CN107132101A - One kind tissue light clarifier and its preparation method and application - Google Patents
One kind tissue light clarifier and its preparation method and application Download PDFInfo
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- CN107132101A CN107132101A CN201710193040.6A CN201710193040A CN107132101A CN 107132101 A CN107132101 A CN 107132101A CN 201710193040 A CN201710193040 A CN 201710193040A CN 107132101 A CN107132101 A CN 107132101A
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- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- ZHNUHDYFZUAESO-UHFFFAOYSA-N Formamide Chemical compound NC=O ZHNUHDYFZUAESO-UHFFFAOYSA-N 0.000 claims abstract description 56
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims abstract description 29
- 239000004202 carbamide Substances 0.000 claims abstract description 29
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 26
- 230000008520 organization Effects 0.000 claims abstract description 16
- 238000003384 imaging method Methods 0.000 claims abstract description 11
- 210000001519 tissue Anatomy 0.000 claims description 130
- 210000005013 brain tissue Anatomy 0.000 claims description 24
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 claims description 21
- 238000000034 method Methods 0.000 claims description 14
- 238000010438 heat treatment Methods 0.000 claims description 10
- 210000004185 liver Anatomy 0.000 claims description 7
- 210000003734 kidney Anatomy 0.000 claims description 6
- 238000007654 immersion Methods 0.000 claims description 5
- 210000005252 bulbus oculi Anatomy 0.000 claims description 4
- 210000003205 muscle Anatomy 0.000 claims description 4
- 230000000968 intestinal effect Effects 0.000 claims description 2
- 210000001161 mammalian embryo Anatomy 0.000 claims description 2
- 210000001672 ovary Anatomy 0.000 claims description 2
- 210000003491 skin Anatomy 0.000 claims description 2
- NSOXQYCFHDMMGV-UHFFFAOYSA-N Tetrakis(2-hydroxypropyl)ethylenediamine Chemical compound CC(O)CN(CC(C)O)CCN(CC(C)O)CC(C)O NSOXQYCFHDMMGV-UHFFFAOYSA-N 0.000 abstract 1
- 238000005516 engineering process Methods 0.000 description 14
- 230000000694 effects Effects 0.000 description 12
- 210000004556 brain Anatomy 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 241000699666 Mus <mouse, genus> Species 0.000 description 8
- 241000699670 Mus sp. Species 0.000 description 7
- 239000003153 chemical reaction reagent Substances 0.000 description 7
- 210000004072 lung Anatomy 0.000 description 7
- 239000008280 blood Substances 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 230000003287 optical effect Effects 0.000 description 5
- 239000008055 phosphate buffer solution Substances 0.000 description 5
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 5
- 229930040373 Paraformaldehyde Natural products 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 229920002866 paraformaldehyde Polymers 0.000 description 4
- 230000010412 perfusion Effects 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000000523 sample Substances 0.000 description 4
- 239000012472 biological sample Substances 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 210000000952 spleen Anatomy 0.000 description 3
- QURLONWWPWCPIC-UHFFFAOYSA-N 2-(2-aminoethoxy)ethanol;3,6-dichloro-2-methoxybenzoic acid Chemical compound NCCOCCO.COC1=C(Cl)C=CC(Cl)=C1C(O)=O QURLONWWPWCPIC-UHFFFAOYSA-N 0.000 description 2
- 206010002091 Anaesthesia Diseases 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
- 229930091371 Fructose Natural products 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- 230000037005 anaesthesia Effects 0.000 description 2
- 230000037396 body weight Effects 0.000 description 2
- 210000000170 cell membrane Anatomy 0.000 description 2
- 230000002490 cerebral effect Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 210000005240 left ventricle Anatomy 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 210000002784 stomach Anatomy 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 241000446313 Lamella Species 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000007844 bleaching agent Substances 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 150000004985 diamines Chemical class 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000003722 extracellular fluid Anatomy 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- -1 formyl Amine Chemical class 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 239000000017 hydrogel Substances 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 238000007912 intraperitoneal administration Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000005036 nerve Anatomy 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000003463 organelle Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000003204 osmotic effect Effects 0.000 description 1
- 230000008823 permeabilization Effects 0.000 description 1
- 229910052698 phosphorus Inorganic materials 0.000 description 1
- 239000011574 phosphorus Substances 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000010410 reperfusion Effects 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 210000000115 thoracic cavity Anatomy 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
- 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
- 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
- G01N2001/302—Stain compositions
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- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Investigating Or Analysing Biological Materials (AREA)
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Abstract
Light clarifier is organized the invention provides one kind, the tissue light clarifier includes each component of following mass fraction：Urea：15 25%, formamide：8 15%, Qula is led to：10 20%, N, N, N', N' tetra- (2 hydroxypropyl) ethylenediamine：20 25%, surplus is water.The tissue light clarifier energy short time effectively reduces biological tissue scatters, realize transparency of organization, the integrality of biological tissue can also be kept well, volume is maintained to be basically unchanged, do not expand or shrink, tissue will not become brittle, imaging depth and signal to noise ratio is greatly improved, and endogenous fluorescence signal intensity is substantially unaffected.Present invention also offers the preparation method and application of the tissue light clarifier.
