CN106525792B - A kind of fluorescence control method of light-operated fluorescent protein labeling biological tissue embedding sample - Google Patents
A kind of fluorescence control method of light-operated fluorescent protein labeling biological tissue embedding sample Download PDFInfo
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- 238000000799 fluorescence microscopy Methods 0.000 claims abstract description 10
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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
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
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Abstract
The invention discloses a kind of fluorescence control methods of light-operated fluorescent protein labeling biological tissue embedding sample, and with the light-operated fluorescin on activation photoactivation sample surface layer, the light-operated fluorescin on the surface layer described in excitation issues fluorescence in turn.By selecting the wave-length coverage of suitable activation light and exciting light, while the angle of suitable activation direction and sample upper surface is controlled, realizes the accurate control of light-operated fluorescent protein labeling biological tissue embedding sample surface layer fluorescence, activation is convenient, and activation skin depth is thin；Fluorescence control method of the invention only activates the fluorescence on biological tissue embedding sample surface layer when activating, when fluorescence tomography applied to biological tissue embedding sample, excitation is activated surface layer and carries out fluorescence imaging, sample be activated surface layer thickness be fluorescence imaging axial resolution, axial resolution is high.
Description
Technical field
The invention belongs to fluorescent microscopic imaging fields, and in particular, to a kind of light-operated fluorescent protein labeling biological tissue packet
Bury the fluorescence control method of sample.
Background technique
The photophysical property of certain fluorescins can accurately be controlled by light, and this kind of fluorescin is light-operated fluorescin.
It includes at least following two class, photoactivation fluorescin and light conversion fluorescence albumen.Photoactivation fluorescin is in the initial state
It does not fluoresce, after specific wavelength activates photoactivation, fluorescence, such as PAGFP can be inspired.Light conversion fluorescence albumen exists
It can fluoresce when original state, but after activating photoactivation, the fluorescence of another color can be inspired, than
Such as mEos3.1, mEos3.2.The biological tissue of light-operated fluorescent protein labeling and the biological tissue of common fluorescent protein labeling, at
Image space method is similar, commonly uses common wide field imaging method or burnt, the two-photon Mapping with copolymerization.For big biological group
It knits, it is often necessary to carry out tomographic imaging after embedding medium embedding.Due to lacking suitable fluorescence control method, the prior art is caused to be adopted
There is a huge defect in imaging with light-operated fluorescent protein labeling biological tissue embedding sample: quick wide field imaging method
Chromatography ability is poor, cannot achieve high-resolution；And high-resolution Mapping method, it is such as copolymerized burnt or two photon imaging, for
Bulky biological tissue embedding sample is then imaged too slow.
Summary of the invention
Aiming at the above defects or improvement requirements of the prior art, the present invention provides a kind of light-operated fluorescent protein labeling biologies
The fluorescence control method of organization embedding sample, its object is to pass through photosensitive light-operated fluorescent protein labeling biological tissue packet
Bury the light-operated fluorescin in activation photoactivation sample surface layer that sample uses specific wavelength, then the excitation for passing through respective wavelength
The fluorescin on sample surface layer realizes the control of the surface layer fluorescence of light-operated fluorescent protein labeling biological tissue embedding sample, and logical
Optimization fluorescence control condition is crossed to keep the activation of its surface layer thinner, and it is applied to fluorescence tomography, surface layer is activated
Axial resolution of thickness when being tomography, thus solve the fluorescent protein labeling biological tissue of the prior art due to lacking
Weary effective fluorescence control method and in quick wide field fluorescence imaging since ability is poor, axial resolution is low for chromatography, and high score
Bulky biological tissue embedding sample is imaged too slow technical problem in the Mapping method distinguished.
To achieve the above object, according to one aspect of the present invention, a kind of light-operated fluorescent protein labeling biology group is provided
The fluorescence control method for knitting embedded samples, with activation photoactivation sample surface layer light-operated fluorescin, described in excitation
The light-operated fluorescin on surface layer issues fluorescence in turn.
