CN106872252B - A kind of resin embedding method of smooth transparence biological tissue - Google Patents
A kind of resin embedding method of smooth transparence biological tissue Download PDFInfo
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- CN106872252B CN106872252B CN201710209343.2A CN201710209343A CN106872252B CN 106872252 B CN106872252 B CN 106872252B CN 201710209343 A CN201710209343 A CN 201710209343A CN 106872252 B CN106872252 B CN 106872252B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/36—Embedding or analogous mounting of samples
- G01N2001/364—Embedding or analogous mounting of samples using resins, epoxy
Abstract
The invention discloses a kind of resin embedding methods of smooth transparence biological tissue.It is used by selection and there is tissue itself embedding medium of the refractive index to match to embed to tissue, to obtain the resin embedding tissue with certain transparency, wherein embedding medium is the polymer that initiator causes that polymerized monomer is polymerize and formed.The resin embedding method of light transparence biological tissue of the invention, on the basis of guaranteeing that resin itself provides solid state support, it is transparent that a degree of light is provided to biological tissue, make group be woven in carry out cutting imaging when, the depth of sample imaging increases, the enlarged in thickness of z-axis cutting, and then greatly shorten the time required for imaging of tissue.
Description
Technical field
The invention belongs to biomedical optical technical field of imaging, and murine brain can be wrapped by specifically designing one kind
It buries, while after polymerization (embedding) occurs for monomer, tissue keeps the resin embedding of the light transparence biological tissue of certain transparency
Method.
Background technique
With flourishing for Biomedical Photonics subject, Modern Optics Technology neural network knot in brain and tissue
The parsing of structure has come into the stage of rapid development.In brain imaging technique, by successively cut imaging can obtain it is high
Resolution ratio, while people have been obtained for the extremely fine full cranial nerve distribution map of mouse brain 3D.However since brain tissue is impermeable
Bright, light penetration depth is insufficient, and z-axis thickness of cutting is smaller, the disadvantage for causing image taking speed excessively slow.Study cranial nerve network structure
Ultimate purpose is in order to obtain the fine structure map of the cranial nerve of primate, and then to the relevant neurological disease of brain, brain
The fields such as activity guidance is provided.Currently, research topic produces a series of during being transitioned into primate monkey brain from mouse brain
New technical problem.Wherein, by mouse brain, (volume is about 1cm3) being transitioned into primate, (volume is about 103cm3) in intuitive volume
Variation leads to the total amount of imaging and sharply increasing for corresponding information amount.Complete mouse Brian Imaging needs to be more than year,
So if still continuing to use the imaging technique of mouse brain, then the time being imaged will become too long, and meaning is lost.It improves layer-by-layer
The speed of cutting technology imaging becomes extremely urgent.
By carrying out certain processing, the transparent biological sample of available light, so that the penetration capacity of light is big to tissue
Big reinforcement, and then greatly shorten the time required for imaging.However, these tissue light transparent technologies are almost without exception
It all concentrates in solution medium, therefore is not used to successively cut in imaging system.And traditional organization embedding technology, packet
It is often opaque because refractive index mismatches between the medium and tissue buried, so that its z-axis imaging depth is smaller, single imaging
Cutting thickness it is less than normal so that imaging time is too long.Light can be effectively reduced in group by means of transparency of organization technology
Light scattering in knitting, can increase the depth of z-axis imaging, and then shortens and the required time is imaged.If group can be woven in
After embedding, certain transparency is still maintained, then by the thickness of the depth and cutting that increase cutting imaging, and then directly
Shorten the time required for imaging of tissue, plays critical technical role for the imaging of the biggish primate monkey brain of volume.Together
When, temperature majority used in the resin embedding technology of current organization all could effectively cause monomer polymerization at 50 DEG C or more.
And fluorescin is at a higher temperature, and denaturation often occurs and makes fluorescent weakening even quenching completely, is unfavorable for being imaged.Cause
This, how to reduce the polymerization temperature that group is woven in embedding process also will preferably protect fluorescin, to improve the matter of imaging
Amount is another problem that tissue resin embedding needs to be concerned about.
