CN106706577B - A kind of photoimaging systems and method - Google Patents
A kind of photoimaging systems and method Download PDFInfo
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- CN106706577B CN106706577B CN201611022248.3A CN201611022248A CN106706577B CN 106706577 B CN106706577 B CN 106706577B CN 201611022248 A CN201611022248 A CN 201611022248A CN 106706577 B CN106706577 B CN 106706577B
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/645—Specially adapted constructive features of fluorimeters
- G01N21/6456—Spatial resolved fluorescence measurements; Imaging
- G01N21/6458—Fluorescence microscopy
Abstract
The invention discloses a kind of photoimaging systems, applied to optical technical field, wherein the system includes: light source, for generating laser, spatial light modulator, for being modulated to the laser, the regional structure light for generating goal stimulus light and being addressed to sample object region, it is radiated on sample, the fluorescence signal that cellular change information is carried after by the goal stimulus light stimulus is generated on target area, detector, for recording the fluorescence signal, control unit, keep goal stimulus light constant for controlling the spatial light modulator, the phase of the mobile regional structure light, so that the regional structure striations on sample is mobile, detector record moves the fluorescence signal generated after the regional structure striations every time, control unit, it is also used to carry out spectrum analysis to the corresponding fluorescent image of all fluorescence signals of record, it obtains by mesh The high-definition picture of target area when mark thorn laser effect.A kind of photoimaging methods are also disclosed, the imaging resolution in Structured Illumination region can be improved.
Description
Technical field
The present invention relates to optical technical field more particularly to a kind of photoimaging systems and methods.
Background technique
The advantages of traditional fluorescence microscope has specific marker, can be to target real time imagery, in life science
Play the role of extensive.However, traditional fluorescent microscopic imaging is influenced by ssystem transfer function, limit system at
As resolution ratio, to limit effect of the fluorescent microscopic imaging in life science.In recent years, with a series of breakthrough diffraction poles
Limit, realizes the proposition of super-resolution imaging method, and super-resolution fluorescence technology becomes the hot spot of research work.Such as: stimulated emission damage
Consume (STED, stimulated emission depletion) technology, Structured Illumination microscope (SIM, structured
Illumination microscopy) technology and based on unimolecule positioning super-resolution imaging technology --- photoactivation positioning
Microscope (PALM, photo-activation localization microscopy) and random optical reconstruct aobvious mirror
(STORM, stochastic optical reconstruction microscopy) etc..
Structured Illumination micro-imaging technique forms modulation image by changing the space structure of illumination light, in modulation image
Just contain the high-frequency information more than ssystem transfer function.By calculation processing, the high-frequency information of modulation image is extracted, is breached
Diffraction limit, reconstruct obtain the image of super-resolution.Structured Illumination micro-imaging technique can be applied directly on fluorescence microscope,
Simple with structure, image taking speed is fast, does not require particularly fluorescent molecule, can be applied to real time imagery, thus receives
It is extensive to pay attention to.
But current Structured Illumination micro-imaging technique is to entire view other than to the illumination of target area cell
The even entire sample in field carries out Structured Illumination, wastes the energy of laser, while causing to other cells outside target area
Photobleaching and phototoxicity.In addition, light channel structure is complicated, laser power requirement also limits greatly its use scope.In research cell
Between interact and environmental stimuli under the conditions of cell effect and many cells selection concurrent observation iuntercellular behavior and structure
Relationship cannot exclude the target under the conditions of the whole audience with the photic influence of noise of exterior domain.
Summary of the invention
The present invention provides a kind of photoimaging methods system and method, to pass through spatial light modulator for Laser Modulation into mesh
Mark stimulation light and regional structure light, are radiated on sample and generate fluorescence signal, record and analyze the fluorescence signal, can get the light
According to the high fdrequency component on direction, the resolution ratio of the illumination region direction can be improved, reduce photic influence of noise, save laser energy
Amount, and simplify light channel structure.
The present invention provides a kind of photoimaging methods system, comprising:
Light source, for generating laser；
Spatial light modulator, for being modulated to the laser, generating goal stimulus light and being addressed to sample object
The regional structure light in region, the goal stimulus light and the regional structure light are radiated on sample, in the target of the sample
It is generated on region by the fluorescence signal for carrying cellular change information after the goal stimulus light stimulus；
Detector, for recording the fluorescence signal；
Control unit keeps the goal stimulus light constant, the mobile region for controlling the spatial light modulator
The phase of structure light, so that the regional structure striations on the sample is mobile；
The detector is also used to record the fluorescence signal for moving generate after the regional structure striations every time；
Described control unit is also used to carry out spectrum analysis to the corresponding fluorescent image of all fluorescence signals of record, obtain
Obtain the high-definition picture of target area when being rung by the goal stimulus shadow.