Preferably, the light-operated fluorescin is photoactivation fluorescin or light conversion fluorescence albumen.
Preferably, the surface layer with a thickness of 0.5~5 micron.
Preferably, the activation light is the activation light with the light-operated matched specific wavelength of fluorescin.
Preferably, the exciting light is the exciting light with the light-operated matched specific wavelength of fluorescin.
Preferably, the direction of the activation light and the angle of sample upper surface are 0~90 °.
Preferably, the direction of the activation light and the angle of sample upper surface are 15 °~30 °.
Preferably, the embedding medium of the biological tissue embedding sample is preferably resin.
Preferably, the resin is acrylic resin.
Preferably, light absorbing substance is added in the embedding medium.
Preferably, the light absorbing substance is sudan black.
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 light-operated fluorescent protein labeling biological tissue embedding sample provided by the invention, passes through choosing
The wave-length coverage of suitable activation light and exciting light is selected, while controlling the angle of suitable activation direction and sample upper surface, it is real
Existing light-operated fluorescent protein labeling biological tissue embedding sample surface layer fluorescence is precisely controlled, and activation is convenient, and control effect is good；
(2) fluorescence that biological tissue embedding sample surface layer is only activated when fluorescence control method of the invention activation, is applied to
When the fluorescence tomography of biological tissue embedding sample, excitation is activated surface layer and carries out fluorescence imaging, and sample is swashed
The thickness on surface layer living is the axial resolution of fluorescence imaging, by optimizing fluorescence control condition, the wave including preferably activating light
Long, control activates the angle of light and sample and controls the thickness on the surface layer that is activated using light absorbing substance is added in embedding medium
Degree, thickness can be even lower down to 1~2 micron, is expected to realize high-resolution tomography；
(3) when with activation photoactivation light-operated fluorescin, only activate the surface layer of embedded samples, and below surface layer not by
Photoactivation is activated, therefore surface layer can not issue fluorescence with undertissue absorbing subsequent exciting light, so as to avoid rear
The interference of non-focal plane fluorescence below tissue surface when continuous wide field imaging；
(4) when fluorescence control method of the invention is applied to the fluorescence imaging of biological tissue embedding sample, since there is no
The interference of the following fluorescence of focal plane, therefore can be used for high-throughput face imaging, i.e., quick wide field imaging activates thick additionally, due to surface layer
Degree is that axial resolution can achieve 1~2 micron, and even more thin therefore of the invention fluorescence control method is applied to fluorescence coating
Analysis imaging when, be provided simultaneously with wide field imaging high speed and with the approximate high-resolution of Mapping, realize large volume
The quick high-resolution imaging of biological tissue embedding sample.
Detailed description of the invention
Fig. 1 is the schematic diagram that light-operated fluorescent protein labeling biological tissue embedding fluorescent is controlled and is imaged, wherein 1. table
Show activation light, 2. indicate exciting light, 3. indicate sample, 4. indicates excitation and image-forming objective lens, 5. indicate to have activated sample surface layer；
Fig. 2 is light-operated fluorescin activation excitation front and back fluorescence intensity comparison diagram used in embodiment 1；Fig. 2 (A) is embodiment
The fluorescence intensity excited before 1 activation；Fig. 2 (B) is the fluorescence intensity excited after embodiment 1 activates, wherein activation light wave is a length of
405nm；
Fig. 3 is light-operated fluorescin activation excitation front and back fluorescence intensity comparison diagram used in embodiment 2；Fig. 3 (A) is embodiment
The fluorescence intensity excited before 2 activation；Fig. 3 (B) is the fluorescence intensity excited after embodiment 2 activates.
In all the appended drawings, identical appended drawing reference is used to denote the same element or structure, wherein the scale bar generation of Fig. 2
20 microns of table.