Summary of the invention
Aiming at the above defects or improvement requirements of the prior art, the present invention provides a kind of trees of smooth transparence biological tissue
Rouge embedding method, its object is to use and organize itself to have the embedding medium of the refractive index to match to tissue by selection
It is embedded, to obtain the resin embedding tissue with certain transparency, thus solves the transparency of organization means of the prior art
It can only be carried out in solution medium, cutting imaging can not be suitable for；And traditional organization embedding technology can be suitable for cutting into
Picture, but it is opaque due to being organized after embedding, so that Z axis imaging depth is smaller when cutting imaging, the too long technology of imaging time
Problem.
To achieve the above object, according to one aspect of the present invention, a kind of resin of smooth transparence biological tissue is provided
Embedding method, the resin embedding of biological tissue is carried out using embedding medium, and the refractive index of the biological tissue and the embedding are situated between
The refractive index of matter matches, to realize the resin embedding of the biological tissue of light transparence, the embedding medium draws for initiator
The polymer sending out polymerized monomer polymerization and being formed.
Preferably, the polymerized monomer be benzyl methacrylate, 2- acrylic acid -2- methyl -2- phenoxyethyl acrylate and/
Or 2- acrylic acid -2- hydroxyl -3- phenoxy-propyl is one or more.
Preferably, the initiator is azobisisoheptonitrile.
Other side according to the invention provides resin embedding method described in one kind, includes the following steps:
(1) degreasing processing will be carried out using biological tissue of the chemical method after fixed, the biological tissue after obtaining degreasing；
(2) biological tissue after the degreasing of step (1) acquisition is soaked in polymerized monomer, initiator is then added, mixed
Conjunction dissolves the initiator sufficiently, then permeates 12~24 hours under the conditions of being protected from light at -20~0 DEG C, after being permeated
Biological tissue；
(3) biological tissue after the infiltration of step (2) acquisition is reacted 12~48 hours at 35~45 DEG C, it is saturating obtains light
Biological tissue's resin of brightization.
Preferably, step (1) described chemical method is fixed as being fixed using formalin.
Preferably, the specific steps of the processing of degreasingization described in step (1) are as follows: mixed in tetrahydrofuran-water of different gradients
It closes in liquid and carries out ungrease treatment.
Preferably, the amount ratio of step (2) polymerized monomer and the initiator is 1 milliliter: 1~6 milligram.
Preferably, step (3) reaction temperature is 40 DEG C.
In general, through the invention it is contemplated above technical scheme is compared with the prior art, can obtain down and show
Beneficial effect.
(1) the present invention provides a kind of resin embedding method that can carry out light transparence to biological tissue, it is guaranteeing
It is transparent to biological tissue's a degree of light of offer on the basis of resin itself provides solid state support, it is woven in group and is cut
When imaging, the depth of sample imaging increases, the enlarged in thickness of z-axis cutting, and then greatly shortens required for imaging of tissue
Time.
(2) polymerized monomer of the present invention by selection with specific refractive index and the initiation with specific initiation polymerization temperature
Agent is used cooperatively, and the initiator polymerization temperature of selection is lower, is carried out low temperature osmotic to biological tissue, is then caused under lower temperature
Polymerization, the embedding medium after polymerization has the refractive index to match with biological tissue, to realize the transparence of biological tissue
Embedding, simultaneously because polymerization temperature is lower, the fluorescence of fluorescin keeps good, therefore, biological tissue's transparence of the invention
Resin embedding method can not only realize the transparence of biological tissue, and can also ensure that the biological group in polymerization process
The fluorescence for the fluorescin knitted keeps good, is not quenched, and creates good condition for fluorescence imaging.
(3) transparent resin embedding method of the invention passes through meticulous selective polymerization monomer, initiator and preferred embedding
Condition, has finally obtained the overall technical architecture of a set of biological tissue's resin embedding and transparence, each parameter coordinated, finally
The transparent resin embedding of biological tissue is realized, biological tissue's fluorescence keeps good, and the biological tissue of transparence is applied to cut
It is whittled into as increasing thickness of cutting, shortens the time required for full Brian Imaging.
