CN106568753B - A kind of fluorescence control method of organic fluorescent dye molecular labeling biological tissue - Google Patents
A kind of fluorescence control method of organic fluorescent dye molecular labeling biological tissue Download PDFInfo
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- CN106568753B CN106568753B CN201610964922.3A CN201610964922A CN106568753B CN 106568753 B CN106568753 B CN 106568753B CN 201610964922 A CN201610964922 A CN 201610964922A CN 106568753 B CN106568753 B CN 106568753B
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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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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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
- 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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- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
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- G—PHYSICS
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- 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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of fluorescence control methods of organic fluorescent dye molecular labeling biological tissue, form hexatomic ring in conjunction with organic fluorescent dye molecule using metal ion, the fluorescence of organic fluorescent dye molecule is quenched；Coordinate bond is formed using metal ion chelation agent and metal ion, destroys the combination of metal ion and organic fluorescent dye molecule, the fluorescence of reactivation organic fluorescent dye molecule.Fluorescence control method fluorescent quenching provided by the invention is thorough, reactivation facilitates controllable, the fluorescence control method combination wide field imaging system of organic fluorescent dye molecular labeling provided by the invention biological tissue can carry out chemical tomography to the sample that luminescent dye molecule marks, and realize the rapid chemical tomography of the large volume biological tissue of organic fluorescence molecular labeling.
Description
Technical field
The invention belongs to bio-imaging fields, more particularly, to a kind of organic fluorescent dye molecular labeling biological tissue
Fluorescence control method.
Background technique
The spatial distribution for obtaining the molecule and albumen in biological tissue in detail, is to understand organism structure and functional relationship
Important foundation.Specific molecule and albumen can be marked in fluorescence probe labelling technique, in order to preferably study
The spatial distribution and function of these molecules and albumen in biological tissues, carry out a variety of fluorescence probe labels in same organism
Method, be widely used in biology and medical domain.
When carrying out optical imagery for the biological tissue that fluorescence probe marks, current existing physics chromatographic technique mainly has
Two kinds: one is optical chromatography technologies, optically inhibit the fluorescence other than focal plane to improve z to resolution ratio, optics
The major limitation of chromatographic technique is that its z is lower to resolution ratio, and optical chromatography technology uses point-scan mode, for polychrome
The biological tissue of fluorescence probe label, sweep time are longer；Another kind is mechanical chromatographic technique, by the way that biological tissue embedding exists
In resin, tissue slice is generated in such a way that physics is sliced, then wide field imaging is carried out to tissue slice, physics chromatographic technique
Major limitation is during being imaged by this method that tissue slice is easily lost, and behaviour does complexity, takes a long time,
Obtained image, post-processing and three-dimensional registration are extremely difficult, are not used to the biological tissue of large volume.
Organic fluorescent dye molecular labeling biological tissue, have following advantage: luminous efficiency is high, stabilization is not easy to be quenched
It goes out, have a wide range of application and can show more neural fine structures.But the physics chromatographic technique of the prior art is to organic glimmering
When photoinitiator dye molecular labeling biological tissue carries out fluorescence imaging, due to lacking suitable fluorescence control method, cause to deposit when imaging
Be quenched be not thorough cause be imaged background fluorescence interference, Image Acquisition flux is low, Z-direction resolution ratio is low, three-dimensional registration is difficult to accurately
Etc. technical problems.
Summary of the invention
Aiming at the above defects or improvement requirements of the prior art, the present invention provides a kind of organic fluorescent dye molecular labelings
The fluorescence control method of biological tissue, its object is to by forming six in conjunction with organic fluorescent dye molecule using metal ion
Member ring reduces the conjugated pi electron density of organic fluorescence molecule, so as to the inverse fluorescence that organic fluorescent dye molecule is quenched；Again
Coordinate bond is formed using metal ion chelation agent and metal ion, destroys the combination of metal ion and organic fluorescent dye molecule,
Restore its conjugated pi electron structure, thus the fluorescence of reactivation organic fluorescent dye molecule, fluorescence controlling party through the invention
Method, and be applied in chromatography imaging technique, it thus solves to be quenched to be not thorough existing for prior art physics chromatographic technique to lead
The interference of cause imaging background fluorescence, Image Acquisition flux is low, Z-direction resolution ratio is low, three-dimensional registration is difficult to the technical problems such as accurate.
