2
- 210000005036 nerve Anatomy 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- 235000021017 pears Nutrition 0.000 description 2
- 238000002834 transmittance Methods 0.000 description 2
- 230000002792 vascular Effects 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000014759 maintenance of location Effects 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
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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
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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
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- 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
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Abstract
The processing method and its application for the rapid tissue transparence based on water-soluble reagent that the present invention provides a kind of, the treating method comprises following steps: the tissue fixed through paraformaldehyde, which is put into tissue bulking inorganic agent, sufficiently to impregnate makes its loose expansion, removes lipid；The tissue that tissue bulking inorganic agent sufficiently impregnated is put into tissue contracts agent, it is made to shrink back reset condition by swelling state, and obtains preliminary transparent effect；The tissue sufficiently handled in tissue contracts agent is put into index matching reagent to impregnate, finally obtains the tissue of the relatively uniform highly transparent of refractive index.Tissue after processed by the invention becomes highly transparent, can be used in the imaging of tissue entirety three-dimensional fluorescence, realizes and carry out three-dimensional high definition imaging to full cranial nerve net and organ blood vessel web frame.
Description
Technical field
The invention belongs to biomedical optical technical field of imaging more particularly to a kind of quick groups based on water-soluble reagent
Knit the processing method and its application of transparence.
Background technique
The whole high-resolution imaging of biological tissue can provide complete, fine three-dimensional tissue's knot for the research of life science
Structure information, to more fully understand the relationship of organization network structure and function.Three-dimensional knot is obtained based on tradition tissue microtomy
Structure, it is time-consuming and laborious, and the loss of structural information is easily led to, image reconstruction difficulty is big.In recent years, a variety of fluorescent labelling techniques and aobvious
The three-dimensional structure information for being combined into acquisition tissue of micro- imaging technique provides new thought.However, the high scattering of biological tissue
Characteristic makes the imaging depth of optical image technology in biological tissues limited, is unable to satisfy the imaging demand of bulk tissue, such as
The depth of laser confocal microscope is about 50-100 μm, and the imaging depth of Two Photon Fluorescence also only has 500-800 μm, tissue
The appearance of light transparent technology provides important method for the whole imaging of big tissue three-dimensional structure.Tissue light transparent technology passes through
Hypertonic, high refractive index chemical reagent is introduced into tissue, index matching degree between each ingredient, reduces tissue in raising tissue
Scattering, become transparent " tissue, thus the microscopical imaging depth of improving optical.In recent years, the transparent development of tissue light
The concern and participation for having attracted numerous neuroscientists constantly have new light clearing method to be suggested.Wherein based on water-soluble examination
Favor of the method for agent with its effective control to the preferable holding of fluorescence signal and to sample deformation, by life scholar.So
And the transparent speed of method based on water-soluble reagent is slow, the time for generally require several weeks implements transparency of organization even several months,
And transparent effect is limited.This disadvantage significantly limits application of the current light clearing method in life science.
Summary of the invention
In view of the above-mentioned drawbacks in the prior art, the main purpose of the present invention is to provide a kind of based on water-soluble reagent
The method and its application of rapid tissue transparence, Various Tissues after processing can become highly transparent in a short time, can be with
For organizing in integral fluorescence micro-imaging, realize to the three-dimensional high definition fluorescence of full cranial nerve net and organ blood vessel web frame at
Picture.
In order to achieve the above object, it is transparent to provide a kind of rapid tissue based on water-soluble reagent for first aspect present invention
Change method, the treating method comprises following steps:
S1, by the tissue fixed through paraformaldehyde be put into tissue bulking inorganic agent sufficiently impregnate make its loose expansion,
Remove lipid；
S2, it will organize to be put into tissue contracts agent obtained by step S1 to handle, it made to shrink back reset condition by swelling state,
And obtain preliminary transparent effect；
S3, tissue obtained by step S2 is put into refractive index matched agent and is impregnated, it is saturating to obtain the relatively uniform height of refractive index
The tissue of brightization.
