CN103543135B - A kind of nano-precision hot spot alignment methods based on Fluorescence lifetime distribution and device - Google Patents
A kind of nano-precision hot spot alignment methods based on Fluorescence lifetime distribution and device Download PDFInfo
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Abstract
The present invention discloses a kind of nano-precision hot spot alignment methods based on Fluorescence lifetime distribution, it is applicable to have the STED super-resolution microscopic system of pulse excitation light and continuous loss light, first sample is carried out horizontal two-dimensional scan, according to obtaining Fluorescence lifetime distribution and fluorescence hot spot carries out horizontal alignment, then single fluorescent grain is carried out axial scanning imagery, analyze with the axial two dimensional image of fluorescence intensity and life distribution, regulate the divergency of continuous loss light, complete axially aligning of hot spot. The invention also discloses a kind of nano-precision hot spot alignment device based on Fluorescence lifetime distribution. Apparatus of the present invention are simple for structure, and fast and easy high precision adjusts, it is not necessary to add additionally detect light path because adopting nm gold particles; Sharpness of regulation height, hot spot alignment precision can reach nanometer scale.
Description
Technical field
The invention belongs to the micro-field of optical ultra-discrimination, in particular in a kind of stimulated emission depletion microscopy based on the nano-precision hot spot alignment methods of Fluorescence lifetime distribution and device.
Background technology
Confocal fluorescent microscopy has the advantages such as Noninvasive, high specific and highly sensitive, is the important research method in life science always. But the diffraction effect due to light wave, about its imaging resolution is limited to half wavelength, cannot meet the research demand of current life science.
In order to break the restriction of the Diffraction of light wave limit, explore the essence of life and the mechanism of disease further, scientists is devoted to the research of super-resolution optical microscopy, from the nineties in 20th century, propose multiple optical ultra-discrimination microscopic method: stimulated emission depletion microscopy (Stimulatedemissiondepletionmicroscopy, STED), photoactivation location microscopy (photoactivatedlocalizationmicroscopy, PALM), random optical rebuilds microscopy (stochasticopticalreconstructionmicroscopy, STORM), saturated structures optical illumination microscopy (saturatedstructuredilluminationmicroscopy, SSIM), etc.. among those methods, STED super-resolution microscopy has the application prospect of the fastest writing speed and most future at present.
First STED super-resolution microscopy is proposed in 1994 by S.W.Hell. In the STED super-resolution microscopic system of a standard, the red shift loss light of a branch of phase encoding is introduced into confocal system, produces an annular hollow hot spot (donutspot) by the focal plane that focuses on of high-NA microcobjective. Loss light consumes the excitation electron of the solid focal beam spot periphery of exciting light by stimulated radiation effect, suppresses the autofluorescence effect of hot spot periphery and then obtains less effective point spread function (PointSpreadFunction, PSF). Therefore, best STED imaging effect needs the solid focal beam spot center of exciting light and the annular hollow focal beam spot center of loss light accurately to overlap. In traditional method, two focal beam spot centers alignment be by two-beam respectively scanning nano gold grain carry out imaging, then by contrast two width pictures adjustment two-beam focal beam spots relative position, repeatedly repeat until spot center overlap. There is following shortcoming in this kind of alignment methods: 1. needs additionally to add scattered light imaging optical path: because STED microscopic system itself is for wavelength of fluorescence, collection unit divides the spectral filter that filtering exciting light and loss light are housed, nm gold particles is then utilize exciting light and loss scattering of light light to carry out imaging, therefore to adopt nm gold particles alignment to need to add extra light path; 2. exciting light and loss light need independent scanning imagery, sample drift therebetween can produce error needs correction: common acquisition system many employings avalanche diode (APD) records scanning intensity, but APD cannot differentiate the wavelength collecting signal, therefore exciting light and loss light need independent scanning imagery, contrast twice sweep result, will certainly introduce sample drift error. 2013, having external seminar to propose adopts fluorescent grain that the focal beam spot center of two-beam in STED is directed at (Auto-aligningstimulatedemissiondepletionmicroscopeusinga daptiveoptics.Opticsletters, 2013), but its method does not overcome Time share scanning introduces the problems such as sample drift error, the method is distributed by fluorescence intensity and determines focal beam spot center in addition, and fluorescence intensity distribution depends on exciting light and the strength ratio of loss light.