To achieve the above object, according to one aspect of the present invention, it is raw to provide a kind of organic fluorescent dye molecular labeling
The fluorescence control method of object tissue forms hexatomic ring in conjunction with organic fluorescent dye molecule using metal ion, is quenched organic glimmering
The fluorescence of photoinitiator dye molecule forms coordinate bond using metal ion chelation agent and metal ion, destroy metal ion with it is organic glimmering
The combination of photoinitiator dye molecule, the fluorescence of reactivation organic fluorescent dye molecule.
Preferably, the fluorescence control method, includes the following steps:
(1) biological tissue of organic fluorescent dye molecular labeling quenching fluorescence: is immersed in the water of metal ion compound
In solution, metal ion forms hexatomic ring in conjunction with organic fluorescent dye molecule, to reduce the conjugated pi electron of fluorescent chromophore
Cloud density obtains the biological tissue of fluorescent quenching so that the fluorescence of organic fluorescent dye molecule is quenched；
(2) biological tissue for the fluorescent quenching that step (1) obtains reactivation fluorescence: is immersed in metal ion chelation agent
In, metal ion chelation agent and metal ion form coordinate bond, destroy the combination of metal ion and organic fluorescent dye molecule, weight
Activate the fluorescence of organic fluorescent dye molecule.
Preferably, the organic fluorescent dye molecule is Alexa line fluorescent dye molecule, including Alexa488, Alexa
514, one of Alexa 532 and/or Alexa 546 or a variety of.
Preferably, the metal ion compound is transition metal ions compound.
Preferably, the transition metal ions compound is Cr2+、Mn2+、Fe2+、Fe3+、Co2+、Ni2+、Cu+And/or Cu2+
One of or a variety of compounds, preferably Fe3+Compound, further preferably Fe2(SO4)3Or FeCl3。
Preferably, the concentration of the transiting state metal ion is 10mmol/L~500mmol/L.
Preferably, the concentration of the transiting state metal ion is 100~400mmol/L.
Preferably, the metal ion chelation agent is EDTA-Na4, 8-hydroxyquinoline, dimethyl dithiocarbamic acid
It sodium, KORANTIN SH, protoporphyrin disodium, diethylene triamine and/or one of desferrioxamines or a variety of.
Preferably, the metal ion chelation agent is to desferrioxamine or EDTA-Na4。
Preferably, the concentration of the metal ion chelation agent is 100mmol/L~500mmol/L.
Preferably, the concentration of the metal ion chelation agent is 200~500mmol/L.
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 fluorescence control method of organic fluorescent dye molecular labeling provided by the invention biological tissue is by utilizing transition
Metal ion and organic fluorescence molecule form hexatomic ring, the conjugated pi electron density of organic fluorescence molecule are reduced, to quench organic
The fluorescence of luminescent dye molecule is quenched reversible, controllable and thorough.
(2) strong by forming coordination using chelating agent and metal ion, binding ability be greater than metal ion with it is organic glimmering
The binding force of optical molecule is restored its conjugated pi electron structure, is swashed again to destroy the combination of metal ion and organic fluorescence molecule
The fluorescence of organic fluorescent dye molecule living, reactivation is convenient, and degree is high, effect is good, and fluorescence intensity contrast greatly increases,
Its fluorescence intensity after reactivating is 10 times or more of the fluorescence intensity of cancellation state, ensure that it was used to further be imaged
Imaging effect.