Detailed description of the invention
After Fig. 1 be mouse brain before tetrahydrofuran-water mixed solution and the methylene chloride processing of different gradients (a) and processing
(b) comparison diagram；
Fig. 2 is that common mouse brain is placed in (a) PBS solution and (b) using benzyl methacrylate monomer embedding (polymerization)
Effect picture afterwards；
Fig. 3 is that common mouse brain is placed in (a) PBS solution and (b) uses 2- acrylic acid -2- hydroxyl -3- phenoxy group propylene
Acid monomers embed the effect picture after (polymerization).
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, and
It is not used in the restriction present invention.As long as in addition, technical characteristic involved in the various embodiments of the present invention described below
Not constituting a conflict with each other can be combined with each other.
It can only be carried out in solution medium for the transparency of organization means of the prior art, cutting imaging can not be suitable for；And
Traditional organization embedding technology can be suitable for cutting imaging, but since investing tissue is opaque, so that Z axis when cutting imaging
The technical problem that imaging depth is small, imaging time is long, the present invention creatively provide a kind of tree of smooth transparence murine brain
Rouge embedding method carries out the resin embedding of biological tissue, the refractive index of biological tissue and the embedding medium using embedding medium
Refractive index match, to realize the resin embedding of the biological tissue of light transparence.
The selection of polymerized monomer is key point of the invention, the condition that the polymerized monomer met the requirements needs to have are as follows: should
Polymerized monomer is under the initiation of initiator, and in the system existing for biological tissue, the polymer formed after polymerization reaction occurs
Refractive index must match with the refractive index of biological tissue.Applicant of the present invention is in a large amount of infrastest and creative labor
After dynamic, benzyl methacrylate, 2- acrylic acid -2- methyl -2- phenoxyethyl acrylate and/or 2- acrylic acid -2- hydroxyl-are obtained
3- phenoxy-propyl be meet the present invention claims feasible polymerized monomer.Initiator is that can draw at a lower temperature
Send out the initiator of polymerization, such as azobisisoheptonitrile.
The resin embedding method of smooth transparence murine brain provided by the invention, specifically comprises the following steps:
(1) degreasing processing will be carried out using biological tissue of the chemical method after fixed, the biological tissue after obtaining degreasing；
Chemical method fixation can be to be fixed using formalin；Degreasingization processing can be in tetrahydrofuran-water of different gradients
It is carried out in mixed liquor.
(2) biological tissue after the degreasing of step (1) acquisition is soaked in polymerized monomer, initiator is then added, mixed
Conjunction dissolves the initiator sufficiently, then handles 12~24 hours, is permeated in -20~0 DEG C of infiltration under the conditions of being protected from light
Biological tissue afterwards；
Wherein, polymerized monomer be preferably benzyl methacrylate, 2- acrylic acid -2- methyl -2- phenoxyethyl acrylate and/or
One of 2- acrylic acid -2- hydroxyl -3- phenoxy-propyl is a variety of；Initiator is preferably azobisisoheptonitrile；Polymerized monomer
Amount ratio with the initiator is 1 milliliter: 1~6 milligram, i.e. 1~6 milligram of initiator of every milliliter of polymerized monomer.
(3) biological tissue after the infiltration of step (2) acquisition is carried out polymerization reaction 12~48 hours at 35~45 DEG C,
So that polymerized monomer carries out polymerization under the initiation of initiator and forms polymer, after wherein initiator causes polymerized monomer polymerization
The refractive index of polymer and the refractive index of the biological tissue match, and thus obtain the biology of the light transparence of different hardness
Tissue.
Under normal circumstances, polymeric reaction temperature is higher, and rate of polymerization is faster, and extent of polymerization is higher, but practical application is also wanted
Consider the irreversible breaking caused by fluorescin in tissue of excessively high temperature.The resin embedding of prior art progress biological tissue
Polymerization temperature is typically chosen at 50~70 DEG C, temperature is higher, is easy to shine into irreversible destruction to fluorescin and drop fluorescence
Low or quenching, is unfavorable for fluorescence imaging.For this problem, the present invention has selected azobisisoheptonitrile as initiator, and
The initiator effectively causes monomer in lower temperature (35~45 DEG C) and is polymerize.Therefore resin embedding side of the invention
Method can not only realize the transparence of biological tissue, and can also ensure that the fluorescence egg of the biological tissue in polymerization process
White fluorescence property keeps good, is not quenched or reduces quenching.