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 light-operated fluorescent protein labeling biological tissue provided by the invention, and be applied to biological tissue and wrap
When burying the tomography of sample, comprising the following steps:
One, sample embeds
The biological tissue of light-operated fluorescent protein labeling is embedded with embedding medium, the detailed process of embedding is referring to embedding medium commodity
The general step of specification or open source literature data.
The embedding medium of the biological tissue embedding sample is preferably resin, and the resin is preferably acrylic resin, including
HM20 (lowicryls acrylates resinoid), HM23 (lowicryls acrylates resinoid), T9100, London white glue (LR
White) etc..
Two, different directions activation excitation
Fig. 1 is the schematic diagram that light-operated fluorescent protein labeling biological tissue embedding fluorescent is controlled and is imaged, wherein 1. table
Show activation light, 2. indicate exciting light, 3. indicate sample, 4. indicates excitation and image-forming objective lens, 5. indicate to have activated sample surface layer.
By the biological tissue embedding sample with the light-operated fluorescence egg on activation photoactivation sample surface layer in non-imaged optical path
White fluorescence, then with the fluorescence of the excitation of the corresponding wavelength fluorescin and imaging on imaging optical path.It is described at
As optical path refers to that the optical path of reception emission spectrum (i.e. fluorescence signal), the non-imaged optical path refer to other light except imaging optical path
Road.
" activation " refers to, light-operated fluorescin hardly sends out some wave band under the excitation of specific wavelength exciting light
Fluorescence open fluorescin only after " activation light " activation of some other wavelength, be allowed to receive specific wavelength
" exciting light " excitation is to issue fluorescence.And " excitation " refers to the process of that fluorescin absorbs the light sending fluorescence of some wavelength.
Label biological tissue light-operated fluorescin include photoactivation fluorescin (Photoactivatable FPs,
PAFPs one of) or a variety of, such as PAGFP, PAmCherry1；Or light conversion fluorescence albumen (photoswitchable
FPs, rsFPs) one of or a variety of, such as Dendra2, mEos3.1.
The activation light is the activation light with the light-operated matched specific wavelength of fluorescin.
The exciting light is the exciting light with the light-operated matched specific wavelength of fluorescin.
The direction of activation light and the angle of the sample upper surface is 0~90 °, preferably 15~30 °.
By adjusting the angle of activation light and sample upper surface (i.e. imaging layer or having activated sample surface layer), conjunction can control
Suitable activation thickness.Activate the angle of light and sample upper surface can be within the scope of 0 degree to 90 degree, 15 °~30 ° are conducive to activate
The surface layer of thinner thickness.When activating the angle of light and sample upper surface to be 0 °, activation light is parallel to sample upper epidermis, needs at this time
Transparency process is carried out to sample, sample surface layer is activated from side using the method for similar mating plate illumination.When activation light and sample
When the angle of product upper surface is 90 °, light vertical irradiation sample upper surface is activated, when activating thickness compared to 15 °~30 ° of angle
Activate depth degree deeper.
Fluorescence control method of the invention passes through selection and the light-operated fluorescin to matched activation light and exciting light
Wavelength activate the direction of light and sample upper surface and by the direction of the suitable activation light of control so that with specific
When the light-operated fluorescin of the activation photoactivation of wavelength, the fluorescin on only sample surface layer is activated.
Activation light can only activate the surface layer of sample, and reason has two: first is that activate light for exciting light, general wavelength
Shorter, the ability for penetrating tissue is weaker, therefore activates light that can only penetrate the fluorescin for activating more surface layer；Second is that activation light
Activation can control activation light and the angle of sample of being activated, angle is smaller, and surface reflection is stronger, the activation light of such deep layer
What is not only entered is few, and the energy density entered is low.
Alternatively, it is also possible to by adding light absorbing substance in the embedding medium of sample, in this way when activation photoactivation sample
When, even if activation light penetrates tissue and enters deep tissues, light absorbing substance can be absorbed and cannot be activated, light absorbing
Substance is preferably sudan black.Sudan black is added in embedding medium, mass percent of the sudan black in embedding medium is preferably
0.05%~0.5%, preferably 0.3%.