(3) the fluorescence control method of the biological tissue of luminescent dye molecule label of the present invention, can be applied to large volume biology
The tomography of tissue farthest inhibits background fluorescence by accurately controlling to be quenched with the fluorescent contrast of reactivation
Interference additionally by control reactivation biological tissue surface layer fluorescence and is imaged, and the activation thickness on biological tissue surface layer is Z-direction layer
The thickness of analysis, Z-direction chromatography with a thickness of 0.5 micron even nanoscale, to improve Z-direction resolution ratio.
(4) the fluorescence control method combination wide field imaging system of luminescent dye molecule label provided by the invention biological tissue
Rapid automatized scanning imagery can be realized to the sample that luminescent dye molecule marks, easy to operate, three-dimensional registration is accurate, the later period
Processing may be implemented to automate, and Image Acquisition flux is high, and organic fluorescence molecular labeling may be implemented substantially in Z-direction high resolution
The rapid chemical tomography of product biological tissue.
Detailed description of the invention
Fig. 1 is the fluorescence control method flow chart of organic fluorescent dye molecular labeling biological tissue of the invention；
Fig. 2 is that 488 luminescent dye molecule of Alexa is quenched-reactivation mechanism；
Fig. 3 is that 514 luminescent dye molecule of Alexa is quenched-reactivation mechanism；
Fig. 4 is that 532 luminescent dye molecule of Alexa is quenched-reactivation mechanism；
Fig. 5 is that 546 luminescent dye molecule of Alexa is quenched-reactivation mechanism；
Fig. 6 is being quenched-activating into for the 488 luminescent dye molecule immunohistochemical markers murine brain of Alexa of embodiment 1
As figure.Fig. 6 (a) is 488 luminescent dye molecule immunohistochemical markers murine brain of Alexa in 10mmol/L FeSO4In solution
Image after being quenched, the image after increasing 10 times for contrast；Fig. 6 (b) is 488 luminescent dye molecule immunohistochemistry of Alexa
Label murine brain is desferrioxamined the reactivation image in solution in 100mmol/L；
Fig. 7 is being quenched-activating into for the 488 luminescent dye molecule immunohistochemical markers murine brain of Alexa of embodiment 2
As figure.Fig. 7 (a) is 488 luminescent dye molecule immunohistochemical markers murine brain of Alexa in 100mmol/L FeCl3In solution
Image after being quenched, the image after increasing 10 times for contrast；Fig. 7 (b) is 488 luminescent dye molecule immunohistochemistry of Alexa
Mark murine brain in 200mmol/L EDTA-Na4Reactivation image in solution；
Fig. 8 is being quenched-activating into for the 514 luminescent dye molecule immunohistochemical markers murine brain of Alexa of embodiment 3
As figure.Fig. 8 (a) is 514 luminescent dye molecule immunohistochemical markers murine brain of Alexa in 200mmol/L NiSO4After being quenched
Image, for contrast increase 10 times after image；Fig. 8 (b) is 514 luminescent dye molecule immunohistochemical markers mouse of Alexa
Reactivation image of the brain tissue in 500mmol/L protoporphyrin disodium solution.
Fig. 9 is being quenched-activating into for the 514 luminescent dye molecule immunohistochemical markers murine brain of Alexa of embodiment 5
As figure.Fig. 9 (a) is 514 luminescent dye molecule immunohistochemical markers murine brain of Alexa in 400mmol/L MnSO4After being quenched
Image, for contrast increase 10 times after image；Fig. 9 (b) is 514 luminescent dye molecule immunohistochemical markers mouse of Alexa
Reactivation image of the brain tissue in 400mmol/L sodium dimethyl dithiocarbamate solution.
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.
