CN104515759B - Nonlinear organization optical illumination micro imaging method and system - Google Patents
Nonlinear organization optical illumination micro imaging method and system Download PDFInfo
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Abstract
The invention discloses a kind of nonlinear organization optical illumination micro imaging method, it is comprised the following steps：1) computed hologram is loaded on the digital micromirror array；2) the first space structure light field for activating fluorescin for meeting Sine distribution, the first space structure light field is produced to be irradiated to sample surfaces, Partial Protein is transformed on state of from dark-state；3) second space light field irradiating sample, makes the fluorescin on state of fluoresce, and collects fluorescence, is imaged in photodetector；2) and 3) 4) repeat the step, the multiple spatial frequencys of collection, multiple initial bit phases of each direction collection, obtain multiple original images, and reconstruct super resolution image according to GPU accelerating algorithms.Meanwhile, the invention also discloses a kind of nonlinear organization optical illumination micro imaging system.The present invention has the advantages that system imaging resolution ratio is higher, anti-fluorescence high is drifted, low phototoxicity, image taking speed are fast.
Description
Technical field
The present invention relates to bio-imaging field, and in particular to a kind of nonlinear organization optical illumination micro imaging method and be
System.
Background technology
Bio-imaging is to be related to the Front Fields of Life Sciences field of multi-crossed disciplines, by molecule-cell-tissue-live body
Interact to study the various imaging tip skills of biological function etc. " seeing " in real time, dynamically in different levels to living cells
Art has turned into the breach that the great achievement of current life science is produced.
Traditional light microscope is limited by optical diffraction limit, and the size of its diffraction pattern (Airy disc) is about light
The half of source wavelength and microscope objective numerical apertures ratio, imaging resolution typically up to is in 200-300nm or so, it is impossible to
Directly observe some important molecules such as gene, structural proteins, immune protein, RNA and its compound, active factors etc. be how
It is intracellular to be expressed, how to constitute the basic structure system of cell, the major vital activity of cell how is adjusted, such as cell increases
Grow, cell differentiation, Apoptosis and cell signal transmission etc..Reflect the characteristic dimension of these system properties all in the amount of nanometer
Level.Electron microscope (EM) can reach the resolution ratio of nanometer scale, can be to organelles such as cell interior vesica, mitochondrias
Positioning, but due to lacking corresponding probe mark, be not suitable for positioning single protein molecule.Electron microscope sample needs
It is fixed, while the irradiation of electron beam can also make biological sample be subject to irradiation damage, therefore be not suitable for carrying out living cells dynamic change
Observation.
The super-resolution optical microtechnic that resolution ratio reaches sub-micron even nanometer resolution can overcome traditional optical to show
The defect of micro mirror and electron microscope, breaks through diffraction limit, realizes super-resolution imaging ability on a cellular level.
Super-resolution optical microtechnic mainly includes three major types：1st, the micro- skill of photosensitive positioning based on fluorescence unimolecule positioning
Art (photo-activated localization microscopy, abbreviation PALM) and random optical reconstruct microtechnic
(stochastic optical reconstructionmicroscopy, abbreviation STORM)；2nd, by changing point spread function
Stimulated emission depletion microtechnic (stimulated emission depletion, abbreviation STED)；3rd, shone using structure light
The bright structured illumination microtechnic (structured illumination microscopy, abbreviation SIM) for exciting fluorescence.
The image-forming principle of PALM/STORM is identical, is all the random excitation-emission fluorescent photon one by one for utilizing fluorescence molecule,
Digitized by point spread function and obtain its center, so as to break through limitation of the Diffraction of light wave phenomenon to imaging resolution.To the greatest extent
Pipe PALM/STORM uses face detection imaging, but every time only to a small amount of fluorescent molecules imaging, it is necessary to activation-quenching fluorescence divides repeatedly
Son is imaged, and to obtain the complete cell super resolution image of a width, generally requires 10,000 images of collection, and data volume is big, imaging
Time is very long, and this causes that experiment is most of and completes on fixed cells, it is impossible to carry out living cells research.