Summary of the invention
The present invention provides a kind of nano-precision hot spot alignment methods based on Fluorescence lifetime distribution and device, changed by the horizontal and vertical Fluorescence lifetime distribution of fluorescence hot spot caused due to loss light in detection STED microscopy, the relative position of adjustment life-span point spread function center with corresponding fluorescent foci spot center, thus the center of two focal beam spots is carried out three-dimensional alignment. Present configuration is simple, it is not necessary to adopt nm gold particles, it is not necessary to add extra light path, it is not necessary to Time share scanning imaging, and alignment precision is in nanometer scale, and to be specially adapted to exciting light be pulse light, loss light is the STED super-resolution microscopic system of continuous light.
Based on a nano-precision hot spot alignment methods for Fluorescence lifetime distribution, it is applicable to have the STED super-resolution microscopic system of pulse excitation light and continuous loss light, comprises following step:
1) enable pulse excitation light and continuous loss light simultaneously, described pulse excitation light and continuous loss light focus on fluorescent samples surface through microcobjective after being converted to rotatory polarization, collect the fluorescence that fluorescent grain sends, obtain focusing on fluorescence intensity and the fluorescence lifetime of point;
2) fluorescent samples described in laterally mobile, repeating step 1), obtain fluorescence intensity information and the fluorescence lifetime information of each scanning spot in corresponding scanning area;
3) to step 2) the fluorescence intensity information that obtains and fluorescence lifetime information analyzes, select the fluorescence intensity hot spot of single fluorescent grain, matching spot center and record hot spot centre coordinate, extract the life distribution that described fluorescence intensity hot spot is corresponding simultaneously, the longest point in matching life-span also records the longest point coordinate, calculates spot center and the distance of life-span the longest point;
4) distance calculated according to step 3), changes the angle of the incident microcobjective of described continuous loss light, spot center is overlapped completely with life-span the longest point, completes the transverse direction alignment of hot spot;
5) to single the fluorescent grain selected in step 3), repeating step 1) in operation, choose the axial slices through particle center to scan, and the fluorescent samples described in moving completes axial two-dimensional scan, obtain fluorescence intensity information and the fluorescence lifetime information of corresponding each point;
6) corresponding according to the oval fluorescence hot spot of single fluorescent grain in step 5) and described fluorescence hot spot life distribution, regulate the divergency of described continuous loss light, make long lifetime region run through the region intermediate of fluorescence hot spot and distribute axisymmetricly along oval fluorescence hot spot major axis and minor axis, complete axially aligning of hot spot.
Using the particle sparse region of fluorescent samples as described step 2) in scanning area, described particle sparse region defining method is: be used alone pulse excitation light, and be rotatory polarization by pulse excitation optical modulation, then sample surfaces is focused on by microcobjective, sample surfaces is carried out two-dimensional scan, collects the fluorescence that fluorescent grain sends, obtain corresponding scan image, distribution according to fluorescent grain on described scan image, chooses described particle sparse region.
Wherein, pulse excitation light had been modulated to parallel light before pulse excitation optical shutter; Continuous loss light had been modulated to parallel light before continuous loss optical shutter, and have passed through phase modulated, the effect of phase modulated be in order to afterwards under the focussing force of microcobjective focal beam spot become hollow bagel shape hot spot.
Wherein, pulse excitation light is through focal beam spot and the final multimode optical fibers end face the collecting signal conjugate points each other in systems in which of microcobjective.
Wherein, described fluorescent samples is 100 nano-fluorescent grains, because 100 nano-fluorescent grains have good bleach-resistant ability, it is also possible to adopt the fluorescent grain of 40 nanometers��80 nano-scales.
In step 2) in, it is also possible to adopt galvanometer scanning system to realize the transverse scan of sample.