Description
Technical field
The present invention relates to biomedical and transparent sample preparing technical field, and in particular to one kind tissue light clarifier and its
Preparation method and application.
Background technology
With continuing to develop for optical image technology, the structure function information of high-resolution acquisition biological tissue turns into can
Can, but the opacity of biological tissue constrains application of the optical image technology in biomedical sector.
The tissue light transparent technology of rising in recent years causes organ to cut into slices, so that it may carries out observation in the intact state and grinds
Study carefully, the core of the technology is that the chemical reagent that hypertonic, high refraction is introduced into biological tissue can effectively reduce biological tissue
Scattering properties, makes sample bleach, is greatly enhanced the depth of imaging.The transparency of organization technology used in the prior art includes
BABB technologies, Scale technologies, SeeDB technologies, CLARITY technologies etc., wherein BABB technologies use group after organic solvent, processing
Though it is good to knit transparent effect, when tissue catastrophic collapse, and causes fluorescent label signal in tissue to be quenched；Scale technologies are by life
Thing tissue is persistently immersed in urea liquid, and this method makes the time of transparency of organization needs longer, can also cause tissue seriously swollen
It is swollen；SeeDB technologies, though tissue morphology keeps good, are organized normal using the ever-increasing fructose soln immersion tissue of concentration
It is changed into brown, and fructose soln is sticky, operation inconvenience；CLARITY technologies remove the fat for causing scattering in tissue using electrophoresis
Class material is achieved transparent, and the transparency of organization effect of this method very well, but first need to fix group in advance using special hydrogel
Knit, and fat melting can cause cell membrane disruption, tissue native texture to disappear.
Tissue light transparent scheme reported above more or less can all change the form of sample, histoclastic native texture
Structure, is destroyed optical signalling, influences imaging results.
The content of the invention
In view of this, light clarifier is organized the invention provides one kind, it effectively can reduce biological tissue scatters the short time,
Realize transparency of organization, moreover it is possible to keep the integrality of biological tissue well, maintain volume to be basically unchanged, make imaging depth and letter
Make an uproar than being greatly improved, and endogenous fluorescence signal intensity is substantially unaffected.
In a first aspect, organizing light clarifier the invention provides one kind, the tissue light clarifier includes following quality point
Several each components：
Urea：15-25%, formamide：8-15%, Qula is led to：10-20%, N, N, N', N'- tetra- (2- hydroxypropyls) second
Diamines：20-25%, surplus is water.
The formula of the tissue light clarifier provided in the present invention is rationally efficient, wherein, urea has hydration, can
The high protein of the refractive index of close fold domain into biological tissue, produces osmotic gradient to allow moisture content to enter, but hold
Easily cause tissue bulking, but the addition of formamide, tissue bulking can be reduced well；Qula is logical can to play permeabilization so that
Solution can enter tissue and space between cells；N, N, N', N'- tetra- (2- hydroxypropyls) ethylenediamine effectively can occupy biological tissue
Space structure is stabilized while the water binding site on surface, more than under the synergy of 4 kinds of main components, life is enabled to
The refractive index of each constituent (collagenous fibres, cell membrane, organelle etc. and interstitial fluid) of thing tissue matches, and is quickly having
Effect ground reduction biological tissue scatters, while tissue is become transparent, moreover it is possible to preferably control biological tissue's volume to be basically unchanged,
Avoid expanding or shrink, tissue will not become brittle, the space structure of biological tissue can be maintained to greatest extent so that into
As depth and quality are greatly improved, and make endogenous optical strength unaffected.