The fluorescence control method of organic fluorescent dye molecular labeling provided by the invention biological tissue, includes the following steps:
(1) NaCl with 0.9% fixed and rinsing biological tissue: is fixed using 4% PFA solution in biological tissue
Solution rinsing processing；
(2) it marks biological tissue: the biological tissue after fixation and rinsing is immunized using organic fluorescent dye molecule
Groupization label.After 100 microns are fixed and are rinsed using the PBS solution of 20%DMSO and 0.2%Triton X-100 first
Biological tissue's piece carries out punching processing 12 hours, and the serum for being then added 10% is closed 12 hours, using PBS rinsing 1 hour/3
Primary antibody is added according still further to the ratio of 500:1 after secondary to be protected from light at 37 DEG C lower vibration incubation 2 days, after 37 DEG C are protected from light lower vibration incubation 2 days
It is added after the secondary antibody with organic fluorescent dye molecule is protected from light at 4 DEG C lower vibration incubation 8 hours and uses according still further to the ratio of 800:1
PBS is rinsed 1 hour/3 times, obtains the biological tissue of organic fluorescent dye molecular immune groupization label；
(3) quenching fluorescence: the biological tissue after the organic fluorescent dye molecular immune groupization label that step (2) are obtained soaks
Stain is in the aqueous solution of transition metal ions compound, and the concentration of transition metal ions is 10mmol/L~500mmol/L, preferably
For 100~400mmol/L, transition metal ions forms hexatomic ring in conjunction with organic fluorescent dye molecule, to be quenched organic glimmering
The fluorescence of photoinitiator dye molecule obtains the biological tissue of fluorescent quenching；
(3) biological tissue for the fluorescent quenching that step (2) obtain reactivation fluorescence: is immersed in metal ion chelation agent
In, the concentration of metal ion chelation agent is 100mmol/L~500mmol/L, preferably 200~500mmol/L, metal ion chela
Mixture and metal ion form coordinate bond, destroy the combination of metal ion and organic fluorescent dye molecule, reactivation organic fluorescence
The fluorescence of dye molecule.
Wherein, organic fluorescent dye molecule be Alexa line fluorescent dye molecule, including Alexa 488, Alexa 514,
One of Alexa 532 and/or Alexa 546 or a variety of.
Transition metal ions compound is Cr2+、Mn2+、Fe2+、Fe3+、Co2+、Ni2+、Cu+And/or Cu2+In one
Kind or a variety of compounds, preferably Fe3+Compound, further preferably FeCl3Or Fe2(SO4)3。
Metal ion chelation agent is EDTA-Na4, 8-hydroxyquinoline, KORANTIN SH, dimethyl dithiocarbamic acid
It sodium, protoporphyrin disodium, diethylene triamine and/or one of desferrioxamines or a variety of, preferably desferrioxamines or EDTA-Na4。
Step (3) is immersed in fluorescent quenching in the aqueous solution of transition metal ions compound to the biological tissue after label
Dip time and step (4) are immersed in the fluorescence in metal ion chelation agent to the biological tissue of the fluorescent quenching of acquisition and swash again
Live time depend on the biological tissue of selection specific thickness and other properties, the biological tissue samples choosing in the embodiment of the present invention
It is 100 microns of mouse brain coronal-plane brain pieces, step (3) fluorescent quenching impregnation time is 10~60min, preferably 10
~30min；Step (4) reactivation fluorescence impregnation time is 10~60min, preferably 10~30min.
Fig. 1 is the fluorescence control method flow chart of organic fluorescent dye molecular labeling biological tissue.Fig. 2, Fig. 3, Fig. 4 and figure
5 be that luminescent dye molecule Alexa 488, Alexa 514, Alexa 532 and Alexa 546 utilize fluorescence control of the invention respectively
- reactivation mechanism schematic diagram is quenched in method processed.