In STED imaging processes, fluorescent material is set to light with a branch of exciting light, while with other high energy pulse laser
Device launches a branch of overlap, ring-like, longer wavelengths of laser by most fluorescent material in the first beam hot spot by being excited
Launch loss process is quenched, so as to reduce the diffraction area of fluorescence luminous point, considerably improves microscopical resolution ratio.But by
Need to use high intensity laser beam that (required peak power is 400~800MW/cm2 during 50-70 nanometer resolutions, and 5.8 receive in STED
Required laser power density reaches GW/cm2 magnitudes during rice resolution ratio), high power laser light easily causes biological tissue's damage, greatly
Constrain the range of application of STED.
SIM illuminates sample using modulated light source, and illumination light and fluorescence occur mixing, shift frequency in frequency domain, will be originally inseparable
The high resolution information distinguished is coded into fluoroscopic image, and high resolution information is obtained with reference to decoding FFT is calculated.
It is affected by the limitation of system diffraction limit in itself yet with illumination light field, SIM maximums can improve resolution ratio in theory
About one times, lateral resolution reaches about 100-150nm.Gustafsson.etal. [PNAS, 102 in 2005：13801(2005)]
A kind of nonlinear organization optical illumination super-resolution imaging method (NL-SIM) is proposed, using the saturation nonlinearity characteristic of fluorescence molecule,
The high-frequency information of sample can be obtained, any high-resolution fluorescence imaging can be realized in theory, but in order to realize saturation non-thread
Property fluorescent emission, it is necessary to excitation light power density very high, easily causes biological tissue's damage, simultaneously because NL-SIM needs to adopt
The data that collection is several times as much as SIM can just reconstruct a super resolution image, and image taking speed is slow, and these problems limit NL-SIM in life
The practical application of thing medical domain.
The content of the invention
In order to solve the above-mentioned technical problem, the invention provides a kind of nonlinear organization optical illumination micro imaging method and it is
System, its space structure light field activation fluorescence labeling sample for passing through low power density reuses the space with activation light field phase
Light field excites fluorescence and gathers imaging, can make to realize that 3 times of optical cut-offs are imaged, and imaging transverse resolution ratio reaches 60 and receives
Rice, system has a good mechanically stable, and wherein Z-direction drift is received m/h less than 5, and IMAQ speed reaches 100Hz, energy
Enough realize to living cells super-resolution imaging.
In order to achieve the above object, technical scheme is as follows：
Nonlinear organization optical illumination micro imaging method, it is comprised the following steps：
1) make digital micromirror array be operated in locking mode, computed hologram is loaded on digital micromirror array, calculate complete
The transmittance function of breath figure is：Wherein γ represents modulation degree, k0Representation space frequency,
Represent initial phase；
2) first laser for making space uniform be distributed is irradiated on digital micromirror array, and first laser is by digital microscope battle array
Holographic grating on row is diffracted into each level, and the light of each level is selected after the Fourier blade face filtering of the system that lens are constituted
A pair therein positive and negative first-order diffraction light are selected, a pair positive and negative first-order diffraction light pass sequentially through lens and microcobjective, are irradiated to sample
Product surface, makes positive and negative first-order diffraction light be interfered in the protein surface of sample, and generation meets the glimmering for activating of Sine distribution
First space structure light field of photoprotein, the first space structure light field is irradiated to sample surfaces, Partial Protein is changed from dark-state
To on state of；
3) second laser of the first laser using wavelength more than the 2) in step carrys out repeat step 2), it is met sinusoidal point
The second space light field for exciting fluorescin of cloth, using second space light field irradiating sample, makes in bright
The fluorescin of state fluoresces, and collects fluorescence, is imaged in photodetector；
4) change the computed hologram loaded on digital microarray, repeat the 2) and 3) step, the multiple spatial frequencys of collection,
The multiple initial bit phases of each direction collection, obtain multiple original images, finally use GPU accelerating algorithms, according to multiple original graphs
As reconstruct super resolution image.