The present invention also provides a kind of photoimaging methods, comprising:
Generate laser；
Spatial light modulator is modulated the laser, generates goal stimulus light and is addressed to sample object region
Regional structure light, the goal stimulus light and regional structure light are radiated on sample, generate on the target area of the sample
By the fluorescence signal for carrying cellular change information after the goal stimulus light stimulus；
Record the fluorescence signal；
Controlling spatial light modulator keeps the goal stimulus light constant, the phase of the mobile regional structure light, so that
Regional structure striations on the sample is mobile；
Record moves the fluorescence signal generated after the regional structure striations every time；
Spectrum analysis is carried out to the corresponding fluorescent image of all fluorescence signals of record, is obtained by the goal stimulus shadow
The high-definition picture of target area when ringing.
From the embodiments of the present invention it is found that by SLM by Laser Modulation at goal stimulus light and regional structure light, irradiation
On sample, in target area, life carries the fluorescence signal of structural change of the cells after by the goal stimulus light stimulus, and remembers
The fluorescence signal is recorded, high fdrequency component can be obtained, that is, the resolution ratio of the illumination region direction can be improved, control SLM keeps the thorn
Laser is constant, and mobile rear direction can be improved so that the regional structure striations on sample is mobile in the phase of the mobile structure light
Resolution ratio, record move the fluorescence signal generated after the regional structure striations every time, carry out frequency spectrum point to these fluorescence signals
Analysis obtains the high-definition picture of target area when being rung by the goal stimulus shadow, after completing the move of stripe of entire plane,
The imaging resolution that all directions illumination region in the plane can be improved, when can be obtained stimulated shadow sound, specific region is thin
The high-definition picture of born of the same parents.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those skilled in the art without any creative labor, can be with root
Other attached drawings are obtained according to these attached drawings.
Fig. 1 is the light channel structure figure of photoimaging systems provided in an embodiment of the present invention；
Fig. 2 is the implementation process schematic diagram of photoimaging methods provided in an embodiment of the present invention；
The schematic diagram of goal stimulus light and regional structure optical illumination mode in Fig. 3 (a)~3 (d) embodiment of the present invention.
Specific embodiment
In order to make the invention's purpose, features and advantages of the invention more obvious and easy to understand, below in conjunction with the present invention
Attached drawing in embodiment, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described reality
Applying example is only a part of the embodiment of the present invention, and not all embodiments.Based on the embodiments of the present invention, those skilled in the art
Member's every other embodiment obtained without making creative work, shall fall within the protection scope of the present invention.
The embodiment of the invention provides a kind of photoimaging systems and method by one-photon excitation structure light, can be used for
The nano-resolution structural information of living cells is studied under the conditions of the light stimulus of any way with the response of light stimulus.The present embodiment benefit
The area one or more goal stimulus Guang Yu needed for being formed with spatial light modulator (SLM, Spatial Light Modulator)
Domain structure optical illumination pattern, is radiated in the specific region of microscopical sample, generates fluorescence letter based on the excitation of single photon effect
Number, and be imaged on detector (EMCCD, Electron-Multiplying CCD) by microcobjective collection.It can be according to quasi-
Be stimulated the stimulation sites and imaging region of target, at the same generate stimulation light (luminous point) and be stimulated object matching regional structure
Light, stimulation luminous point can be one, be also possible to multiple points, the position of point can according to need any adjusting.Imageable target can
To be one, it is also possible to multiple, can according to need any selection.When being stimulated light stimulus shadow by surrounding target cell
It rings, which contains the high-frequency information of the correspondence illumination region cell more than ssystem transfer function at stimulation moment.Control
SLM moves different structure light, obtains corresponding modulation image, and calculating can get by goal stimulus light stimulus time domain or cell
High lateral resolution image therefrom can analyze out the super-resolution knot of the cell under cell interaction and incentive condition
Structure information.It is further explained below.
Referring to Fig. 1, Fig. 1 is a kind of light channel structure schematic diagram of photoimaging systems.The photoimaging systems include:
Light source 1, for generating laser.