The fluorescence control method of organic fluorescent dye molecular labeling provided by the invention biological tissue is by utilizing transition gold
Belong to ion and organic fluorescent dye molecule forms hexatomic ring, the conjugated pi electron density of organic fluorescence molecule is reduced, thus by organic
Luminescent dye molecule marks the fluorescent quenching in biological tissue, is quenched controllable and thorough；Again by utilizing chelating agent and metal
Ion forms coordination and is good for, and binding ability is greater than the binding force of metal ion and organic fluorescence molecule, to destroy metal ion
And the combination of organic fluorescence molecule restores its conjugated pi electron structure, the fluorescence of reactivation organic fluorescent dye molecule, reactivation
Convenient, effect is good.
Using fluorescence control method of the invention, reagent and its concentration and glimmering are preferably quenched or activated by many experiments
Photocontrol condition can be such that its fluorescence intensity contrast greatly increases, and the fluorescence intensity after reactivating is cancellation state
10 times or more of fluorescence intensity ensure that its imaging effect for being used to further be imaged.
The fluorescence control method of organic fluorescent dye molecular labeling provided by the invention biological tissue can in conjunction with wide field at
As system realizes the quick wide field imaging of large volume biological tissue, and Z-direction high resolution.Using fluorescent quenching method of the invention
The fluorescence of luminescent dye molecule label biological tissue is quenched completely first, fluorescence method of reactivation of the invention is then recycled to swash
The surface layer fluorescence of living biological tissue is simultaneously imaged, since fluorescence is quenched completely when imaging, no background fluorescence interference；Reactivation is raw
Z-direction resolution ratio when the skin depth of object tissue is tomography, Z-direction chromatography with a thickness of 0.5 micron even nanoscale, from
And improve Z-direction resolution ratio.
By the following method to sample (100 microns of mouse brain coronal-plane brain pieces) fluorescence intensity of activation front and back in following embodiment
It is tested:
Sample: 100 microns of mouse brain coronal-plane brain pieces.
Instrument: the German bis- femto-second laser multiphoton microscopes of Zeiss.
Test condition: Laser 488nm, 4%；pinhole 40；gain master 550；Pixel 1024 × 1024,
16bit；60 microns of imaging depth, × 20W.
The following are embodiments:
Embodiment 1
(1) the mouse brain after perfusion is fixed using 4% PFA solution and carries out rinsing processing with PBS solution；
(2) 100 microns of murine brain pieces after fixation and rinsing are subjected to immunohistochemical markers using Alexa 488 and adopted
Rinsing processing is carried out with PBS solution: solid to 100 microns using the PBS solution of 20%DMSO and 0.2%Triton X-100 first
Murine brain piece after fixed and rinsing carries out punching processing 12 hours, and the serum for being then added 10% is closed 12 hours, using PBS
Ratio after rinsing 1 hour/3 times according still further to 500:1 is added primary antibody and is protected from light at 37 DEG C lower vibration incubation 2 days, and 37 DEG C are protected from light lower vibration
After secondary antibody of the ratio addition with Alexa 488 after dynamic incubation 2 days according still further to 800:1 is protected from light lower vibration incubation 8 hours at 4 DEG C
It is rinsed 1 hour/3 times using PBS, obtains 100 microns of murine brain pieces of 488 immunohistochemical markers of Alexa；
(3) 100 microns of murine brain pieces of 488 immunohistochemical markers of Alexa are immersed under room temperature environment
10mmol/L FeSO4Solution be quenched handling and is imaged after twenty minutes；
(4) the murine brain piece of 488 immunohistochemical markers of Alexa after being quenched is immersed under room temperature environment
100mmol/L desferrioxamine solution carry out reactivation carry out fluorescence imaging after twenty minutes.
Fig. 6 is being quenched-activating into for the 488 luminescent dye molecule immunohistochemical markers murine brain of Alexa of embodiment 1
As figure.Fig. 6 (a) is 488 luminescent dye molecule immunohistochemical markers murine brain of Alexa in 10mmol/L FeSO4In solution
Image after being quenched increases 10 times of treated images for contrast, and the fluorescence intensity after being convenient for and activating compares；Figure
6 (b) be 488 luminescent dye molecule immunohistochemical markers murine brain of Alexa desferrioxamines swashing in solution again in 100mmol/L
Image living.As can be seen that after activation processing, fluorescence intensity and the effect phase after 10 times of fluorescence intensity contrast amplification before activation
When even higher, i.e. fluorescence intensity is enhanced 10 times or more by activation processing.