Key technology of the invention is that embedding medium matches with the refractive index of itself is organized.The refractive index of resin is excessively high
Or it is too low can all cause embedding biological tissue it is opaque, embedding medium be initiator cause polymerized monomer polymerize and shape
At polymer.The prior art carries out the light transparency process of biological tissue usually in solution medium, need to only find and biology
The transparence of biological tissue can be realized in the chemical reagent that tissue refractive index matches.However, solution medium is not used to cut
In fluorescence imaging.And the present invention is not the transparence biological tissue in solution medium, but for the characteristics of being imaged is cut, it finds
A kind of resin embedding method of smooth transparence biological tissue is imaged, in order to realize the life after resin embedding in order to which fluorescence is cut
The light transparence of object tissue, if selecting the reagent to match with Refractive Index of Biotissue as polymerized monomer, since polymerization is single
Refractive index can change after body polymerize under the initiation of initiator, therefore polymerization list is found according to the thinking of the prior art
Body and initiator be it is unworkable, the polymerized monomer and initiator that the present invention selects can guarantee the embedding formed after the polymerization
Medium and the refractive index of biological tissue itself match, therefore the final transparence embedding treatment for guaranteeing biological tissue, are applied to
Fluorescence imaging is cut, thickness of cutting increases, and imaging time greatly increases compared with the existing technology.
The following are embodiments:
Embodiment 1
A kind of resin embedding method of smooth transparence murine brain, includes the following steps:
(1) the mouse brain by formalin after fixed, is placed in tetrahydrofuran-water mixed liquid (content of tetrahydrofuran 50%)
12 hours；Then solution is replaced in the mixed solution that tetrahydrofuran ratio is 80% 1 hour；Continue replacement into pure THF 1
Hour, twice.Then by treated, mouse brain is placed in methylene chloride half an hour (such as Fig. 1)；
(2) monomer methacrylic acid benzyl ester is added dropwise and is getting out alkali alumina (200-300 mesh) column chromatographic column in advance
It on son, is flowed out under the pressurization of air pump, removes polymerization inhibitor therein；
(3) in the serum bottle that volume is 10ml, will treated mouse brain soaking in benzyl methacrylate monomer (2ml)
In (polymerized monomer), initiator azobisisoheptonitrile (4mg) then is added, is allowed to sufficiently dissolve.Then by bottle masking foil
Shading is wrapped up, is placed in save 24 hours in -10 DEG C and carries out infiltration processing, heating in 40 DEG C of baking ovens is transferred to and polymerize 1 day, can see
Polymerization occurs to monomer and becomes solid, when the refractive index of tissue and embedding medium matches, tissue becomes transparence.
After Fig. 1 be mouse brain before tetrahydrofuran-water mixed solution and the methylene chloride processing of different gradients (a) and processing
(b) comparison diagram；Mouse brain is after tetrahydrofuran-water and the methylene chloride processing of different gradients as can be seen from Figure 1, mouse brain
There is apparent variation in volume.
Fig. 2 is that treated mouse brain is soaked in polymerize in benzyl methacrylate monomer before and polymerization after comparison diagram.From
Fig. 2 can be seen that mouse brain can not be penetrated to handle preceding light；After embedding, the mouse brain in resin has fabulous transmitance to light.
The transparence mouse brain that the embodiment is prepared can greatly improve the depth of imaging, increase the thickness of cutting, and then shorten
Time required for full Brian Imaging.
Embodiment 2
2- acrylic acid -2- hydroxyl -3- phenoxy group acrylic acid is used to carry out transparent embedding as monomer.