Each light-operated fluorescin has a matched optimal activation wavelength, when selecting the shorter activation light of wavelength,
It needs to select the activation light of the shorter wavelength near optimal activation wavelength, in this way while can be realized high efficiency and optimum thickness
The activation of sample surface layer.
For light-operated fluorescin PAGFP and mEos3.1, activate wavelength all near ultraviolet band, most optimum wavelengths all exist
405nm.When using both fluorescent protein labeling biological tissues and using fluorescence control method activation sample surface layer of the invention
When, optimal wavelength is all the activation light of 405nm.
The present invention select in the non-imaged optical path of sample, with activation photoactivation sample surface layer light-operated fluorescin, so
With the excitation of the corresponding wavelength fluorescin and be imaged on imaging optical path afterwards, be achieved in the surface layer of sample at
Picture.
For photoactivation fluorescin, itself does not fluoresce, and can pass through control activation optical wavelength, activation light and sample
It adds the light absorbing substances such as Sudan black B in product angle and resin to be activated to control only surface layer, therefore excitation
When surface layer below will not issue fluorescence, background fluorescence is not present below surface layer when such surface layer fluorescence imaging, i.e., below focal plane
The problem of fluorescence interferes.
For light conversion fluorescence albumen, itself issues the fluorescence of specific wavelength, such as mEos3.1 fluoresced green, leads to
It crosses light conversion and is allowed to aobvious red, it can be by adding the Sudan in control activation optical wavelength, activation light and sample angle and resin
The light absorbing substance such as black B makes this light conversion only occur in sample surface layer, surface layer is made to issue red fluorescence.When red to surface layer
When the fluorescence of color is imaged, due to green still aobvious below surface layer, background is just not present for red band signal for receiving
Green fluorescence interference the problem of.
Three, tomography
When fluorescence control method of the invention is applied to chromatography imaging technique, since fluorescence control method of the invention can
Accurately to control the thickness that sample surface layer is activated, the surface layer being activated carries out surface layer fluorescence imaging, table through the excitation of exciting light
It, can be by machining away the sample surface layer being imaged, then the surface layer new with activation photoactivation, then after layer fluorescent image obtains
It with the activated new surface layer fluorescence of excitation and acquires the fluorescence signal of transmitting again and is imaged, repeat " cut later
Cut ", the process of " activation photoactivation ", " excitation imaging ", tomographic imaging is until obtain all two dimensions of entire sample repeatedly
Image.Acquired two dimensional image can be superimposed to obtain light-operated fluorescent protein labeling biology group by the methods of autoregistration
The complete three dimensional image knitted.
Fluorescence control method of the invention only has the surface layer of sample to be activated when activating, and the thickness that surface layer is activated is as glimmering
The axial resolution of optical tomography can pass through control activation optical wavelength, the angle and packet of activation light and sample upper surface
It buries and adds the light absorbing substances such as Sudan black B in resin to realize skin depth as thin as possible.Fluorescence controlling party of the invention
Method obtain being activated surface layer thickness can be even lower down to 1~2 micron therefore of the invention light-operated fluorescent protein labeling
When the fluorescence control method of biological tissue embedding sample is applied to the tomography of sample, it is even lower micron order can be obtained
Axial resolution, the problem of additionally, due to background interference is not present, can be realized using the mode of wide field imaging it is high-throughput at
The advantage of picture, high throughput this for the biological sample of large volume more highlights, therefore solves existing optical image technology point
Resolution is low, bulky biological tissue's resin embedding sample is imaged too slow technical problem.
The following are embodiments:
Embodiment 1
MEos3.1 is a kind of common light conversion fluorescence albumen.Its optical characteristics is not activate in ultraviolet 405nm wavelength
Before, fluoresced green (excitation wavelength 488nm), does not send out red fluorescence；After the activation of ultraviolet 405nm wavelength short time,
Under 561nm exciting light, (emission spectrum is in 550nm or more) for hair red fluorescence.