Embodiment 2
(1) the mouse brain after perfusion is fixed using 4% PFA solution and carries out rinsing processing with PBS solution；
(2) 100 microns of murine brain pieces after fixation and rinsing are subjected to immunohistochemical markers using Alexa 488 and adopted
Rinsing processing is carried out with PBS solution: solid to 100 microns using the PBS solution of 20%DMSO and 0.2%Triton X-100 first
Murine brain piece after fixed and rinsing carries out punching processing 12 hours, and the serum for being then added 10% is closed 12 hours, using PBS
Ratio after rinsing 1 hour/3 times according still further to 500:1 is added primary antibody and is protected from light at 37 DEG C lower vibration incubation 2 days, and 37 DEG C are protected from light lower vibration
After secondary antibody of the ratio addition with Alexa 488 after dynamic incubation 2 days according still further to 800:1 is protected from light lower vibration incubation 8 hours at 4 DEG C
It is rinsed 1 hour/3 times using PBS, obtains 100 microns of murine brain pieces of 488 immunohistochemical markers of Alexa；
(3) 100 microns of murine brain pieces of 488 immunohistochemical markers of Alexa are immersed under room temperature environment
100mmol/L FeCl3Solutions Solution be imaged after processing being quenched 30 minutes；
(4) the murine brain piece of 488 immunohistochemical markers of Alexa after being quenched is immersed under room temperature environment
200mmol/L EDTA-Na4Solution carries out fluorescence imaging after carrying out reactivation 30 minutes.
Fig. 7 is being quenched-activating into for the 488 luminescent dye molecule immunohistochemical markers murine brain of Alexa of embodiment 2
As figure.Fig. 7 (a) is 488 luminescent dye molecule immunohistochemical markers murine brain of Alexa in 100mmol/L FeCl3In solution
Image after being quenched increases 10 times of treated images for contrast, and the fluorescence intensity after being convenient for and activating compares；Figure
7 (b) be 488 luminescent dye molecule immunohistochemical markers murine brain of Alexa in 200mmol/L EDTA-Na4Weight in solution
Activate image.As can be seen that after activation processing, fluorescence intensity and the effect phase after 10 times of fluorescence intensity contrast amplification before activation
When fluorescence intensity is enhanced 10 times or more by i.e. activation processing.
Embodiment 3
(1) 100 microns of murine brains after perfusion are fixed using 4% PFA solution and are rinsed with PBS solution
Processing；
(2) 100 microns of murine brain pieces after fixation and rinsing are subjected to immunohistochemical markers using Alexa 514 and adopted
Rinsing processing is carried out with PBS solution: solid to 100 microns using the PBS solution of 20%DMSO and 0.2%Triton X-100 first
Murine brain piece after fixed and rinsing carries out punching processing 12 hours, and the serum for being then added 10% is closed 12 hours, using PBS
Ratio after rinsing 1 hour/3 times according still further to 500:1 is added primary antibody and is protected from light at 37 DEG C lower vibration incubation 2 days, and 37 DEG C are protected from light lower vibration
After secondary antibody of the ratio addition with Alexa 514 after dynamic incubation 2 days according still further to 800:1 is protected from light lower vibration incubation 8 hours at 4 DEG C
It is rinsed 1 hour/3 times using PBS, obtains 100 microns of murine brain pieces of 514 immunohistochemical markers of Alexa；
(3) 100 microns of murine brain pieces of 514 immunohistochemical markers of Alexa are used into 200mmol/L Fe2(SO4)3It is molten
Liquid be quenched handling and is imaged after ten minutes；
(4) the murine brain piece of 514 immunohistochemical markers of Alexa after being quenched uses 500mmol/L EDTA-Na4
Solution carries out reactivation and carries out fluorescence imaging after ten minutes.