Specifically, light source 1 can be used visible laser, such as blue laser, and the wavelength of the blue laser can be with
For 405nm (nanometer), or 488nm.In another example ultraviolet laser, the wavelength of the ultraviolet laser can be
350nm.It can be used for the laser or mercury lamp and ultraviolet LD (Laser Diode, semiconductor laser) of single photon fluorescence imaging
It is ok with LED, the present embodiment relaxes the requirement to light source selection, so that the optional face of light source is wider.
Spatial light modulator (SLM) 5, the laser for generating to light source 1 are modulated, and are generated goal stimulus light and can be sought
Location is to the regional structure light of the target area of sample, and the goal stimulus light and regional structure light are radiated on sample 15, in sample
The fluorescence signal after by the goal stimulus light stimulus is generated on 15 target area, the cell of the target area is by the target
After stimulation, eucaryotic cell structure changes, and carries cellular change information in the fluorescence signal of generation.The spatial light modulator
It can be reflective pure phase type spatial light modulator.It is modulated, is reflected required using phase of the SLM5 to incident light
Goal stimulus light and regional structure light pattern.
Sample 15 is placed under microscopical microcobjective 14, includes eucaryotic cell structure in sample 15 in the present embodiment.
The goal stimulus light, for stimulating specific region or the specific cells of sample 15, the regional structure light, for illuminating
The cell of specific region around the stimulation light, the goal stimulus light and regional structure light are radiated on sample 15, are generated by the thorn
The specific region carries the fluorescence signal of cellular change information when laser effect.
Detector 18, for recording the fluorescence signal.
Control unit 19, can be computer, a control module being also possible in computer, in Fig. 1 by taking computer as an example.Control
Unit 19 processed is connected with SLM5, and controllable SLM5 generates the goal stimulus light and regional structure light, also SLM5 can control to keep
The goal stimulus light is constant, the phase of the mobile regional structure light, so that the regional structure striations on sample 15 is mobile.Control
Unit 19 can control SLM5 to keep stimulation light constant, and moving area structural light stripes in one direction,
Detector 18 is also used to record the fluorescence signal for moving generate after the regional structure striations every time.
The every movement of regional structure striations is primary, and detector 18 records first order fluorescence signal.The imaging of moving structure striations,
The resolution ratio of rotation rear direction just can be improved.And so on, after completing the move of stripe in a target area, so that it may
Improve the imaging resolution of the illumination zone in the plane in all directions target area.
The fluorescence signal that detector 18 records is stored in control unit 19.
Control unit 19 is also used to carry out spectrum analysis to the corresponding fluorescent image of all fluorescence signals of record, obtain
The high-definition picture of target area when being rung by the goal stimulus shadow.
All fluorescence signals that detector 18 records all form fluorescent image, and detector 18 is connect with control unit 19, will
The fluorescent image of record passes in control unit 19, and control unit 19 carries out frequency spectrum point to these fluorescent images according to Predistribution Algorithm
Analysis, high-definition picture when obtaining by goal stimulus shadow sound, in target area.
Further, it is also successively arranged between light source 1 and SLM5: beam-expanding collimation system and a half-wave plate.Wherein,
The beam-expanding collimation system includes two lens, i.e. lens 2 and lens 3, which is used for the laser for issuing light source 1
Expand the collimated light beam to form preset size.Half-wave plate 4 changes for changing the polarization state of the collimated light beam after beam-expanding collimation
For the linearly polarized light polarized in the target direction.
Further, photoimaging systems further include: spatial filter.
The spatial filter includes two lens and a diaphragm, and two lens are lens 6 and lens 8, and a diaphragm is
Diaphragm 7.Two lens form 4f system, the rear focus of the first lens 6 in two lens and the front focus weight of the second lens 8
It closes, diaphragm 7 is located in the focus of two lens coincidence, for that will pass through the zero-order diffraction light cut-off after SLM5.It can mention
The signal-to-noise ratio for rising imaging, efficiently uses the dynamic range of detector.
Further, photoimaging systems further include: fourier lense 9, reflecting mirror 10, Guan Jing 11.
Wherein, fourier lense 9 focus on the mirror 10 for that will pass through the light after the spatial filter；
Reflecting mirror 10, the light for focusing fourier lense 9 are reflected into pipe mirror 11；
Pipe mirror 11, the light for reflecting reflecting mirror 10 form parallel hot spot.