Preferably, in step S1, the component of the tissue bulking inorganic agent includes m-xylene diamine and sorbierite, remaining is
Water.Double NH that m-xylene diamine carries3With tissue aquation can occur for group, and fine and close fiber in disintegrated tissue dissolves rouge
Matter makes the rapid water swelling of tissue, to reduce the difference of each ingredient refractive index of tissue, speed up processing.Sorbierite is then
Good institutional framework retention agent avoids institutional framework and some albumen from being destroyed.
As a further preference, in step S1, the quality of m-xylene diamine, sorbierite in the tissue bulking inorganic agent
Concentration is respectively 5%-20%, 5%-15%.
Preferably, in step S2, the component of the tissue contracts agent includes m-xylene diamine, sorbierite and phosphate-buffered
Liquid.
As a further preference, in step S2, the mass concentration of m-xylene diamine, sorbierite in the tissue contracts agent
Respectively 20%-40%, 15%-30%, the concentration of the phosphate buffer are 0.01M- 0.05M.Promote isophthalic diformazan
Amine, sorbierite concentration can be to the organizing and implementing fast transparent after expanded, and the tissue for controlling expansion bounces back, together
When phosphate buffer introducing further increase the osmotic pressure of solution, shrink tissue to initial form.
Preferably, in step S3, the component of the refractive index matched agent includes m-xylene diamine and sorbierite, remaining is
Water.
As a further preference, in step S3, m-xylene diamine, the quality of sorbierite are dense in the refractive index matched agent
Degree is respectively 40%-60%, 35%-50%.The maximized concentration for promoting m-xylene diamine and sorbierite, it is high to obtain refractive index
In 1.50 aqueous solution, so that tissue is finally reached the relatively uniform degree of refractive index, transparency of organizationization processing is completed.
Preferably, the tissue includes brain section, Mouse Whole Brain, mouse heart, liver, kidney, the lung of mouse 1-2mm thickness
Dirty, spleen, stomach, intestines or mice embryonic.
As a further preference, in step S1, the time of the immersion is respectively as follows: 1-2mm thickness according to organization type
Brain section 0.5h-6h, Mouse Whole Brain 24-36h, mouse heart 12-24h, liver 24- 36h, kidney 12-24h, lungs 12-
For 24 hours, spleen 12-24h, stomach 6-12h, intestines 3-6h, mice embryonic 4- 8h；
In step S2, the time of the processing is respectively as follows: the brain section 0.5h-3h of 1-2mm thickness, mouse according to organization type
Full brain 8-12h, mouse heart 4-8h, liver 8-12h, kidney 8-12h, lungs 8- 12h, spleen 4-8h, stomach 4-8h, intestines 1-2h,
Mice embryonic 1-2h；
In step S3, the time of the immersion is respectively as follows: the brain section 0.5h-3h of 1-2mm thickness, mouse according to organization type
Full brain 8-12h, mouse heart 4-8h, liver 8-12h, kidney 8-12h, lungs 8- 12h, spleen 4-8h, stomach 4-8h, intestines 1-2h,
Mice embryonic 1-2h.
Preferably, in step S1, the fixation is that tissue is fixed using 4% paraformaldehyde.
Second aspect of the present invention provides a kind of application of rapid tissue transparence method based on water-soluble reagent, will be upper
The processed tissue of processing method is stated for being copolymerized burnt, two-photon or mating plate fluorescent microscopic imaging.
The beneficial effects of the present invention are:
(1) tissue fixed through paraformaldehyde first is put into impregnate in tissue bulking inorganic agent and go by processing method of the present invention
Rouge obtains the tissue of loose expansion；Then expanding tissue is put into tissue contracts agent, shrinks back it by swelling state original
State, and obtain preliminary transparent effect；Preliminary transparent tissue is put into refractive index matched agent again to impregnate, is finally obtained
The tissue of the relatively uniform highly transparent of refractive index.