Fig. 8 is being quenched-activating into for the 514 luminescent dye molecule immunohistochemical markers murine brain of Alexa of embodiment 3
As figure.Fig. 8 (a) is 514 luminescent dye molecule immunohistochemical markers murine brain of Alexa in 200mmol/L Fe2(SO4)3It quenches
Image after going out increases 10 times of treated images for contrast, and the fluorescence intensity after being convenient for and activating compares；Fig. 8
It (b) is 514 luminescent dye molecule immunohistochemical markers murine brain of Alexa in 500mmol/L EDTA-Na4Weight in solution
Activate image.As can be seen that fluorescence intensity contrast amplifies the effect after 10 times before fluorescence intensity even ratio activates after activation processing
Fruit is taller, that is, illustrates that fluorescence intensity is enhanced 10 times or more by activation processing, ensure that when it is used to further be imaged
Imaging effect.
Embodiment 4
(1) 100 microns of murine brains after perfusion are fixed using 4% PFA solution and are rinsed with PBS solution
Processing；
(2) 100 microns of murine brain pieces after fixation and rinsing are subjected to immunohistochemical markers using Alexa 514 and adopted
Rinsing processing is carried out with PBS solution: solid to 100 microns using the PBS solution of 20%DMSO and 0.2%Triton X-100 first
Murine brain piece after fixed and rinsing carries out punching processing 12 hours, and the serum for being then added 10% is closed 12 hours, using PBS
Ratio after rinsing 1 hour/3 times according still further to 500:1 is added primary antibody and is protected from light at 37 DEG C lower vibration incubation 2 days, and 37 DEG C are protected from light lower vibration
After secondary antibody of the ratio addition with Alexa 514 after dynamic incubation 2 days according still further to 800:1 is protected from light lower vibration incubation 8 hours at 4 DEG C
It is rinsed 1 hour/3 times using PBS, obtains 100 microns of murine brain pieces of 514 immunohistochemical markers of Alexa；
(3) 100 microns of murine brain pieces of 514 immunohistochemical markers of Alexa are used into 500mmol/L NiSO4Solution
Be quenched handling and be imaged after forty minutes；
(4) the murine brain piece of 514 immunohistochemical markers of Alexa after being quenched is molten using 500mmol/L protoporphyrin disodium
Liquid carries out reactivation and carries out fluorescence imaging after forty minutes.
After activation processing, the fluorescence intensity after fluorescence intensity contrast amplifies 10 times before fluorescence intensity ratio activates is high, guarantees
Imaging effect when it is used to further be imaged.
Embodiment 5
(1) 100 microns of murine brains after perfusion are fixed using 4% PFA solution and are rinsed with PBS solution
Processing；
(2) 100 microns of murine brain pieces after fixation and rinsing are subjected to immunohistochemical markers using Alexa 514 and adopted
Rinsing processing is carried out with PBS solution: solid to 100 microns using the PBS solution of 20%DMSO and 0.2%Triton X-100 first
Murine brain piece after fixed and rinsing carries out punching processing 12 hours, and the serum for being then added 10% is closed 12 hours, using PBS
Ratio after rinsing 1 hour/3 times according still further to 500:1 is added primary antibody and is protected from light at 37 DEG C lower vibration incubation 2 days, and 37 DEG C are protected from light lower vibration
After secondary antibody of the ratio addition with Alexa 514 after dynamic incubation 2 days according still further to 800:1 is protected from light lower vibration incubation 8 hours at 4 DEG C
It is rinsed 1 hour/3 times using PBS, obtains 100 microns of murine brain pieces of 514 immunohistochemical markers of Alexa；
(3) 100 microns of murine brain pieces of 514 immunohistochemical markers of Alexa are used into 400mmol/L MnSO4Solution
Be quenched handling and be imaged after sixty minutes；
(4) the murine brain piece of 514 immunohistochemical markers of Alexa after being quenched uses 400mmol/L dimethyl disulfide
Reactivation, which is carried out, for carbamic acid sodium solution carries out fluorescence imaging after sixty minutes.