The tissue light clarifier is applied to the transparence of the biological tissue rich in collagenous fibres such as brain, eyeball, muscle,
To biological tissue's deep structure imaging to rebuild 3D institutional frameworks, its structure function is studied significant.
Preferably, in the tissue light clarifier, the logical mass fraction of Qula is 15-20%.
Preferably, in the tissue light clarifier, the mass fraction of formamide is 8-12%.Further it is chosen as 8-
10%.
Further, in the tissue light clarifier, the mass ratio of the urea and formamide is (1.2-2.5)：1.It is excellent
Elect as (1.3-2.0)：1.
In one embodiment of the invention, the tissue light clarifier includes each component of following mass fraction：Urea：
25%, formamide：10%, Qula is led to：20%, N, N, N', N'- tetra- (2- hydroxypropyls) ethylenediamine：10%, surplus is water.
In another embodiment of the present invention, the tissue light clarifier includes each component of following mass fraction：Urea：
20%, formamide：15%, Qula is led to：10%, N, N, N', N'- tetra- (2- hydroxypropyls) ethylenediamine：20%, surplus is water.
In another embodiment of the present invention, the tissue light clarifier includes each component of following mass fraction：Urea：
15%, formamide：8%, Qula is led to：15%, N, N, N', N'- tetra- (2- hydroxypropyls) ethylenediamine：20%, surplus is water.
In another embodiment of the present invention, the tissue light clarifier includes each component of following mass fraction：Urea：
25%, formamide：12%, Qula is led to：20%, N, N, N', N'- tetra- (2- hydroxypropyls) ethylenediamine：15%, surplus is water.
Second aspect, the invention provides a kind of preparation method of tissue light clarifier, comprises the following steps：
By urea, formamide, Qula be logical, N, N, N', N'- tetra- (2- hydroxypropyls) ethylenediamine is mixed with water, and in 35-47
Dissolved by heating at DEG C, until solution clear, that is, obtain the tissue light clarifier, wherein, in the tissue light clarifier
The mass fraction of each component is：Urea：15-25%, formamide：8-15%, Qula is led to：(the 2- of 10-20%, N, N, N', N'- tetra-
Hydroxypropyl) ethylenediamine：20-25%, surplus is water.
During the tissue light clarifier is prepared, the temperature of heating for dissolving can not be too high, in order to avoid urea and formyl
Amine is decomposed.Preferably, the temperature of the heating for dissolving is 35-40 DEG C.
In the embodiment of the present invention, each component of tissue light clarifier first can be added to the water together, reheat dissolving；
The urea of powder can be added to the water after heating for dissolving, then be proportionally added into other solution compositions, mixing.The present invention the
The preparation method for the tissue light clarifier that two aspects are provided is simple, with low cost.
The third aspect, the invention provides described in a kind of tissue light clarifier or second aspect as described in relation to the first aspect
Organize application of the preparation method of light clarifier in transparency of organization processing and/or imaging of tissue.Wherein, it is described be organized as from
Body biological tissue.The tissue can be the biological tissue that brain, eyeball, muscle etc. are rich in collagenous fibres.
Transparent organism tissue after being handled through above-mentioned tissue light clarifier, can be used for various light microscopes it is biological into
Picture, such as wide field fluorescence microscope, laser lamella flying-spot microscope, laser confocal microscope, Two Photon Fluorescence, based on double light
Son excites multiphoton microscope of fluorescence and second harmonic etc..
Make the method for transparency of organization there is provided a kind of in an embodiment of the present invention, comprise the following steps：
(1) biological tissue is fixed and separated；
(2) above-mentioned gained biological tissue is immersed in the tissue light clarifier described in first aspect present invention, until raw
Thing tissue becomes transparent.
In one embodiment of the invention, after biological sample is handled through perfusion, remove blood, at low temperature using quality
Fraction takes out with after phosphate buffer solution (PBS) rinsing, is fixed to be fixed in 4% paraformaldehyde after 8-12 hours
Good biological sample, then to be soaked in tissue light clarifier.
Specifically, the step (1) is carried out using the method for heart perfusion：After intraperitoneal anesthesia, thoracic cavity, careful folder are opened
Go out and syringe needle for transfusion device is penetrated into left ventricle, perfusion physiological saline or PBS, right auricle of heart is cut off rapidly, rinse body circulation blood, treat blood
Reperfu- sion mass fraction is fixed 8-12 hours for 4% paraformaldehyde solution after the completion of liquid is rinsed.