(2) tissue bulking inorganic agent, tissue contracts agent and refractive index matched agent are selected in processing method of the present invention, are to pass through
The chemical reagent of strong aquation is introduced into tissue, so that rapidly feltwork in open texture, removes high refractive index
Lipid then introduces high refractive index reagent and uniforms organization internal refractive index, reduces organization internal scattering, make its change
Highly transparent, greatly improve imaging depth, and by control reagent concentration can guarantee it is transparent after sample without significant shape
Become.
In conclusion processing method of the present invention has the advantages that (i) simple and stable；(ii) sufficiently transparent, use mating plate
Lighting fluorescent microscope may be implemented to carry out three-dimensional high definition imaging to full cranial nerve net and blood vessel web frame；(iii) transparent
Time is short, transparent speed is fast；(iiii) without significant deformation.
Detailed description of the invention
Fig. 1 is that present invention is implemented as the flow diagrams of year mouse tissue transparency process method.
Fig. 2 a-2b is isolated mouse 2mm brain piece through visual image captured by light transparent processing front and back.
Fig. 2 c is transmittance curve of the brain section through transparent front and back of statistics.
Fig. 3 a-3b is captured cortex cone neurone fluorescence signal comparison diagram before and after light transparent processing, and Fig. 3 a is
Before light transparent processing, Fig. 3 b is after light transparent processing.
Fig. 3 c is fluorescence imaging depth map of the captured brain No microglial before and after light transparent processing.
Fig. 4 a-4b gives each organ of isolated mouse through visual image captured by light transparent processing front and back.
Fig. 5 gives the Mouse Whole Brain after the present invention is transparent and illuminates the full brain three-dimensional nerve distribution that imaging obtains through mating plate
Image.
Fig. 6 gives the mouse liver after the present invention is transparent and illuminates the whole three-dimensional vascular distribution that imaging obtains through mating plate
Image.
Specific embodiment
The rapid tissue transparence method and its application based on water-soluble reagent that invention broadly provides a kind of, success gram
Take that external existing smooth clearing method transparent rate is slow, transparent effect is poor, the defect of fluorescence poor compatibility.
In order to solve drawbacks described above, the main thought of the embodiment of the present invention is:
Rapid tissue transparence method of the embodiment of the present invention based on water-soluble reagent, the treating method comprises following step
It is rapid:
The tissue fixed through paraformaldehyde is put into tissue bulking inorganic agent sufficiently to impregnate and makes its loose expansion, is removed
Lipid；
The tissue that tissue bulking inorganic agent sufficiently impregnated is put into tissue contracts agent, shrinks back it by swelling state
Reset condition, and obtain preliminary transparent effect；
The tissue sufficiently handled in tissue contracts agent is put into refractive index matched agent to impregnate, finally obtains refractive index
The tissue of relatively uniform highly transparent；
The processed tissue of the processing method is used to be copolymerized in burnt and mating plate fluorescent microscopic imaging；It can greatly promote glimmering
Light imaging depth is realized and the three-dimensional high definition of Mouse Whole Brain nerve net and organ blood vessel web frame is imaged.
After processing method of the embodiment of the present invention handles mouse tissue, tissue can be made to become highly transparent in a short time,
To effectively enhance the penetration depth of light in biological tissues, completely new solution is provided to implement to organize greatly whole three-dimensional fluorescence to be imaged
Certainly scheme.And method simple and stable clearing time is short, transparent speed is fast, and without significant deformation.
In order to which above and other purpose, feature and the advantage of the present invention can be clearer and more comprehensible, several implementations are cited below particularly
Example, to illustrate the rapid tissue transparence method and its application based on water-soluble reagent of the present invention.