Present invention also offers a kind of nano-precision hot spot alignment device based on Fluorescence lifetime distribution, comprising:
The pulse excitation photogenerated device arranged successively along pulse excitation light road and pulse excitation optical shutter;
Along continuous loss photogenerated device, continuously loss optical shutter and dichroscope that continuous loss light light path is arranged successively;
Quarter-wave plate, for being rotatory polarization by described pulse excitation light and continuous loss optical modulation;
Microcobjective, for focusing to fluorescent samples by described rotatory polarization and collect fluorescence;
Information collecting device, collects strength information and the life information of fluorescence for gathering described microcobjective;
And the computer being connected with described information collecting device.
Wherein, described pulse excitation photogenerated device comprises pulsed laser, the first single-mode polarization maintaining fiber, the first collimating lens, first polarizer, the first quarter-wave plate and the 1/1st wave plate arranged successively along light path.
Wherein, described continuous loss photogenerated device comprises continuous wave laser, the 2nd single-mode polarization maintaining fiber, the 2nd collimating lens, the 2nd polarizer, the 2nd quarter-wave plate, the 1/2nd wave plate and 0��2 �� vortex phase plate arranged successively along light path.
Wherein, described information collecting device comprises spectral filter, lens, multimode optical fibers, avalanche diode and the time correlation single photon counting system arranged successively along the light path collecting fluorescence, and time correlation single photon counting system is connected with described computer and pulse excitation photogenerated device respectively.
Spectral filter is used for the light of filtering other any wavelength except spontaneous radiation wavelength of fluorescence, lens be used for will fluorescent foci that collect in multimode optical fibers end face, multimode optical fibers is connected with avalanche diode (APD), avalanche diode (APD) time correlation single photon counting (TCSPC) system is connected, time correlation single photon technique system is connected with computer, and time correlation single photon counting system is responsible for the number of photons of avalanche diode record and photon life information are supplied to computer.
The principle of work of the present invention is as follows:
Pulse excitation light and continuous loss light through phase modulated are laterally forming solid circles focal beam spot and annular hollow focal beam spot respectively under the focussing force of microcobjective, are axially forming solid oval focal beam spot (oval major axis is along optical axis direction) and bottleneck shape hollow hot spot respectively.
Fluorescence lifetime refers to that fluorescence molecule decays to, at the molecule number of excited state, the time that original 1/e experiences, i.e. " mean residence time " of fluorescence molecule on upper energy level. If only opening exciting light, then within the scope of exciting light focal beam spot, fluorescence molecule all will be excited to energy level and spontaneous transition emitting fluorescence, then whole under not considering other environmental factorss excite the expression formula of any point fluorescence lifetime within the scope of hot spot to be write as
��(r)=1/kf
Wherein, kfIt is the speed of fluorescence molecule spontaneous transition emitting fluorescence, for k given fluorescence molecule kindfValue is determined, if then only opening exciting light, then the fluorescence lifetime that finally single fluorescent grain is corresponding is equal one, as shown in Figure 3.
If open exciting light and loss light simultaneously, then within the scope of exciting light focal beam spot, fluorescence molecule all will be excited to energy level, from only to open exciting light different, excite the expression formula of fluorescence lifetime within the scope of hot spot to need to be changed to
��(r)=1/(kf+��ISTED(r))
Wherein, kfIt is the speed of fluorescence molecule spontaneous transition emitting fluorescence, for k given fluorescence molecule kindfValue is determined, �� is the absorption cross section of fluorescence molecule stimulated radiation, ISTEDR () is the loss light intensity at r place in focal beam spot, namely the region fluorescence lifetime that there is loss light shortens, and it is relevant with the intensity of loss light to shorten degree, if when then annular hollow loss light focal beam spot overlaps with solid exciting light focusing circle spot, the coincidence spot center life-span is the longest, as shown in Figure 4.