Further, photoimaging systems further include: exciter filter 12, dichroic mirror 13, microscope.
The parallel hot spot come out from pipe mirror 11 enters microscope by exciter filter 12, dichroic mirror 13, micro- at this
Under the focussing force of the microcobjective 14 of mirror, focuses on and form goal stimulus light and regional structure on the focal plane of microcobjective 14
Light, the focal plane of microcobjective 14 are overlapped with the sample face of sample 15.
Further, photoimaging systems further include: transmitting optical filter 16, the third lens 17；
Transmitting optical filter 16 is bandpass filter, for instead, being connected to fluorescence signal to laser height, only allowing fluorescence signal
Pass through.
The third lens 17, for by by the fluorescence signal generated on the target area of sample 15 focus on detection
On device 18.
In the present embodiment, Laser Modulation is radiated on sample at goal stimulus light and regional structure light by SLM,
Target area, which is generated, is carried the fluorescence signal of cellular change information later by the goal stimulus light stimulus, and records fluorescence letter
Number, high fdrequency component can be obtained, that is, the resolution ratio of the illumination region direction can be improved, control SLM keeps the stimulation light constant, moves
The phase of the regional structure light is moved, so that the regional structure striations on sample is mobile, the resolution ratio of mobile rear direction can be improved,
Record moves the fluorescence signal generated after the regional structure striations every time, carries out spectrum analysis to these fluorescence signals, obtains
The high-definition picture of target area can be improved after completing the move of stripe of entire plane when being rung by the goal stimulus shadow
The imaging resolution of all directions illumination region in the plane, when can be obtained stimulated shadow sound, the height of specific region cell
Image in different resolution.Meanwhile light channel structure is simplified, save the cost.
Referring to Fig. 2, Fig. 2 is a kind of implementation process schematic diagram of photoimaging methods.The photoimaging methods include:
S201, laser is generated；
Laser is generated by light source.The light source be can single photon fluorescence imaging laser.
S202, spatial light modulator are modulated the laser, generate goal stimulus light and are addressed to sample object area
The regional structure light in domain, the goal stimulus light and regional structure light are radiated on sample, generate on the target area of the sample
By the fluorescence signal for carrying cellular change information after the goal stimulus light stimulus；
To Laser Modulation, the regional structure light for generating goal stimulus light and being addressed to sample object region is radiated at SLM
On sample, fluorescence signal is generated in the target area of sample, which is on target area by goal stimulus light
The fluorescence signal generated after stimulation, after the cell of the target area is by the goal stimulus, eucaryotic cell structure changes, and is generating
The fluorescence signal in carry cellular change information.
Specifically, the beam-expanding collimation system including two lens carries out the laser to expand the directional light to form preset size
The polarization state of the collimated light beam after expanding is changed to the linear polarization polarized in the target direction by beam, half-wave plate by half-wave plate
Light, reflective pure phase type spatial light modulator are modulated the phase of the laser after changing polarization state, generate initial mesh
Mark stimulation light and regional structure light.
After successively carrying out space filtering, focusing, reflection to the initial goal stimulus light and regional structure light, pass through Guan Jing
Form parallel hot spot.Wherein, space filtering is realized by spatial filter, which includes two lens and one
A diaphragm, two lens form 4f system, the rear focus of the first lens in two lens and the front focus weight of the second lens
It closes, the relationship of the first lens and the second lens in optical path before and after forming position, diaphragm is located at the focus of two lens coincidence
On, for the zero-order diffraction light cut-off after SLM will to be passed through.Later, by fourier lense focus the light on the mirror,
Goal stimulus light is formed on the mirror surface of reflecting mirror and the light that fourier lense focuses is reflected by regional structure light pattern, reflecting mirror
On Guan Jing, parallel hot spot is formd by Guan Jing.
The parallel hot spot successively passes through exciter filter, dichroic mirror, microcobjective, gathers on the focal plane of the microcobjective
Coke forms illumination excitation hot spot, i.e. the goal stimulus light and regional structure light.The goal stimulus light and regional structure light are radiated at
On sample, the fluorescence that cellular change information is carried after by the goal stimulus light stimulus is generated on the target area of the sample
Signal.
S203, the fluorescence signal is recorded；
The fluorescence signal successively passes through dichroic mirror, transmitting optical filter, lens after being collected by the microcobjective, by detector
It receives and records.