Embodiment 1
Biological tissue of the embodiment of the present invention is derived from the brain tissue of C57 mouse, the slice of its 2mm is passed through the embodiment of the present invention
1 transparent processing method is handled, and is specifically comprised the following steps:
Slice is immersed in tissue bulking inorganic agent (m-xylene diamine 10%, sorbierite 5%) first, when processing
Between be 4-6h；Slice is immersed in tissue contracts agent (m-xylene diamine 30%, sorbierite 20%, phosphate buffer again
0.03M), time 2-3h is handled；Slice is finally immersed in refractive index matched agent (m-xylene diamine 40%, sorbierite 40%)
In, handle time 1-2h；
Fig. 2 a-2b gives isolated mouse 2mm brain piece through visual image captured by light transparent processing front and back.Wherein Fig. 2 a
For the situation before transparent processing, due to the muddy characteristic of tissue, the line lattice of beneath tissue are completely covered；Fig. 2 b is that implementation light is saturating
Situation after the reason of daylight, tissue becomes highly transparent at this time, and the line lattice of beneath tissue are high-visible；Fig. 2 c is the brain section of statistics
Transmittance curve through transparent front and back chooses 3 groups of mm mouse brain brain piece tissues, to its light within the scope of wavelength 400nm-800nm
Transmitance measures statistics, it can be found that transmitance greatly increases in measurement wave-length coverage after transparent, shows in tissue
Portion's refractive index high degree of homogenous after transparent, scattering reduce.
Embodiment 2
Biological tissue of the embodiment of the present invention is derived from the brain tissue of Thy-1EGFP, Cx3cr1-EGFP mouse, by cutting for its 2mm
Piece is handled by 1 transparent processing method of the embodiment of the present invention, is specifically comprised the following steps:
Slice is immersed in tissue bulking inorganic agent (m-xylene diamine 10%, sorbierite 5%) first, when processing
Between be 4-6h；Slice is immersed in tissue contracts agent (m-xylene diamine 30%, sorbierite 20%, phosphate buffer again
0.05M), time 2-3h is handled；Slice is finally immersed in refractive index matched agent (m-xylene diamine 40%, sorbierite
40%) time 1-2h, is handled；By the 2mm brain section of transparent completion be immersed in refractive index matched agent carry out confocal fluorescent at
Picture.
Fig. 3 a-3c gives mouse brain slice through cortex cone captured by the smooth transparent processing of the embodiment of the present invention 2 front and back
Neuron and the fluorescent image of microglia.Wherein Fig. 3 a-3b is captured cortex cone neurone in light transparent processing
Front and back fluorescence signal comparison diagram, Fig. 3 a is before light transparent processing, Fig. 3 b is after light transparent processing；Fig. 3 c is small in captured brain
Fluorescence imaging depth map of the spongiocyte before and after light transparent processing.The result shows that the transparent processing to tissue does not have an impact
The fluorescence signal of the green fluorescent protein of neuron expression, and the brain tissue slice after transparent is under Laser Scanning Confocal Microscope
Fluorescence imaging depth greatly increase.
Embodiment 3
Biological tissue of the embodiment of the present invention is derived from the various organs of C57 mouse, and entire organ is passed through the embodiment of the present invention 3
Transparent processing method is handled, and is specifically comprised the following steps:
Various organs are immersed in tissue bulking inorganic agent (m-xylene diamine 20%, sorbierite 10%) first, are impregnated
Time is respectively as follows: Mouse Whole Brain 24-36h, mouse heart 12-24h, liver 24-36h, kidney 12-24h, lung according to organization type
Dirty 12-24h, spleen 12-24h, stomach 6-12h；Each organ is immersed in tissue contracts agent (m-xylene diamine 40%, sorb again
Alcohol 30%, phosphate buffer 0.01M), soaking time is respectively as follows: Mouse Whole Brain 8-12h, mouse heart 4- according to organization type
8h, liver 8-12h, kidney 8- 12h, lungs 8-12h, spleen 4-8h, stomach 4-8h；Each organ is finally immersed in refractive index
In ingredients (m-xylene diamine 50%, sorbierite 40%), Mouse Whole Brain 8-12h, mouse heart 4-8h, liver 8-12h, kidney 8-
12h, lungs 8-12h, spleen 4-8h, stomach 4-8h；
Fig. 4 a-4b gives each organ of isolated mouse through visual image captured by light transparent processing front and back.Wherein Fig. 4 a
For the situation before transparent processing, due to the muddy characteristic of tissue, the line lattice of beneath tissue are completely covered；Fig. 4 b is that implementation light is saturating
Situation after the reason of daylight, organ becomes highly transparent at this time, and the line lattice of beneath tissue are high-visible；
Embodiment 4
Biological tissue of the embodiment of the present invention is derived from the full brain of Thy-1EGFP mouse, and Mouse Whole Brain is implemented by the present invention
4 transparent processing method of example is handled, and is specifically comprised the following steps:
Full brain is immersed in tissue bulking inorganic agent (m-xylene diamine 20%, sorbierite 10%) first, handles the time
For 24-36h；Full brain is immersed in tissue contracts agent (m-xylene diamine 40%, sorbierite 30%, phosphate buffer again
0.02M), the processing time is Mouse Whole Brain 8-12h；Full brain is finally immersed in refractive index matched agent (m-xylene diamine 50%, mountain
Pears alcohol 40%) in, the processing time is 8-12h.The full brain sample of transparent completion is immersed in refractive index matched agent and carries out mating plate
Lighting fluorescent imaging.