Preferably, it is described to be organized as brain tissue, skin, eyeball, muscle, embryo, kidney, liver, spleen, lung, stomach, enteron aisle
Tissue, ovary tissue etc..
In the application, it can adjust used in appropriate scope according to the source of different biological tissues, age, body weight etc.
The ratio of each component in tissue light clarifier and the time of immersion.For example, the mouse for embryonic period, embryonic phase or being just born, its brain
The lipid components of tissue are in the majority, and moisture is very big, can suitably turn down urea and formamide ratio, time control was at 2-3 days
Transparent effect is can reach, and form size is held essentially constant.Now, it is adaptable to the full brain of embryonic period, embryonic phase or the mouse being just born
The formula of the clarifier of tissue is：Urea：15%, formamide：8%, Qula is led to：(the 2- hydroxyls third of 15%, N, N, N', N'- tetra-
Base) ethylenediamine：20%, surplus is water.
For another example the transparency of organization reagent soaks to complete tissues such as kidney, liver, spleen, lung, stomach, intestinal tissues
4-5 days are steeped with regard to good transparent effect can be reached.
Especially, when the biological tissue is kidney, liver, spleen, relative to lung tissue, its transparency process compared with
It is difficult, it is preferable that after biological tissue is placed in above-mentioned transparency of organization reagent, it is placed in carrying out in 35-47 DEG C of water-bath transparent
Processing, can so improve transparence speed and transparent effect.
In an embodiment of the present invention, when it is described be organized as biological tissue's thin slice when, time of the immersion is 5-8min.
It is observed that tissue becomes transparent, form size is held essentially constant.It is transparent suitable for the tissue light of the tissue slice
Agent, includes each component of following mass fraction：Urea：20%, formamide：10%, Qula is led to：(the 2- of 20%, N, N, N', N'- tetra-
Hydroxypropyl) ethylenediamine：20%, surplus is water.
Making in the method for transparency of organization for above-mentioned offer of the invention, it is described to organize light clarifier effectively to be reduced in the short time
Biological tissue scatters, realize transparency of organization, moreover it is possible to keep the integrality of biological tissue well, maintain biological tissue's volume base
This is constant, does not expand or shrinks, and tissue will not become brittle, not destroy in tissue collagen structure, can maximum limit
Degree ground maintains the space structure of biological tissue, depth and signal to noise ratio of imaging etc. is greatly improved, and make endogenous
Optical strength is unaffected.
Brief description of the drawings
Fig. 1 be C57/BL6 Mouse Whole Brains in transparent front and rear comparison diagram, wherein before the first behavior transparent processing, the second row
After transparent processing；
The comparison diagram that Fig. 2 cuts into slices before and after transparent 8min for the C57/BL6 Mice brain tissues of 500 μ m-thicks, wherein the first row
Before transparent processing, after the second behavior transparent processing；
Fig. 3 is the transparent front and rear two photon imaging figure (Z=405 μm) of Thy1-GFP Mice brain tissues section, and wherein A is
Bright before processing, B is after transparent processing.
Embodiment
As described below is the preferred embodiment of the present invention, it is noted that for those skilled in the art
For, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications are also considered as
Protection scope of the present invention.
Unless otherwise specified, each reagent used in the present invention is commercial reagents.
Wherein, Qula used is led to for Value 3608 (octylphenylpolyethylene
Glycol), its general entitled triton x-100 (triton X-100), can pass through conventional Reagent Company Alfa, Sigma etc.
Buy.It has following structural formula：
Wherein, n is 9-10 integer.
Embodiment 1
A kind of preparation method of tissue light clarifier, comprises the following steps：
By urea, formamide, triton x-100 (Sigma-Aldrich reagents), N, N, N', N'- tetra- (2- hydroxypropyls)
Ethylenediamine is mixed with water, and in being dissolved by heating at 35 DEG C, up to solution clear, that is, obtains the tissue light clarifier, its
In, the mass fraction of each component is in the tissue light clarifier：Urea：25%, formamide：10%, Qula is led to：20%, N,
N, N', N'- tetra- (2- hydroxypropyls) ethylenediamine：25%, water：20%.The tissue light clarifier is suitable for young mice brain tissue,
Transparent effect is preferable when transparent to whole brain tissue, and tissue morphology is held essentially constant, especially can be very fast to thin slice brain tissue
Preferable transparent effect is reached, transparency of organization can be realized in 5-8min.