Transmitting optical filter only allows fluorescence signal to pass through.
Angle between dichroic mirror and incident light can be 45 degree.
S204, control spatial light modulator keep the goal stimulus light constant, the phase of the mobile regional structure light, so that
Regional structure striations on the sample is mobile；
SLM and detector are connected to a computer respectively, and computer can control SLM by the control unit of its own and produce
The raw goal stimulus light and regional structure light, also can control SLM to keep the goal stimulus light constant, the mobile regional structure light
Phase, so that the regional structure striations on sample is mobile.Control unit can control SLM to keep stimulation light constant, and towards one
Direction moving area structural light stripes.
S205, record move the fluorescence signal generated after the regional structure striations every time；
The every movement of regional structure striations is primary, and detector just records first order fluorescence signal.The imaging of moving structure striations,
The resolution ratio of rotation rear direction just can be improved.And so on, after completing the move of stripe in a target area, so that it may
Improve the imaging resolution of the illumination zone in the plane in all directions target area.
S206, spectrum analysis is carried out to the corresponding fluorescent image of all fluorescence signals of record, obtained by the goal stimulus
The high-definition picture of target area when shadow is rung.
All fluorescence signals of detector record all form fluorescent image, and the fluorescent image of record is passed to calculating by detector
In machine, control unit carries out spectrum analysis to these fluorescent images according to Predistribution Algorithm, when acquisition receives goal stimulus shadow sound,
High-definition picture in target area.
In one example, Fig. 3 (a)~Fig. 3 (d) is referred to, Fig. 3 (a)~Fig. 3 (d) is target thorn in the embodiment of the present invention
The schematic diagram of laser and regional structure optical illumination mode, specifically, Fig. 3 (a) are unicellular by single stimulation light, many cells matching
Excitation structure light has in figure a cell to be stimulated the luminous point stimulation of light by one, and Fig. 3 (b) is unicellular by thorniness laser,
Many cells match excitation structure light, have in figure a cell to be stimulated the luminous point stimulation of light by 4, Fig. 3 (c) be many cells by
Single stimulation light, many cells match excitation structure light, and having 3 cells in figure respectively is stimulated the luminous point stimulation of light by 1；Figure
3 (d) be many cells by thorniness laser, and many cells match excitation structure light, have 3 cells respectively by 2,3,4 in figure
The luminous point stimulation of a stimulation light.In Fig. 3 (a)~Fig. 3 (d), structure light illuminates multiple cells, and structure light illuminates range not
Become.
In the present embodiment, Laser Modulation is radiated on sample at goal stimulus light and regional structure light by SLM,
The raw fluorescence signal after by the goal stimulus light stimulus in target area, and the fluorescence signal is recorded, high fdrequency component can be obtained,
The resolution ratio of the illumination region direction can be improved, control SLM keeps the stimulation light constant, and the phase of the mobile structure light makes
The regional structure striations obtained on sample is mobile, and the resolution ratio of mobile rear direction can be improved, and record moves the regional structure every time
The fluorescence signal generated after striations carries out spectrum analysis to these fluorescence signals, obtains mesh when being rung by the goal stimulus shadow
All directions lighting area in the plane can be improved after completing the move of stripe of entire plane in the high-definition picture for marking region
The imaging resolution in domain, when can be obtained stimulated shadow sound, the high-definition picture of specific region cell.
In the above-described embodiments, it all emphasizes particularly on different fields to the description of each embodiment, there is no the portion being described in detail in some embodiment
Point, it may refer to the associated description of other embodiments.
The above are the descriptions to photoimaging systems provided by the present invention and method, for those skilled in the art, according to
According to the thought of the embodiment of the present invention, there will be changes in the specific implementation manner and application range, to sum up, in this specification
Appearance should not be construed as limiting the invention.
Claims (9)
1. a kind of photoimaging systems, which is characterized in that the system comprises:
Light source, for generating laser；
Spatial light modulator, for being modulated to the laser, generating goal stimulus light and being addressed to sample object region
Regional structure light, the goal stimulus light and the regional structure light are radiated on sample, in the target area of the sample
It is upper to generate by the fluorescence signal for carrying cellular change information after the goal stimulus light stimulus；
Detector, for recording the fluorescence signal；
Control unit keeps the goal stimulus light constant, the mobile regional structure for controlling the spatial light modulator
The phase of light, so that the regional structure striations on the sample is mobile；
The detector is also used to record the fluorescence signal for moving generate after the regional structure striations every time；
Described control unit, be also used to the corresponding fluorescent image of all fluorescence signals of record carry out spectrum analysis, obtain by
The high-definition picture of target area when the goal stimulus shadow is rung.