Fig. 5 gives the Mouse Whole Brain after the present invention is transparent and illuminates the full brain three-dimensional nerve distribution that imaging obtains through mating plate
Image.
Embodiment 5
Biological tissue of the embodiment of the present invention is derived from the liver organization of C57 mouse, is marked by tail vein injection dextran
One leaf liver is handled by 5 transparent processing method of the embodiment of the present invention, is specifically comprised the following steps: by blood vessel structure
Liver is immersed in tissue bulking inorganic agent (m-xylene diamine 20%, sorbierite 10%) first, handles the time
For 24-36h；Liver is immersed in tissue contracts agent (m-xylene diamine 40%, sorbierite 30%, phosphate buffer again
0.04M), the processing time is Mouse Whole Brain 8-12h；Liver is finally immersed in refractive index matched agent (m-xylene diamine 50%, mountain
Pears alcohol 40%) in, the processing time is 8-12h.The liver samples of transparent completion are immersed in refractive index matched agent and carry out mating plate
Lighting fluorescent imaging.
Fig. 6 gives the mouse liver after the present invention is transparent and illuminates the whole three-dimensional vascular distribution that imaging obtains through mating plate
Image.
Technical solution in above-mentioned the embodiment of the present application, at least have the following technical effects or advantages:
(1) tissue fixed through paraformaldehyde will be first put into tissue bulking inorganic agent sufficiently by processing method of the present invention
Immersion makes its loose expansion, removes lipid；The tissue that tissue bulking inorganic agent sufficiently impregnated is put into tissue contracts agent again,
So that it is shunk back reset condition by swelling state, and obtains preliminary transparent effect；
The tissue sufficiently handled in tissue contracts agent is finally put into refractive index matched agent to impregnate, finally obtains folding
Penetrate the tissue of the relatively uniform highly transparent of rate；Method stabilization easy to operate, clearing time are short, transparent speed is fast, and without aobvious
Write deformation.
(2) refractive index of tissue bulking inorganic agent, tissue contracts agent and high refractive index is selected in processing method of the present invention
Ingredients can make rapidly organization internal refractive index uniform, and reduce organization internal scattering, it is made to become highly transparent, will be described
The processed tissue of processing method can greatly promote fluorescence imaging depth, in fact for being copolymerized in burnt and mating plate fluorescent microscopic imaging
Now the three-dimensional high definition of Mouse Whole Brain nerve net and organ blood vessel web frame is imaged.
In conclusion processing method of the present invention has the advantages that (i) simple and stable；(ii) sufficiently transparent, it can be achieved that right
The imaging of the three-dimensional high definition of Mouse Whole Brain nerve net and organ blood vessel web frame；(iii) clearing time is short, transparent speed is fast；
(iiii) without significant deformation.
Although preferred embodiments of the present invention have been described, it is created once a person skilled in the art knows basic
Property concept, then additional changes and modifications may be made to these embodiments.So it includes excellent that the following claims are intended to be interpreted as
It selects embodiment and falls into all change and modification of the scope of the invention.Obviously, those skilled in the art can be to the present invention
Carry out various modification and variations without departing from the spirit and scope of the present invention.If in this way, these modifications and changes of the present invention
Within the scope of the claims of the present invention and its equivalent technology, then the present invention is also intended to encompass these modification and variations and exists
It is interior.