Embodiment 2
A kind of preparation method of tissue light clarifier, comprises the following steps：
By urea, formamide, triton x-100, N, N, N', N'- tetra- (2- hydroxypropyls) ethylenediamine is mixed with water, and in
Dissolved by heating at 37 DEG C, until solution clear, that is, obtain the tissue light clarifier, wherein, the tissue light clarifier
The mass fraction of middle each component is：Urea：20%, formamide：15%, Qula is led to：(the 2- hydroxyls third of 10%, N, N, N', N'- tetra-
Base) ethylenediamine：20%, surplus is water.The tissue light clarifier is suitable for the higher tissue of young mice water content, such as lung group
Knit.Apply also for thin slice brain tissue.When transparent to thin slice brain tissue, transparent speed is relatively slow, but final transparent effect is still
It is so also more satisfactory.The transparency temperature of clarifier can be properly increased in transparent processing, transparent effect can be accelerated.
Embodiment 3
A kind of preparation method of tissue light clarifier, comprises the following steps：
By urea, formamide, triton x-100, N, N, N', N'- tetra- (2- hydroxypropyls) ethylenediamine is mixed with water, and in
Dissolved by heating at 35 DEG C, until solution clear, that is, obtain the tissue light clarifier, wherein, the tissue light clarifier
The mass fraction of middle each component is：Urea：25%, formamide：8%, Qula is led to：(the 2- hydroxyls third of 2015%, N, N, N', N'- tetra-
Base) ethylenediamine：15%, surplus is water.The tissue light clarifier is more suited to transparent, its global tissue to liver, nephridial tissue
Clearing time can be controlled in 3-4 days.
Embodiment 4
A kind of preparation method of tissue light clarifier, comprises the following steps：
By urea, formamide, triton x-100, N, N, N', N'- tetra- (2- hydroxypropyls) ethylenediamine is mixed with water, and in
Dissolved by heating at 37 DEG C, until solution clear, that is, obtain the tissue light clarifier, wherein, the tissue light clarifier
The mass fraction of middle each component is：Urea：25%, formamide：12%, Qula is led to：(the 2- hydroxyls third of 20%, N, N, N', N'- tetra-
Base) ethylenediamine：15%, surplus is water.The tissue light clarifier is equally applicable to the various tissues of previous examples, including brain, liver,
Lung, intestines, kidney.Transparent efficiency wherein to brain tissue does not have the effect of example 1 preferable, and the clearing time of brain tissue thin slice is needed
More than 20min.
Embodiment 5
The transparency process of Mice brain tissues, brain tissue thin slice, bag are carried out using the obtained tissue light clarifier of embodiment 1
Include following steps：
1. materials
Healthy male Thy1-GFP mouse and C57/BL6 mouse, the body weight about 10-15g of about 4 week old are taken, its abdominal cavity is noted
160-200 μ l arcotic (the Ethylurethanm aqueous solution of 2% chloraldurate+10%) is penetrated, after being fixed after mouse anesthesia.
2. fix
Heart perfusion：Above-mentioned tissue is carefully pressed from both sides out syringe needle for transfusion device is penetrated into left ventricle, irrigate the PBS of precooling, rapidly
Right auricle of heart is cut off, body circulation blood is rinsed；
After blood to be cleaned, after the paraformaldehyde solution that the mass fraction for continuing to irrigate precooling is 4% fixes 8-12 hours,
The tissue such as whole brain tissue, liver, lung, kidney needed for taking out, 4% paraformaldehyde continues 4 DEG C of fixations and stayed overnight, taking-up phosphorus before transparent experiment
After hydrochlorate cushioning liquid (PBS) rinsing, the biological sample (such as whole brain tissue, full lung tissue etc.) being fixed；
3. section：
For chip sample (such as brain tissue slice), using brain mould by the brain tissue fixed be cut into 1mm, 500 μm
Coronal thin slice, used for subsequent clear.
4th, transparence：The brain tissue or brain tissue slice of above-mentioned acquisition are immersed in the obtained tissue light of embodiment 1 transparent
In agent, until becoming transparent.
Fig. 1 is contrast of the C57/BL6 Mouse Whole Brains before and after the tissue light clarifier through embodiment 1 carries out transparency process
Figure.The tissue light clarifier of existing literature report all destroys cerebral morphology to a certain extent when carrying out transparent to full brain,
Or to become expansion frangible, or shrink, such as classical CUBIC and ScaleA2.It is of the invention then reaching the same of preferable transparent effect
When, the integrality of cerebral morphology can be kept well, and significant change does not occur for volume.