Cerebral cortex injects the embedding for having the Mouse Whole Brain of Rv-dg-mEos3.1 and mating plate fluorescence control method and fluorescence coating
Analysis imaging, comprising the following steps:
(1) sample is fixed
The Mouse Whole Brain of Rv-dg-mEos3.1 infection is fixed with chemical fixing means, obtains fixed mEos3.1 label
Biological tissue.Specific step is as follows:
At 4 deg. celsius, the Mouse Whole Brain that after cardiac perfusion, solution is cut is impregnated through the PFA solution that mass fraction is 4%
About 12 hours, PFA solution usage was 20ml every, and then three times with PBS solution rinsing, every uses PBS solution 40ml every time,
Rinsing four hours every time.
(2) sample is dehydrated
The Mouse Whole Brain ethanol replacement that fixed mEos3.1 is marked, so that the biological tissue dewatering, is dehydrated
The Mouse Whole Brain of mEos3.1 label afterwards.Specific steps are as follows:
At 4 deg. celsius, the fixed mEos3.1 Mouse Whole Brain marked is passed sequentially through into graded ethanol double steaming solution
20ml is impregnated 2 hours, is dehydrated.It according to percent by volume is 50% that ethyl alcohol double steaming solution concentration gradient, which is ethyl alcohol,
75%, 95%, 100%, 100%.
(3) embedding medium infiltration is handled
The Mouse Whole Brain that dewatered mEos3.1 is marked carries out HM20 embedding medium infiltration processing, obtains HM20 embedding medium
The Mouse Whole Brain of the mEos3.1 label of working solution filling.Specific steps are as follows:
At 4 deg. celsius, the dimethylbenzene for the dewatered mEos3.1 Mouse Whole Brain marked being passed sequentially through gradient HM20 is molten
Liquid 5ml or more carries out embedding medium infiltration.It according to percent by volume is 50%, 75% that the xylene solution gradient of HM20, which is HM20,
100%, 100%, 100%, 100%, it is small that each gradient immersion of first three gradient respectively impregnates 24 in 2 hours, the 4th group and the 5th group
When, the 6th group immersion 14 hours.
(4) embedding medium polymerize
Make the HM20 embedding medium that polymerization reaction occur, obtains the resin embedding sample of the biological tissue of mEos3.1 label.
Specific steps are as follows:
The gelatine capsule for the 9mm bore that the HM2O embedding medium working solution injection of 1.1mL is mounted on the base, then will
The Mouse Whole Brain of the mEos3.1 label of HM20 embedding medium working solution filling is put into capsule, is adjusted good position and is covered capsule lid
Son, move into vacuum oven in carry out gradient increased temperature polymerization: 37 degrees Celsius 12 hours, 42 degrees Celsius 3 hours, it is 45 degree Celsius 12 small
When, 50 degrees Celsius 3 hours.
(5) activation excites light-operated fluorescin
By the cutting of the surface layer of the resin embedding Mouse Whole Brain sample it is smooth after, activate photoactivation with ultraviolet 405nm wavelength
Sample surface layer, activation skin depth are 3~4um, then with 561nm excitation fluorescence and are imaged, activate light direction and sample
The angle of product is 30 °.
The fluorescent brightness excited respectively before activation photoactivation processing and after activation processing is as shown in Figure 2.Fig. 2A is not use
405nm activation photoactivation and the fluorescence signal result observed using 561nm exciting light deexcitation, it can be seen that if do not used
405nm activates photoactivation, that is, uses 561nm exciting light deexcitation, and also without any true fluorescence signal, (brightness of distribution is only dry
Disturb noise)；Fig. 2 B is to reuse the fluorescence signal result that 561nm excitation obtains after 405nm activates photoactivation
Figure, it can be seen that after 405nm activates photoactivation, it is glimmering that mEos3.1 can be gone out bright red by 561nm excitation
Light, Fig. 2 B show the cell space form an of neuron.