2. system according to claim 1, which is characterized in that between the light source and the spatial light modulator also successively
It is equipped with: beam-expanding collimation system and a half-wave plate；
The beam-expanding collimation system includes two lens, and the laser beam expanding for issuing the light source forms the flat of preset size
Row light beam；
The half-wave plate, for the polarization state of the collimated light beam after beam-expanding collimation to be changed to the line polarized in the target direction
Polarised light.
3. system according to claim 1 or 2, which is characterized in that the system also includes: spatial filter；
The spatial filter includes two lens and a diaphragm, and described two lens form 4f system, described two lens
In the rear focuses of the first lens be overlapped with the front focus of the second lens, the diaphragm is located at the focus that described two lens are overlapped
On, for the zero-order diffraction light cut-off after the spatial light modulator will to be passed through.
4. system according to claim 3, which is characterized in that the system also includes: fourier lense, reflecting mirror, pipe
Mirror；
The fourier lense, for the light passed through after the spatial filter to be focused on the reflecting mirror；
The reflecting mirror, the light for focusing the fourier lense are reflected into the Guan Jing；
The Guan Jing, the light for reflecting the reflecting mirror form parallel hot spot.
5. system according to claim 4, which is characterized in that the system also includes: exciter filter, is shown dichroic mirror
Micro mirror；
The parallel hot spot enters microscope by the exciter filter, dichroic mirror, in the microscopical microcobjective
Under focussing force, focuses on the focal plane of the microcobjective and form the goal stimulus light and regional structure light, the coke
Plane is overlapped with the sample face of the sample.
6. system according to claim 5, which is characterized in that the system also includes: transmitting optical filter, the third lens；
The transmitting optical filter is bandpass filter, for high anti-to laser, to fluorescence signal conducting；
The third lens, for by by the fluorescence signal generated on the target area of the sample focus on institute
It states on detector.
7. a kind of photoimaging methods, which is characterized in that the described method includes:
Generate laser；
Spatial light modulator is modulated the laser, generates goal stimulus light and is addressed to the region in sample object region
Structure light, the goal stimulus light and regional structure light are radiated on sample, are generated on the target area of the sample by institute
State after goal stimulus light stimulus and carry the fluorescence signal of cellular change information；
Record the fluorescence signal；
Controlling spatial light modulator keeps the goal stimulus light constant, the phase of the mobile regional structure light, so that described
Regional structure striations on sample is mobile；
Record moves the fluorescence signal generated after the regional structure striations every time；
Spectrum analysis is carried out to the corresponding fluorescent image of all fluorescence signals of record, when obtaining by goal stimulus shadow sound
The high-definition picture of target area.
8. the method according to the description of claim 7 is characterized in that the spatial light modulator is modulated the laser,
The regional structure light for generating goal stimulus light and being addressed to sample object region, the goal stimulus light and regional structure illumination
It penetrates on sample, is generated on the target area of the sample and carry cellular change after by the goal stimulus light stimulus
The fluorescence signal of information includes:
The laser is carried out to expand the collimated light beam to form preset size；
The polarization state of the collimated light beam after expanding is changed to the linearly polarized light polarized in the target direction by half-wave plate；
Reflective pure phase type spatial light modulator is modulated the phase of the laser after changing polarization state, generates initial mesh
Mark stimulation light and regional structure light；
After successively carrying out space filtering, focusing, reflection to initial the goal stimulus light and regional structure light, pass through pipe mirror shape
At parallel hot spot；
The parallel hot spot successively passes through exciter filter, dichroic mirror, microcobjective, gathers on the focal plane of the microcobjective
Coke forms the goal stimulus light and regional structure light；
The goal stimulus light and regional structure light are radiated on sample, are generated on the target area of the sample by the mesh
The fluorescence signal of cellular change information is carried after mark stimulation light stimulus.
9. according to the method described in claim 8, it is characterized in that, described record the fluorescence signal and include:
The fluorescence signal successively passes through dichroic mirror, transmitting optical filter, lens after being collected by the microcobjective, by detector
It receives and records.
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