Claims (10)
1. a kind of processing method of the rapid tissue transparence based on water-soluble reagent, it is characterised in that: the processing method packet
Include following steps:
S1, by the tissue fixed through paraformaldehyde be put into tissue bulking inorganic agent sufficiently impregnate make its loose expansion, remove
Lipid；
S2, it will organize to be put into tissue contracts agent obtained by step S1 to handle, so that it is shunk back reset condition by swelling state, and obtain
Obtain transparent effect tentatively；
S3, tissue obtained by step S2 is put into refractive index matched agent and is impregnated, obtain the relatively uniform highly transparent of refractive index
Tissue.
2. the rapid tissue transparency process method according to claim 1 based on water-soluble reagent, it is characterised in that: step
In rapid S1, the component of the tissue bulking inorganic agent includes m-xylene diamine and sorbierite, remaining is water.
3. the processing method of the rapid tissue transparence according to claim 2 based on water-soluble reagent, it is characterised in that:
In step S1, m-xylene diamine in the tissue bulking inorganic agent, sorbierite mass concentration be respectively 5%-20%, 5%-
15%.
4. the processing method of the rapid tissue transparence according to claim 1 based on water-soluble reagent, it is characterised in that:
In step S2, the component of the tissue contracts agent includes m-xylene diamine, sorbierite and phosphate buffer.
5. the processing method of the rapid tissue transparence according to claim 4 based on water-soluble reagent, it is characterised in that:
In step S2, m-xylene diamine in the tissue contracts agent, sorbierite mass concentration be respectively 20%-40%, 15%-
30%, the concentration of the phosphate buffer is 0.01M-0.05M.
6. the processing method of the rapid tissue transparence according to claim 1 based on water-soluble reagent, it is characterised in that:
In step S3, the component of the refractive index matched agent includes m-xylene diamine and sorbierite, remaining is water.
7. the processing method of the rapid tissue transparence according to claim 6 based on water-soluble reagent, it is characterised in that:
In step S3, m-xylene diamine in the refractive index matched agent, sorbierite mass concentration be respectively 40%-60%, 35%-
50%.
8. the processing method of the rapid tissue transparence according to claim 1 based on water-soluble reagent, it is characterised in that:
The tissue includes brain section, Mouse Whole Brain, mouse heart, liver, kidney, lungs, spleen, stomach, the intestines of mouse 1-2mm thickness,
Or mice embryonic.
9. the processing method of the rapid tissue transparence according to claim 8 based on water-soluble reagent, it is characterised in that:
In step S1, the time of the immersion is respectively as follows: the brain section 0.5h-6h of 1-2mm thickness, Mouse Whole Brain 24- according to organization type
36h, mouse heart 12-24h, liver 24-36h, kidney 12-24h, lungs 12-24h, spleen 12-24h, stomach 6-12h, intestines 3-
6h, mice embryonic 4-8h；
In step S2, the time of the processing is respectively as follows: the brain section 0.5h-3h of 1-2mm thickness, Mouse Whole Brain according to organization type
8-12h, mouse heart 4-8h, liver 8-12h, kidney 8-12h, lungs 8-12h, spleen 4-8h, stomach 4-8h, intestines 1-2h, mouse
Embryo 1-2h；
In step S3, the time of the immersion is respectively as follows: the brain section 0.5h-3h of 1-2mm thickness, Mouse Whole Brain according to organization type
8-12h, mouse heart 4-8h, liver 8-12h, kidney 8-12h, lungs 8-12h, spleen 4-8h, stomach 4-8h, intestines 1-2h, mouse
Embryo 1-2h.
10. the processing method of such as described in any item rapid tissue transparences based on water-soluble reagent of claim 1-9 is answered
With, it is characterised in that: the processed tissue of the processing method is used for co-focusing imaging, two photon imaging or mating plate fluorescence and is shown
In micro- imaging.
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