Fig. 2 is that the C57/BL6 Mice brain tissues section of 500 μ m-thicks is transparent in the tissue light clarifier progress through embodiment 1
Change the comparison diagram of 10min before and after the processing.Compared with existing other method (such as CUBIC and ScaleA2), the tissue of embodiment 1
It is fully transparent that light clarifier can enable the brain tissue slice of 500 μ m-thicks be realized in 5-8min, and preferably keeps biological tissue's body
Product is basically unchanged, it is to avoid is expanded or is shunk；And existing method is after transparency process 10min, Mice brain tissues section
Still it is not up to all-transparent.
Fig. 3 is the brain tissue slice of Thy1-GFP mouse before and after the same depth of same position (being specifically 405 μm) is transparent
Two photon imaging comparison diagram (wherein excitation wavelength be 920nm, a length of 360~540nm of received wave).
From figure 3, it can be seen that brain tissue slice is after fast transparent is realized, fluorescence at relatively deep 405 μm of degree into
As signal preservation well, imaging depth is deeper, image quality is higher, and tiny nerve is even more clear and legible.
Embodiment described above only expresses the several embodiments of the present invention, and it describes more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (10)
1. one kind tissue light clarifier, it is characterised in that the tissue light clarifier includes each component of following mass fraction：
Urea：15-25%, formamide：8-15%, Qula is led to：10-20%, N, N, N', N'- tetra- (2- hydroxypropyls) ethylenediamine：
20-25%, surplus is water.
2. light clarifier is organized as claimed in claim 1, it is characterised in that in the tissue light clarifier institute, formamide
Mass fraction is 8-12%.
3. tissue light clarifier as claimed in claim 1, it is characterised in that the mass ratio of the urea and formamide is
(1.2-2.5)：1.
4. light clarifier is organized as claimed in claim 1, it is characterised in that the tissue light clarifier includes following quality point
Several each components：Urea：25%, formamide：10%, Qula is led to：20%, N, N, N', N'- tetra- (2- hydroxypropyls) ethylenediamine：
10%, surplus is water.
5. a kind of preparation method of tissue light clarifier, it is characterised in that comprise the following steps：
By urea, formamide, Qula be logical, N, N, N', N'- tetra- (2- hydroxypropyls) ethylenediamine is mixed with water, and at 35-47 DEG C
Dissolve by heating, until solution clear, that is, obtain the tissue light clarifier, wherein, each group in the tissue light clarifier
Point mass fraction be：Urea：15-25%, formamide：8-15%, Qula is led to：(the 2- hydroxyls of 10-20%, N, N, N', N'- tetra-
Propyl group) ethylenediamine：20-25%, surplus is water.
6. the preparation method of tissue light clarifier as claimed in claim 5, it is characterised in that the temperature of the heating for dissolving is
30-38℃。
7. the tissue light clarifier as described in claim any one of 1-4 is in transparency of organization processing and/or imaging of tissue
Using.
8. a kind of make the method for transparency of organization, it is characterised in that comprises the following steps：
(1) biological tissue is fixed and separated；
(2) above-mentioned gained biological tissue is immersed in the tissue light clarifier as described in claim any one of 1-4, until raw
Thing tissue becomes transparent.
9. make the method for transparency of organization as claimed in claim 8, it is characterised in that described to be organized as brain tissue, skin, eye
Ball, muscle, embryo, kidney, liver, intestinal tissue or ovary tissue.
10. make the method for transparency of organization as claimed in claim 9, it is characterised in that when it is described be organized as complete tissue when, institute
The time for stating immersion is 4-5 days；When it is described be organized as thin sectioned tissue when, time of the immersion is 5-8min.
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| CN111610078A (en) * | 2020-07-03 | 2020-09-01 | 中国科学技术大学 | Reagent and method for transparentizing biological tissue |
| CN113324819A (en) * | 2021-04-19 | 2021-08-31 | 任政宇 | Composite accelerator for biological tissue transparentization and preparation method and application thereof |
| WO2023010845A1 (en) * | 2021-08-02 | 2023-02-09 | 西湖大学 | Degreasing composition for clearing biological tissue |
| CN113781848A (en) * | 2021-08-14 | 2021-12-10 | 西安电子科技大学 | Optical projection tomography device, system and method based on smart phone |
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