Nano-precision alignment is carried out according to the corresponding coincidence hot spot Fluorescence lifetime distribution of 100 nano-fluorescent grains, as shown in the table:
In transverse direction, the focal beam spot of exciting light is solid circles focal beam spot, and the focal beam spot of loss light is annular hollow focal beam spot, if when only opening exciting light, corresponding life distribution is the round spot of equal; If two-beam is opened simultaneously, then when the horizontal misalignment of two-beam, then life-span the longest point is not in fluorescent grain spot center, and when two-beam focal beam spot laterally overlaps, the Fluorescence lifetime distribution center of circle that fluorescent grain hot spot is corresponding is the highest.
In axis, the focal beam spot of exciting light is solid oval focal beam spot (oval major axis is along optical axis direction), and loss light focal beam spot is bottleneck shape hollow hot spot, if when only opening exciting light, corresponding life distribution is the oval spot of equal; If two-beam is opened simultaneously, then when two-beam axial misalignment, the long lifetime does not run through whole ellipse light spot and does not distribute axisymmetricly along the life distribution of oval spot major axis and minor axis; When two-beam focal beam spot axially overlaps, long lifetime distribution runs through the axial spot center of fluorescent grain and distributes axisymmetricly along oval fluorescence hot spot major axis and minor axis.
If the light path of exciting light is adjusted (namely ensureing focal beam spot and the final multimode optical fibers end face of the collecting signal in systems in which each other conjugate points of pulse excitation light through microcobjective), then the transverse direction of two-beam is overlapped and can be realized by the incident angle regulating the angle of the 2nd dichroscope to realize loss light; The axis of two-beam overlaps and the converging and diverging degree of loss light can be regulated to realize according to axial hot spot life distribution. The alignment of nano-precision can be reached by setting the scanning resolution (nano-precision) of nanometer translation stage.
Present configuration is simple, and alignment precision is in nanometer scale, it is possible to the degree of registration of three-dimensional regulation two light beam, and to be specially adapted to exciting light be pulse light, loss light is the STED super-resolution microscopic system of continuous light.
Compared with prior art, the present invention has following useful technique effect:
(1) systematic error is little, it is not necessary to introduce Time share scanning cause sample drift error because adopting nm gold particles;
(2) sharpness of regulation height, hot spot alignment precision can reach nanometer scale;
(3) apparatus structure is succinct, and fast and easy high precision adjusts, it is not necessary to add additionally detect light path because adopting nm gold particles.
Accompanying drawing explanation
Fig. 1 is the device schematic diagram of the nano-precision hot spot alignment methods based on life distribution;
Fig. 2 is one group of generating apparatus schematic diagram for the twin-beam of life distribution nano-precision hot spot alignment methods;
Horizontal exciting light point spread function intensity distribution and corresponding Fluorescence lifetime distribution sectional view when Fig. 3 is only open exciting light;
Lateral loss light point spread function intensity distribution and corresponding Fluorescence lifetime distribution sectional view when Fig. 4 is open exciting light and loss light simultaneously;
The different light beam of (a��j) graph representation in Fig. 5 focuses on the light spot image that sample surfaces is formed.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in detail, but the present invention is not limited to this.
As shown in Figure 1, the device of a kind of nano-precision hot spot alignment methods based on life distribution, comprising: pulse excitation optical shutter 1, first dichroscope the 2, two dichroscope 3, quarter-wave plate 4, microcobjective 5, nanometer translation stage 6, first spectral filter 8, first lens 9, multimode optical fibers 10, avalanche diode (APD) 11, time correlation single photon counting (TCSPC) system 12, continuous loss optical shutter 13, computer 14.