Preferably, in step 4) in, before super resolution image is reconstructed according to multiple original images using GPU accelerating algorithms
Also include step：Judge whether whole gratings have loaded and be imaged：If it is not, return to step 2)；If so, judging multiple wavelength windows
Whether mouth has been imaged：If it is not, still return to step 2)；If so, closing digital micro-analysis lens array, stop collection image, GPU adds
Fast reconstructed image.
Preferably, in above-mentioned step 2) in, the system that lens are constituted is 4F systems, and the light of each level is in 4F systems
Fourier blade face is filtered.
Preferably, the first above-mentioned space structure light field and second space light field have identical position phase.
Preferably, above-mentioned a pair positive and negative first-order diffraction light are a pair of coherent beams pair.
Nonlinear organization optical illumination micro imaging system, it includes：
Light-source system (1), it includes the different laser of at least two wavelength (11,12), at least two light-balancing filters
(15,16), at least two half-wave plates (19,110), at least two dichroscopes (113,114), acousto-optic tunable filter (117),
Fiber coupler (118) and high-frequency vibrating table (119), in laser (11,12) at least one as activating light source, at least
Another sends as excitation source, the laser sent as the laser of activating light source and from the laser as excitation source
Laser, light-balancing filter (15,16), half-wave plate (19,110) are passed sequentially through respectively, dichroscope (113,114), acousto-optic can
Tunable filter (117), fiber coupler (118) and high-frequency vibrating table (119), finally give and are propagated extremely in same optical fiber
Few a branch of activation light beam and at least a branch of excitation beam；
Diffraction system (2), at least a branch of activation light beam and at least a branch of excitation beam are sequentially passed through in diffraction system respectively
The fiber coupler (21) that is sequentially placed along light path, lens (22), speculum (23), digital micromirror array (24), lens (25),
Intensity mask (26), lens (27), speculum (28) and lens (29), a pair corresponding positive and negative first-order diffractions are obtained per Shu Guangjun
Light；
Imaging system (3), the corresponding first positive and negative first-order diffraction light of activation light beam and exciting light that diffraction system (2) is produced
The corresponding second positive and negative first-order diffraction light of beam passes sequentially through dichroscope (31) and microcobjective (32) in imaging system (3),
And sample surfaces are irradiated to, the first positive and negative first-order diffraction light and the second positive and negative first-order diffraction light is sent out on sample protein surface respectively
Raw interference, produces space structure light field, wherein the first positive and negative first-order diffraction light produces at least one to meet being used for for Sine distribution
The first space structure light field of fluorescin is activated, the second positive and negative first-order diffraction light produces at least one use for meeting Sine distribution
In the second space light field for exciting fluorescin, the first space structure light field and second space light field make electromagnetic radiation
Fluorescence, and the fluorescence launched is by object lens (32) collection, by dichroscope (31), dichroscope (34), interferometric filter (35),
Repeatedly it is imaged on the test surface of photodetector through pipe lens (36), data is accelerated eventually through the GPU in imaging system (3)
Processing subsystem uses GPU accelerating algorithms, and the original image gathered according to multiple photodetectors reconstructs the oversubscription of fluorescent samples
Distinguish image.
Preferably, above-mentioned GPU acceleration data process subsystems include data collecting card, graphics workstation and graphics process
Video card, GPU accelerates the initial data that data process subsystem is gathered from photodetector, by GPU accelerating algorithms, reconstructs sample
The super-resolution micro-image of product.
Preferably, above-mentioned nonlinear organization optical illumination micro imaging system also sees control system including electronics, for reality
The synchronous triggering of existing acousto-optic tunable filter (117), digital micromirror array (24) and photodetector.