(1) the mouse brain by formalin after fixed, is placed in tetrahydrofuran-water mixed liquid (content of tetrahydrofuran 50%)
12 hours；Then solution is replaced in the mixed solution that tetrahydrofuran ratio is 80% 1 hour；Continue replacement into pure THF 1
Hour, twice.Then by treated, mouse brain is placed in methylene chloride half an hour (such as Fig. 1)；
(2) monomer 2- acrylic acid -2- hydroxyl -3- phenoxy group acrylic acid is added dropwise and is getting out alkali alumina in advance
(200-300 mesh) column chromatographs on pillar, flows out under the pressurization of air pump, removes polymerization inhibitor therein；
(3) the mouse brain after transparent processing is placed in the serum bottle that capacity is 10ml.Will treated mouse brain soaking in 2- third
In olefin(e) acid -2- hydroxyl -3- phenoxy group acrylic monomers (2ml) (polymerized monomer), initiator azobisisoheptonitrile is then added
(4mg) is allowed to sufficiently dissolve.Then bottle masking foil is wrapped up into shading, is placed in save 24 hours in -10 DEG C and carries out at infiltration
Reason.It under water bath condition, is gradually warmed up from 20 DEG C (increasing extent of temperature is 5 DEG C), step heating to 25,30,35,40 DEG C, per stage dimension
Constant temperature 6h is held, continues to heat 6h later under the conditions of 40 DEG C.It can thus be seen that monomer occur polymerization and become solid, tissue and
When the refractive index of embedding medium matches, tissue becomes transparence, while mouse brain is from the impermeable of the milky white shape before polymerization
It is bright to become transparent, as shown in Figure 3.
In the transparent embedding techniques of mouse brain, key is the refractive index phase so that the medium embedded and tissue itself
Match.The refractive index of resin it is excessively high or it is too low can all cause embedding mouse brain it is opaque.Refractive index, which matches, refers to that refractive index is identical
Or it is close, the refractive index of the two is closer, and transparency is higher.Therefore, all design structure and thinking of the invention do one
A little simple designs for changing or changing fall within protection scope of the present invention.
As it will be easily appreciated by one skilled in the art that the foregoing is merely illustrative of the preferred embodiments of the present invention, not to
The limitation present invention, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should all include
Within protection scope of the present invention.
Claims (5)
1. a kind of resin embedding method of smooth transparence biological tissue, which is characterized in that carry out biological tissue using embedding medium
Resin embedding, the refractive index of the biological tissue and the refractive index of the embedding medium match, to realize light transparence
Biological tissue resin embedding, the embedding medium be initiator cause polymerized monomer polymerization and formed polymer；
The polymerized monomer is benzyl methacrylate, 2- acrylic acid -2- methyl -2- phenoxyethyl acrylate and/or 2- acrylic acid -
2- hydroxyl -3- phenoxy-propyl it is one or more；
The initiator is azobisisoheptonitrile；
The resin embedding method, includes the following steps:
(1) degreasing processing will be carried out using biological tissue of the chemical method after fixed, the biological tissue after obtaining degreasing；
(2) biological tissue after the degreasing of step (1) acquisition is soaked in polymerized monomer, initiator is then added, mixing makes
The initiator sufficiently dissolves, and then permeates 12~24 hours under the conditions of being protected from light at -20~0 DEG C, the biology after being permeated
Tissue；
(3) biological tissue after the infiltration of step (2) acquisition is reacted 12~48 hours at 35~45 DEG C, obtains resin embedding
Light transparence biological tissue.
2. resin embedding method as described in claim 1, which is characterized in that step (1) described chemical method is fixed as using
Formalin is fixed.
3. resin embedding method as described in claim 1, which is characterized in that degreasingization described in step (1) is handled specific
Step are as follows: carry out ungrease treatment in tetrahydrofuran-water mixed liquid of different gradients.
4. resin embedding method as described in claim 1, which is characterized in that step (2) polymerized monomer and the initiation
The amount ratio of agent is 1 milliliter: 1~6 milligram.
5. resin embedding method as described in claim 1, which is characterized in that step (3) reaction temperature is 40 DEG C.
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| WO2019009300A1 (en) * | 2017-07-06 | 2019-01-10 | 公立大学法人大阪府立大学 | Method for making biological tissue transparent and reagent for same |
| CN109445249A (en) | 2018-10-23 | 2019-03-08 | 武汉华星光电半导体显示技术有限公司 | The preparation method of Photosensitve resin composition, display equipment and Photosensitve resin composition |
| CN110068559B (en) * | 2019-04-08 | 2020-08-18 | 华中科技大学 | Biological tissue transparentizing imaging method |
| CN115728101A (en) * | 2021-09-01 | 2023-03-03 | 西湖大学 | Transparent expansion method and imaging method for biological tissue |
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