Wherein, pulse excitation optical module A transponder pulse exciting light, the optical axis of pulse excitation light arranges pulse excitation optical shutter 1 and the first dichroscope 2 successively, pulse excitation optical shutter 1 is for the switch of setting pulse exciting light, first dichroscope 2 is negative 45 �� with the angle of pulse excitation light optical axis, and the effect of the first dichroscope 2 is used for echo pip exciting light and transmission fluorescence, pulse excitation light is reflected as echo pip exciting light through the first dichroscope 2, echo pip exciting light optical axis and pulse excitation light optical axis are in 90 ��, the optical axis of echo pip exciting light is placed the 2nd dichroscope 3 successively, quarter-wave plate 4, microcobjective 5 and nanometer translation stage 6, 2nd dichroscope 3 is positive 45 �� with the angle of echo pip exciting light, the effect of the 2nd dichroscope 3 is Transflective pulse excitation light, fluorescence and the continuous loss light of reflection, the effect of quarter-wave plate 4 ensures that the light inciding microcobjective 5 is rotatory polarization, microcobjective 5 is for focusing on exciting light, loss light and collection fluorescence, nanometer translation stage 6 is for completing the scanning to the horizontal axial two dimension of two peacekeepings of sample, the reverse extending line of echo pip exciting light is placed the first spectral filter 8, first lens 9 and multimode optical fibers 10 successively, the effect of the first spectral filter 8 only allows the fluorescence of sample spontaneous transition to pass through, and the end face of multimode optical fibers 10 is placed on the burnt face of the first lens 9, multimode optical fibers 10 is connected with avalanche diode (APD) 11, and the effect of avalanche diode (APD) 11 detects the fluorescent photon collected by microcobjective 5, avalanche diode (APD) 11 is connected with time correlation single photon counting (TCSPC) system 12 with pulse excitation light generation module A, and time correlation single photon counting (TCSPC) system is used for the photon signal come from APD conduction is converted into strength information and life information, computer 14 is connected with nanometer translation stage 6 with time correlation single photon counting (TCSPC) system 12, and computer completes sample horizontal and vertical two dimension intensity and the imaging in life-span by period correlated single photon counting (TCSPC) system 12 and nanometer translation stage 6, continuous loss optical module B launches continuous loss light, the optical axis of continuous loss light arranges continuous loss optical shutter 13 and the first dichroscope 3 successively, continuous loss light reflects and echo pip exciting light optical axis coincidence through the 2nd dichroscope 3, and continuous loss optical shutter 13 is for controlling the switch of continuous loss light.
The present embodiment gives one group of pulse excitation light and the generating apparatus of continuous loss light, but generation method is not limited in this.
Such as Fig. 2 A, the generating apparatus of a kind of pulse excitation light for life distribution nano-precision hot spot alignment methods, comprises pulsed laser 15, first single-mode polarization maintaining fiber 16, first collimating lens 17, first polarizer 18, first quarter-wave plate 19 and the 1/1st wave plate 20.
Wherein, laser launched by pulsed laser 15, outgoing after the first single-mode polarization maintaining fiber 16 is coupled, successively through the first collimating lens 17, first polarizer 18, first quarter-wave plate 19 and the 1/1st wave plate 20, the outgoing end face of the first single-mode polarization maintaining fiber 16 is at the front focus place of the first collimating lens 17, the effect of the first collimating lens 17 is used for collimated light beam, the effect of first polarizer 18 is for ensureing that collimation light is line polarisation, first quarter-wave plate 19 and the 1/1st wave plate 20 for compensating the phase differential of pulse excitation light light path and then ensure that final focal beam spot is rotatory polarization.
Such as Fig. 2 B, the generating apparatus of a kind of continuous loss light for life distribution nano-precision hot spot alignment methods, comprise continuous wave laser 21,2nd single-mode polarization maintaining fiber 22,2nd collimating lens 23,2nd polarizer the 24, two quarter-wave plate the 25, the 1/2nd wave plate 26 and 0��2 �� vortex phase plate 27.
Wherein, continuous wave laser 21 launches laser, and outgoing after the 2nd single-mode polarization maintaining fiber 22 is coupled, successively through the 2nd collimating lens the 23, two polarizer the 24, two quarter-wave plate the 25, the 1/2nd wave plate 26 and 0��2 �� vortex phase plate 27. The outgoing end face of the 2nd single-mode polarization maintaining fiber 22 is at the front focus place of the 2nd collimating lens 23, the effect of the 2nd collimating lens 23 is used for collimated light beam, the effect of the 2nd polarizer 24 is for ensureing that collimation light is line polarisation, 2nd quarter-wave plate 25 and the 1/2nd wave plate 26 for compensating the phase differential of continuous loss light light path and then ensure that final focal beam spot is rotatory polarization, and 0��2 �� vortex phase plate 27 is for the formation of be laterally annular hollow, be axially bottleneck shape hollow hot spot.