Preferably, above-mentioned light-source system (1) is placed on a shockproof experiment porch of air supporting, diffraction system (2) and imaging
System (3) is placed on the shockproof experiment porch of another air supporting, is transferred by band standard FC between two shockproof experiment porch of air supporting
The multimode fibre wire jumper connection of mouth, and a part for multimode fibre wire jumper is fixed on bumper, continue in imaging process
Random vibration.
Preferably, the first above-mentioned positive and negative first-order diffraction light and the second positive and negative first-order diffraction light are a pair of coherent beams pair.
The beneficial effects of the present invention are：
1) the more traditional SIM of system imaging resolution ratio is significantly increased；The present invention is glimmering using structure photoactivation by the first step
Light sample, second step excites fluorescence, this two steps structure photoactivation-be excited into using the light field with activation light field same-phase
Image space method so that system maximum useful space frequency reaches three times of traditional optical cut-off frequency, imaging resolution is most in theory
Three times can be improved greatly, and traditional SIM imaging methods excite direct imaging using a step structure light, in theory the maximum useful space
Frequency is only capable of reaching the twice of cut-off frequency, and imaging resolution is also only capable of improving twice；
2) anti-fluorescence high drift, low phototoxicity, the activation light field that uses of the present invention is low power density with light field is excited
Light field (representative value is W/cm2), far below traditional NL-SIM and STED (representative value is MW/cm2), relatively low power density
Weaken blanching effect of the laser to fluorescence molecule, beneficial to the extension fluorescent emission time, while can avoid to cell or biological tissue
Cause to damage, reduce phototoxicity, this is significant for biomedical micro-imaging；
3) image taking speed is fast, is capable of achieving to living cells super-resolution imaging；The present invention is constructed based on digital micromirror array
The light field of (digital micro-mirror device, abbreviation DMD) produces light path, shows that calculating is holographic on DMD
Figure, its transmittance function is represented by：Wherein γ represents modulation degree, k0Representation space is frequently
Rate (determines the direction of diffraction and the size of the angle of diffraction),Represent initial phase (determining the intensity distribution of light field) t, design
One group of computed hologram with different space frequency and initial bit phase, can obtain one group of diffraction with different relative position differences
Light field.The laser of space uniform distribution is irradiated on DMD, each level is diffracted into by holographic grating, by 4f systems, in Fourier
Blade face filters, and selects positive and negative 1 order diffraction light therein, and by the combination of ITRF lens and microcobjective, positive and negative one-level is spread out
Penetrate light to be interfered in sample surfaces, generation meets the space structure light field of Sine distribution；It is worthy of note that, in patent
It is mostly by imaging system that DMD is straight when being related to produce light field using DMD in CN102540446A and other Patents
Connect and be imaged onto sample surfaces, in theory its produce fringe distribution be not strictly to meet Sine distribution, and useful space frequency compared with
It is low.And the light field that the mode based on the DMD holographic grating diffraction interference of light that the present invention is set is produced strictly meets sinusoidal point
Cloth, and spatial frequency higher can be realized, the optical cut-off (using TIRF illuminations) of system, DMD works are can reach in theory
Make in pixel locking mode, switch speed is higher than 1KHz, while using high speed scientific research level SCOMS cameras, its highest frame frequency is same
Reach 1KHz so that present system totally adopts figure speed higher than 100Hz, can meet living cells imaging demand.
Brief description of the drawings
Fig. 1 is the schematic flow sheet of nonlinear organization optical illumination micro imaging method of the invention.
Fig. 2 is the structural representation of nonlinear organization optical illumination micro imaging system of the invention.
Fig. 3 is involved image reconstruction algorithm in nonlinear organization optical illumination micro imaging method of the invention and system
Schematic diagram.
Specific embodiment
The preferred embodiment that the invention will now be described in detail with reference to the accompanying drawings.