Fig. 9 is being quenched-activating into for the 514 luminescent dye molecule immunohistochemical markers murine brain of Alexa of embodiment 5
As figure.Fig. 9 (a) is 514 luminescent dye molecule immunohistochemical markers murine brain of Alexa in 400mmol/L MnSO4After being quenched
Image, contrast increases 10 times；Fig. 9 (b) is that Alexa514 luminescent dye molecule immunohistochemical markers murine brain exists
Reactivation image in 400mmol/L sodium dimethyl dithiocarbamate solution.After activation processing, before fluorescence intensity ratio activation
Fluorescence intensity after fluorescence intensity contrast amplifies 10 times is high, ensure that imaging effect when it is used to further be imaged.
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 (9)
1. a kind of fluorescence control method of the organic fluorescent dye molecular labeling biological tissue applied to chromatography imaging technique, special
Sign is, forms hexatomic ring in conjunction with organic fluorescent dye molecule using metal ion, the glimmering of organic fluorescent dye molecule is quenched
Light forms coordinate bond using metal ion chelation agent and metal ion, destroys the knot of metal ion and organic fluorescent dye molecule
It closes, the fluorescence of reactivation organic fluorescent dye molecule；The organic fluorescent dye molecule is Alexa line fluorescent dye molecule；
The fluorescence control method comprising following steps:
(1) biological tissue of organic fluorescent dye molecular labeling quenching fluorescence: is immersed in the aqueous solution of metal ion compound
In, metal ion forms hexatomic ring in conjunction with organic fluorescent dye molecule, so that the conjugated pi electron cloud for reducing fluorescent chromophore is close
It spends so that the fluorescence of organic fluorescent dye molecule is quenched, obtains the biological tissue of fluorescent quenching；
(2) reactivation fluorescence: the biological tissue for the fluorescent quenching that step (1) obtains is immersed in metal ion chelation agent, gold
Belong to ion chelating agent and metal ion forms coordinate bond, destroys the combination of metal ion and organic fluorescent dye molecule, reactivation
The fluorescence of organic fluorescent dye molecule；Its fluorescence intensity after reactivating can reach 10 times of the fluorescence intensity of cancellation state
More than.
2. fluorescence control method as described in claim 1, which is characterized in that the organic fluorescent dye molecule is Alexa
488, one of Alexa 514, Alexa 532 and/or Alexa 546 or a variety of.
3. fluorescence control method as described in claim 1, which is characterized in that the metal ion compound be transition metal from
Sub- compound.
4. fluorescence control method as claimed in claim 3, which is characterized in that the transition metal ions compound is Cr2+、
Mn2+、Fe2+、Fe3+、Co2+、Ni2+、Cu+And/or Cu2+One of or a variety of compounds.
5. fluorescence control method as claimed in claim 3, which is characterized in that the transition metal ions compound is Fe3+'s
Compound.
6. fluorescence control method as claimed in claim 3, which is characterized in that the transition metal ions compound is Fe2
(SO4)3Or FeCl3。
7. fluorescence control method as claimed in claim 3, which is characterized in that the concentration of the transiting state metal ion is
10mmol/L~500mmol/L.
8. fluorescence control method as described in claim 1, which is characterized in that the metal ion chelation agent is EDTA-Na4、8-
It oxyquinoline, sodium dimethyl dithiocarbamate, KORANTIN SH, protoporphyrin disodium, diethylene triamine and/or desferrioxamines
It is one or more.
9. fluorescence control method as claimed in claim 8, which is characterized in that the concentration of the metal ion chelation agent is
100mmol/L~500mmol/L.
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