Adopting a kind of nano-precision hot spot alignment methods based on life distribution shown in Fig. 1, its process is as follows:
(1) suitable transverse scan region is chosen;
Pulse excitation optical shutter 1 is opened, and continuous loss optical shutter 13 is closed, and pulse excitation light is reflected as echo pip exciting light through the first dichroscope 2, and echo pip exciting light light path is orthogonal with pulse excitation light light path, echo pip exciting light, after the 2nd dichroscope 3 transmission, is modulated to rotatory polarization through quarter-wave plate 4, finally focuses on sample surfaces by microcobjective 5, 100 nano-fluorescent grain samples 7 are fixed on nanometer translation stage 6, fluorescent grain ground state electronics by the supreme energy level of echo pip excitation, spontaneous transition emitting fluorescence, fluorescence is collected through same microcobjective 5, successively successively by quarter-wave plate 4, 2nd dichroscope 3, first dichroscope 2 and the first spectral filter 8, multimode optical fibers 10 end face is focused on by the first lens 9, and avalanche diode (APD) 11 record connected by multimode optical fibers 10 the other end, avalanche diode 11 is connected with time correlation single photon counting (TCSPC) system 12 with pulse excitation light generation module A, time correlation single photon counting system 12 is responsible for the number of photons that recorded by avalanche diode 11 and photon life information is supplied to computer 14, computer 14 can calculate fluorescence intensity and the fluorescence lifetime of this scanning spot, after completing this some record, enter scanning and the record of subsequent point, computer completes horizontal two-dimensional scan by control nanometer translation stage 6, the fluorescence intensity information obtaining each point by once scanning and fluorescence lifetime information, namely obtaining the horizontal two-dimensional intensity map picture of composition with life information by once scanning, image closes pulse excitation optical shutter 1 after obtaining simultaneously, scan image is chosen sample particle sparse region and carries out hot spot calibration.
(2) laterally two dimension fluorescence intensity figure and life distribution image after opening loss light is obtained;
The region that step (1) is chosen is carried out scanning imagery, open pulse excitation optical shutter 1 and continuous loss optical shutter 13 simultaneously, continuous loss light is reflected as the continuous loss light of reflection through the 2nd dichroscope 3, reflects that continuous loss light path is orthogonal with continuous loss light light path originally to be overlapped with echo pip excitation light roadbed, reflect continuous loss light and it is modulated to rotatory polarization through quarter-wave plate 4, finally focus on sample surfaces through microcobjective 5, reflect continuous loss light focal beam spot substantially to overlap with echo pip exciting light focal beam spot, wherein the focal beam spot of echo pip exciting light is filled circles spot, and the focal beam spot reflecting continuous loss light is annular hollow circle spot, fluorescent grain ground state electronics is by the supreme energy level of echo pip excitation, deplete in region harmless, upper energy level electronics spontaneous transition emitting fluorescence, deplete region damaging, the excited radiation light that upper energy level electronics stimulated radiation transition transmitting is identical with loss optical wavelength, the fluorescence and the excited radiation light that produce are collected through same microcobjective 5, successively successively by quarter-wave plate 4, 2nd dichroscope 3, first dichroscope 2, fluorescence focuses on multimode optical fibers 10 end face through the first spectral filter 8 by the first lens 9, and avalanche diode (APD) 11 record connected by multimode optical fibers 10 the other end, excited radiation light is reflected and the first spectral filter 8 filtering by the 2nd dichroscope 3, avalanche diode 11 is connected with time correlation single photon counting (TCSPC) system 12 with pulse excitation light generation module A, time correlation single photon counting system 12 is responsible for number of photons and the photon life information of avalanche diode record are supplied to computer 14, and computer 14 can calculate fluorescence intensity and the fluorescence lifetime of this scanning spot, after completing this some record, enter scanning and the record of subsequent point, computer 14 completes two-dimensional scan by control nanometer translation stage 6, the fluorescence intensity information obtaining each point by once scanning and fluorescence lifetime information, namely obtaining the horizontal two-dimensional intensity map picture of composition with life information by once scanning, image closes pulse excitation optical shutter 1 and continuous loss optical shutter 13 after obtaining.