Fig. 1 is a kind of flow chart of nonlinear organization optical illumination super-resolution fluorescence imaging method.For convenience, at this
In embodiment, activation light refers in particular to the continuous solid body laser that wavelength is 405nm, and exciting light refers in particular to wavelength for 488nm continuous solid bodies
Laser, the power output of two beam laser is adjustable between 0 to 100mW.
In order to reach the purpose of the present invention, as shown in figs. 1 and 3, in nonlinear organization optical illumination micro-imaging of the invention
In some implementation methods of method, it includes step：
Step S101：With exciting light Uniform Illumination sample, traditional Both wide field illumination image is gathered by SCOMS, by moving
Dynamic object stage, selection needs the region of super-resolution imaging；
Step S102：Computer locks Jiao automatically；
Step S103：Excitation light power is heightened, fluorescent protein molecule is completely in dark-state, do not launch fluorescence；
Step S104：Control system changes the working frequency of AOTF, only allows activation light to pass through AOTF, and then be irradiated to
On DMD, a width computed hologram is loaded on DMD, the intensity transmittance function of computed hologram is：Wherein γ represents modulation degree, k0Representation space frequency (determines direction and the diffraction of diffraction
The size at angle),Represent initial phase (determining the intensity distribution of light field)；Activation light is diffracted into by computed hologram first
Each level, is then placed in a 4f systematic collection being made up of two lens after DMD, in the Fourier of 4f systems
On face, by an intensity mask frequency-selecting, only allow positive and negative first-order diffraction light to pass through, from after this 4f system exit, then pass through
A 4f system being made up of TIRF lens and microcobjective is crossed, sample surfaces are interfered, produce the cycle of cosine distribution to shine
Mingguang City, after activation light irradiation 2ms, makes AOTF cut off activation light；
Step S105, control system adjusts the working frequency of AOTF, only allows exciting light by AOTF, and be irradiated to DMD
On, exciting light is computed after hologram diffraction, and the cycle for producing cosine distribution in sample surfaces illuminates light field, makes in step
The fluorescent protein molecule transmitting fluorescence being activated in S104, now fluorescent intensity distribution is expressed as：
Wherein S (r) represents fluorescent protein molecular spatially distributed functions,The spatial frequency of activation light is represented,Represent
The spatial frequency of exciting light, it can be seen that be comprising spatial frequency in I (r)Composition, it is same it is micro- into
As in systemWherein kCThe optics of expression system is by frequency, therefore the imaging system maximum can be realized
Super-resolution imaging of 3 times of optics by frequency.SCOMS gathered datas are triggered simultaneously, and store data in graphics workstation
Among internal memory, excite and terminate after light irradiation 2ms data acquisition, AOTF cut-out exciting lights；
Step S106：Judge whether to complete 9 imagings of the exciting light of stripe direction, 9 stripe direction 9 differences of correspondence
K0Value, 9 azimuths of the exciting light of stripe direction and the angle in x-axis direction are respectively 2N π/9, wherein N=0, and 1,
2 .., 8, corresponding 7 of the initial bit of the exciting light of each stripe direction is differentValue, respectively M π/7, wherein M=0,
1,2 ..., 6,63 width original images are gathered altogether；If not collecting 63 width original images, step S103 is returned to；If collection
To 63 width original images, then step S107 is carried out；
Step S107：On graphics workstation, nonlinear organization optical illumination image processing algorithm is accelerated by GPU, passed through
The 63 width original image reconstructs the super-resolution fluorescence image of sample, and algorithm structure is as shown in Figure 3；Activation light and the exciting light
Wavelength can be changed through row according to the absorption-characteristic spectral emission of switchable protein, it is also possible to set that two beam wavelength are different to be excited
Light, to using two kinds of biological samples of switchable proteins mark through row dual colour imaging.
Wherein, in step S103, the Partial Protein of sample surfaces is transformed on state of from dark-state, and dark-state can not be swashed by laser
Send out and launch the steric configuration of fluorescence, on state of by laser excitation and can launch the steric configuration of fluorescence.Positive and negative traces diffraction
In lens and microcobjective that light is passed sequentially through, lens can use ITRF lens.