Embodiment 2
Fluorescence control method after the Hela cell embedding of overexpression PAGFP, comprising the following steps:
(1) sample is fixed
The Hela cell for being overexpressed PAGFP is fixed with chemical fixing means, obtains biological group of fixed PAGFP label
It knits.Specific step is as follows:
At 4 deg. celsius, Hela cell is impregnated about 12 hours through the PFA solution that mass fraction is 4%, then uses PBS
Solution rinses 3 times, and every uses PBS solution 40ml every time, every time rinsing 1 hour.
(2) sample is dehydrated
The Hela cell ethanol replacement that fixed PAGFP is marked obtains dewatered so that the sample is dehydrated
The cell sample of PAGFP label.Specific steps are as follows:
At 4 deg. celsius, the fixed PAGFP Hela cell marked is passed sequentially through into graded ethanol double steaming solution
20ml is impregnated 2 hours, is dehydrated.It according to percent by volume is 50% that ethyl alcohol double steaming solution concentration gradient, which is ethyl alcohol,
75%, 95%, 100%, 100%.
(3) embedding medium infiltration is handled
The Mouse Whole Brain that dewatered PAGFP is marked carries out HM20 embedding medium infiltration processing, obtains HM20 embedding medium work
Make the cell sample of the PAGFP label of liquid filling.Specific steps are as follows:
At 4 deg. celsius, the Hela cell that dewatered PAGFP is marked is passed sequentially through to the xylene solution of gradient HM20
5ml carries out embedding medium infiltration.It according to percent by volume is 50%, 75%, 100% that the xylene solution gradient of HM20, which is HM20,
100%, 100%, 100%, each gradient of first three gradient, which is impregnated, respectively impregnates 24 hours for 2 hours, the 4th group and the 5th group, and the 6th
Group is impregnated 14 hours.
(4) embedding medium polymerize
Make the HM20 embedding medium that polymerization reaction occur, obtains the cell sample of the resin embedding of PAGFP label.Specific step
Suddenly are as follows: HM2O embedding medium working solution is dripped to above the slide with Hela cell, then by the slide covered, is moved into
In vacuum oven carry out gradient increased temperature polymerization: 37 degrees Celsius 12 hours, 42 degrees Celsius 3 hours, 45 degrees Celsius 12 hours, 50 take the photograph
Family name's degree 3 hours.
(5) activation excites light-operated fluorescin
Then the Hela cell sample of the resin embedding is used into excitation with activation light 405nm laser active sample surface layer
Light 504nm LASER Excited Fluorescence is simultaneously imaged, and activates light direction and sample angular separation is 15 °.
After exciting PAGFP using laser active, fluorescent brightness increase multiple is significant, from almost without fluorescence to all tables
Cell up to PAGFP is all very bright, activate surface layer with a thickness of 1~2um.Embodiment 3
Fluorescence control method after the Hela cell embedding of overexpression PAGFP, comprising the following steps:
(1) sample is fixed
The Hela cell for being overexpressed PAGFP is fixed with chemical fixing means, obtains biological group of fixed PAGFP label
It knits.Specific step is as follows:
At 4 deg. celsius, Hela cell is impregnated about 12 hours through the PFA solution that mass fraction is 4%, then uses PBS
Solution rinses 3 times, and every uses PBS solution 40ml every time, every time rinsing 1 hour.
(2) sample is dehydrated
The Hela cell ethanol replacement that fixed PAGFP is marked obtains dewatered so that the sample is dehydrated
The cell sample of PAGFP label.Specific steps are as follows:
At 4 deg. celsius, the fixed PAGFP Hela cell marked is passed sequentially through into graded ethanol double steaming solution
20ml is impregnated 2 hours, is dehydrated.It according to percent by volume is 50% that ethyl alcohol double steaming solution concentration gradient, which is ethyl alcohol,
75%, 95%, 100%, 100%.