In order to reach the purpose of the present invention, as shown in Figures 2 and 3, in nonlinear organization optical illumination micro-imaging of the invention
In some implementation methods of system, it includes：
Light-source system 1, it includes the different laser 11 of two or more wavelength, 12, two or more neutrality filters
Wave plate 15,16, two or more half-wave plates 19,110, two or more dichroscopes 113,114, acousto-optic tunable filter
(AOTF) 117, fiber coupler 118 and high-frequency vibrating table 119, high-frequency vibrating table 119 are held in imaging process with 5KHz frequencies
Continuous vibration, eliminates the spatial coherence of laser, makes illumination light field space uniform.There is one in the laser 11,12 of different wave length
As activating light source, another as excitation source, from the laser sent as the laser of activating light source and from as exciting
The laser that the laser of light source sends, pass sequentially through respectively light-balancing filter 15,16, half-wave plate 19,110, dichroscope 113,
114th, acousto-optic tunable filter (AOTF) 117, fiber coupler 118 and high-frequency vibrating table 119, finally give in same optical fiber
A branch of activation light beam of middle propagation and a branch of excitation beam；
Diffraction system 2, a branch of activation light beam and a branch of excitation beam that light-source system 1 is produced sequentially passes through diffraction system respectively
The fiber coupler 21 that is sequentially placed along light path in system 2, lens 22, speculum 23, digital micromirror array DMD 24, lens 25,
Intensity mask 26, lens 27, speculum 28 and TIRF lens 29, a pair corresponding positive and negative first-order diffraction light are obtained per Shu Guangjun,
Wherein, positive and negative first-order diffraction light is a pair of coherent beams pair；
Imaging system 3, the corresponding positive and negative first-order diffraction light of activation light beam of the generation of diffraction system 2, excitation beam are corresponding
Positive and negative first-order diffraction light, passes sequentially through the dichroscope 31 and microcobjective 32 (100x, NA1.49, oil) in imaging system 3,
Sample surfaces are irradiated to, sample is fixed on three-dimensional precise motorized subject table 33, make positive and negative first-order diffraction light respectively in sample protein
Surface interferes, and produces space structure light field, wherein, a branch of corresponding positive and negative first-order diffraction light of light beam that activates produces one completely
The space structure light field for activating fluorescin of sufficient Sine distribution, the corresponding positive and negative first-order diffraction light of a branch of excitation beam is produced
A raw space structure light field for exciting fluorescin for meeting Sine distribution, the space structure light field light field makes sample
Product launch fluorescence, and the fluorescence of transmitting is collected by object lens 32, by dichroscope 31, dichroscope 34, interferometric filter 35, warp
Pipe lens 36 are repeatedly imaged on photodetector test surface, and data processing subsystem is accelerated eventually through the GPU in imaging system 3
System uses GPU accelerating algorithms, and the original image gathered according to multiple photodetectors reconstructs the super resolution image of fluorescent samples.
Wherein, above-mentioned GPU accelerates data process subsystem aobvious including data collecting card, graphics workstation and graphics process
Card, GPU accelerates the initial data that data process subsystem is gathered from photodetector, by GPU accelerating algorithms, reconstructs sample
Super-resolution micro-image.
Preferably, above-mentioned nonlinear organization optical illumination micro imaging system also sees control system including electronics, the electronics
See that control system can include that single-chip microcomputer and Switching Power Supply are used to realize acousto-optic tunable filter (117), digital micromirror array
(24) triggered with the synchronous of photodetector.
Above-mentioned light-source system (1) is placed on a shockproof experiment porch of air supporting, diffraction system (2) and imaging system (3)
The shockproof experiment porch of another air supporting is placed on, by the multimode with standard FC converting interfaces between two shockproof experiment porch of air supporting
Optical patchcord is connected, and a part for multimode fibre wire jumper is fixed on 5KHz bumpers, is continued in imaging process random
Vibrations.
For the light source subsystem of light-source system, its light source for being used to activate fluorescin can be that wavelength is 405nm
(wavelength selection depends on the fluorescence that experiment is used to the laser or LED light source of continuous solid body laser, or other wavelength
The type of albumen).
System of the invention be used for excite fluorescent protein emission fluorescence excitation source can be 488nm, 514nm,
561nm, 647nm continuously export solid state laser, or the laser of other wavelength, as shown in Fig. 2 at most may be used in system
To set No. 4 lasers 11 simultaneously, 12,13,14, often increase laser all the way, such as 13,14, need to correspondingly increase neutral filter
Mating plate 17,18, half-wave plate 111,112, dichroscope 115,116.
As shown in Fig. 2 imaging system at least includes 1 photodetector of SCMOS 1, photodetector can also be herein
EMCCD, and according to the difference of fluorescent marker protein, 4 SCMOS photodetectors can be at most set, often increase all the way
SCMOS detects light path, and such as SCMOS2 need to accordingly increase dichroscope 38, interferometric filter 39, pipe lens (Tube Lens)
310。
It is used to activate the light source of fluorescin in light-source system 1, can is the continuous solid body laser that wavelength is 405nm,
Also according to the laser of other wavelength of the type selecting of fluorescin of experiment use.
Above-described is only the preferred embodiment of the present invention, it is noted that for one of ordinary skill in the art
For, without departing from the concept of the premise of the invention, various modifications and improvements can be made, these belong to the present invention
Protection domain.
Claims (9)
1. nonlinear organization optical illumination micro imaging method, it is characterised in that comprise the following steps：
1) make digital micromirror array be operated in locking mode, computed hologram, the meter are loaded on the digital micromirror array
Calculate hologram transmittance function be：Wherein γ represents modulation degree, k0Representation space is frequently
Rate,Initial bit phase is represented, r represents transmitance；
2) first laser for making space uniform be distributed is irradiated on the digital micromirror array, and the first laser is by the numeral
Holographic grating on micro mirror array is diffracted into each level, the Fourier blade face of the system that the light of each level is constituted in lens
After filtering, a pair positive and negative first-order diffraction light therein is selected, the pair of positive and negative first-order diffraction light passes sequentially through lens and micro-
Object lens, are irradiated to sample surfaces, the positive and negative first-order diffraction light is interfered in the protein surface of sample, produce and meet sinusoidal
The first space structure light field for activating fluorescin of distribution, the first space structure light field is irradiated to sample surfaces,
Partial Protein is set to be transformed on state of from dark-state；
3) second laser of the first laser using wavelength more than the 2) in step carrys out repeat step 2), it is met sinusoidal point
The second space light field for exciting fluorescin of cloth, using the second space light field irradiating sample, makes place
Fluoresced in the fluorescin of on state of, and collect fluorescence, be imaged in photodetector；
4) change the computed hologram loaded on the digital micromirror array, repeat the 2) and 3) step, collection is multiple empty
Between frequency, the multiple initial bit phases of each stripe direction collection obtain multiple original images, GPU accelerating algorithms are finally used, according to institute
Multiple original images reconstruct super resolution image stated.
2. nonlinear organization optical illumination micro imaging method according to claim 1, it is characterised in that in the step 4)
In, also included step before using the GPU accelerating algorithms multiple original images reconstruct super resolution image according to：Judge
Whether whole gratings have loaded and have been imaged：If it is not, return to step 2)；If so, judging whether multiple wavelength windows have been imaged：
If it is not, still return to step 2)；If so, closing the digital micromirror array, stop collection image, GPU accelerates reconstructed image.
3. nonlinear organization optical illumination micro imaging method according to claim 1, it is characterised in that in the step 2)
In, the system that the lens are constituted is 4F systems, and the light of each level is filtered on the Fourier blade face of the 4F systems.