(3) embedding medium infiltration is handled
The Mouse Whole Brain that dewatered PAGFP is marked carries out HM20 embedding medium infiltration processing, wherein contains in embedding medium
0.3% mass fraction Sudan black B obtains the cell sample of the PAGFP label of HM20 embedding medium working solution filling.Specific steps
Are as follows:
At 4 deg. celsius, the Hela cell that dewatered PAGFP is marked is passed sequentially through to the xylene solution of gradient HM20
5ml carries out embedding medium infiltration.It according to percent by volume is 50%, 75%, 100% that the xylene solution gradient of HM20, which is HM20,
100%, 100%, 100%, each gradient of first three gradient, which is impregnated, respectively impregnates 24 hours for 2 hours, the 4th group and the 5th group, and the 6th
Group is impregnated 14 hours, the Sudan black B that wherein third is 0.3% containing mass fraction to 100%HM20 solution used in the 6th group.
(4) embedding medium polymerize
Make the HM20 embedding medium that polymerization reaction occur, obtains the cell sample of the resin embedding of PAGFP label.Specific step
Suddenly are as follows:
HM2O embedding medium working solution is dripped to above the slide with Hela cell, then by the slide covered,
Move into vacuum oven in carry out gradient increased temperature polymerization: 37 degrees Celsius 12 hours, 42 degrees Celsius 3 hours, 45 degrees Celsius 12 hours,
50 degrees Celsius 3 hours.
(5) activation excites light-operated fluorescin
Then the Hela cell sample of the resin embedding is used into excitation with activation light 405nm laser active sample surface layer
Light 504nm LASER Excited Fluorescence is simultaneously imaged, and activates light direction and sample angular separation is 15 °.
The fluorescent brightness excited respectively before activation photoactivation processing and after activation processing is as shown in Figure 3A and Figure 3B.Using sharp
After light device activation excitation PAGFP, fluorescent brightness increase multiple is significant, from the cells almost without fluorescence to all expression PAGFP
It is all very bright, activate surface layer with a thickness of 1~2um.
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 fluorescence control method of the light-operated fluorescent protein labeling biological tissue embedding sample suitable for wide field imaging, special
Sign is, with the light-operated fluorescin on activation photoactivation sample surface layer, the light-operated fluorescin on the surface layer described in excitation
And then issue fluorescence；
The light-operated fluorescin is photoactivation fluorescin or light conversion fluorescence albumen；
The activation light is the activation light with the light-operated matched specific wavelength of fluorescin；The exciting light be and the light
Control the exciting light of the matched specific wavelength of fluorescin；The wavelength of the activation light is shorter than the wavelength of the exciting light；
The surface layer with a thickness of 0.5~5 micron；
The direction of the activation light and the angle of sample upper surface are 0~90 °；
Light absorbing substance is added in the embedding medium of the biological tissue embedding sample；
In the fluorescence control method, when activation, only activates the fluorescence on biological tissue embedding sample surface layer, is applied to biological tissue and wraps
When burying the fluorescence tomography of sample, excitation is activated surface layer and carries out fluorescence imaging, and sample is activated the thickness on surface layer
Degree is the axial resolution of fluorescence imaging；
When with the light-operated fluorescin on activation photoactivation sample surface layer, due to only activating the surface layer of embedded samples, and surface layer with
Under be not activated photoactivation, therefore surface layer can not issue fluorescence with undertissue absorbing subsequent exciting light, thus
The interference of non-focal plane fluorescence below tissue surface when avoiding the imaging of subsequent wide field.
2. fluorescence control method as described in claim 1, which is characterized in that the direction and sample upper surface of the activation light
Angle is 15 °~30 °.
3. fluorescence control method as described in claim 1, which is characterized in that the embedding medium of the biological tissue embedding sample is
Resin.
4. fluorescence control method as claimed in claim 3, which is characterized in that the resin is acrylic resin.
5. fluorescence control method as described in claim 1, which is characterized in that the light absorbing substance is sudan black.
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