4. nonlinear organization optical illumination micro imaging method according to claim 1, it is characterised in that first space
Light field and the second space light field have identical position phase.
5. nonlinear organization optical illumination micro imaging method according to claim 1, it is characterised in that the pair of positive and negative
First-order diffraction light is a pair of coherent beams pair.
6. nonlinear organization optical illumination micro imaging system, it is characterised in that including：
Light-source system (1), it include the different laser of at least two wavelength (11,12), at least two light-balancing filters (15,
16), at least two half-wave plates (19,110), at least two dichroscopes (113,114), acousto-optic tunable filter (117), optical fiber
Coupler (118) and high-frequency vibrating table (119), in the laser (11,12) at least one as activating light source, at least
Another sends as excitation source, the laser sent as the laser of activating light source and from the laser as excitation source
Laser, pass sequentially through respectively the light-balancing filter (15,16), the half-wave plate (19,110), the dichroscope (113,
114), the acousto-optic tunable filter (117), the fiber coupler (118) and the high-frequency vibrating table (119), final
To at least a branch of activation light beam and at least a branch of excitation beam propagated in same optical fiber；
Diffraction system (2), at least a branch of activation light beam and at least a branch of excitation beam sequentially pass through diffraction system respectively
Fiber coupler (21), the first lens (22), digital micromirror array (24), the first speculum being sequentially placed along light path in system
(23), the second lens (25), intensity mask (26), the 3rd lens (27), the second speculum (28) and the 4th lens (29), per beam
Light obtains a pair corresponding positive and negative first-order diffraction light；
Imaging system (3), the corresponding first positive and negative first-order diffraction light of activation light beam and exciting light that the diffraction system (2) produces
The corresponding second positive and negative first-order diffraction light of beam passes sequentially through the first dichroscope (31) and microcobjective in imaging system (3)
(32), and sample surfaces are irradiated to, make the described first positive and negative first-order diffraction light and the second positive and negative first-order diffraction light respectively in sample
Protein surface is interfered, and produces space structure light field, wherein the first positive and negative first-order diffraction light produces at least one to meet
The first space structure light field for activating fluorescin of Sine distribution, the second positive and negative first-order diffraction light produces at least one
The individual second space light field for exciting fluorescin for meeting Sine distribution, the first space structure light field and second
Space structure light field makes electromagnetic radiation fluorescence, and the fluorescence of transmitting is collected by object lens (32), by the first dichroscope (31), the
Two dichroscopes (34), interferometric filter (35), are repeatedly imaged, finally through pipe lens (36) on the test surface of photodetector
Accelerate data process subsystem to use GPU accelerating algorithms by the GPU in imaging system (3), adopted according to multiple photodetectors
The original image of collection reconstructs the super resolution image of fluorescent samples.
7. nonlinear organization optical illumination micro imaging system according to claim 6, it is characterised in that the GPU accelerates
Data process subsystem includes data collecting card, graphics workstation and graphics process video card, and the GPU accelerates data processing subsystem
The initial data united from photodetector collection, by GPU accelerating algorithms, reconstructs the super-resolution micro-image of sample.
8. nonlinear organization optical illumination micro imaging system according to claim 6, it is characterised in that also including electronics control
System processed, the synchronization for realizing the acousto-optic tunable filter (117), digital micromirror array (24) and photodetector is touched
Hair.
9. nonlinear organization optical illumination micro imaging system according to claim 6, it is characterised in that the light-source system
(1) a shockproof experiment porch of air supporting is placed on, the diffraction system (2) and the imaging system (3) are placed on another gas
Shockproof experiment porch is floated, is connected by the multimode fibre wire jumper with standard FC converting interfaces between two shockproof experiment porch of air supporting,
And a part for the multimode fibre wire jumper is fixed on bumper, random vibration is continued in imaging process.
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