(3) horizontal alignment is carried out according to Fluorescence lifetime distribution and fluorescence hot spot;
The fluorescence intensity figure and the Fluorescence lifetime distribution figure that obtain step (2) analyze. Select the fluorescence intensity hot spot of single fluorescent grain, matching spot center and record corresponding coordinate, extract the life distribution that this fluorescence hot spot is corresponding simultaneously, the longest point in matching life-span also records corresponding coordinate, calculating spot center and life-span the longest some distance, calculating the 2nd dichroscope according to distance needs the angle rotated and rotates the 2nd dichroscope 3 until spot center point the longest with the life-span overlaps completely.
(4) the axial two dimensional image of the fluorescence intensity after opening loss light and life distribution is obtained;
Single the fluorescent grain that step (3) is selected is carried out axial scanning imagery. Open pulse excitation optical shutter 1 and continuous loss optical shutter 13 simultaneously, choose the axial slices through particle center to scan, computer completes axial two-dimensional scan by a control nanometer translation stage, the fluorescence intensity information of each point obtained and fluorescence lifetime information form the axial two-dimensional intensity map picture with life information, and image closes pulse excitation optical shutter 1 and continuous loss optical shutter 13 after obtaining.
(5) axially align according to axial Fluorescence lifetime distribution and axial fluorescence hot spot;
Fluorescence intensity distribution step (4) obtained and Fluorescence lifetime distribution are analyzed. Select the fluorescence intensity hot spot of single fluorescent grain, extract the life distribution that this fluorescence hot spot is corresponding simultaneously, regulate the converging and diverging degree of loss light to make long lifetime region run through fluorescence hot spot region intermediate.
Wherein, the effect of the first dichroscope 2 is echo pip exciting light and the fluorescence of transmission spontaneous transition generation; The effect of the 2nd dichroscope 3 is the fluorescence that transmitted pulse exciting light and spontaneous transition produce, and reflects continuous loss light simultaneously; The reason adopting 100 nano-fluorescent grains is because 100 nano-fluorescent grains have good bleach-resistant ability, it is also possible to adopt the fluorescent grain of 40 nanometers��80 nano-scales; The effect of the first spectral filter 8 is filtering pulse excitation light, continuously loss light and excited radiation light, only allows autofluorescence to pass through.
Wherein, pulse excitation light had been modulated to parallel light before pulse excitation optical shutter; Continuous loss light had been modulated to parallel light before continuous loss optical shutter, and have passed through phase modulated, the effect of phase modulated be in order to afterwards under the focussing force of microcobjective focal beam spot become hollow bagel shape hot spot.
Wherein, pulse excitation light is through focal beam spot and the final multimode optical fibers end face the collecting signal conjugate points each other in systems in which of microcobjective.
Claims (4)
1., based on a nano-precision hot spot alignment methods for Fluorescence lifetime distribution, it is applicable to have the STED super-resolution microscopic system of pulse excitation light and continuous loss light, it is characterised in that, comprise following step:
1) enable pulse excitation light and continuous loss light simultaneously, described pulse excitation light and continuous loss light focus on fluorescent samples surface through microcobjective after being converted to rotatory polarization, collect the fluorescence that fluorescent grain sends, obtain focusing on fluorescence intensity and the fluorescence lifetime of point;
2) fluorescent samples described in laterally mobile, repeating step 1), obtain fluorescence intensity information and the fluorescence lifetime information of each scanning spot in corresponding scanning area;
3) to step 2) the fluorescence intensity information that obtains and fluorescence lifetime information analyzes, select the fluorescence intensity hot spot of single fluorescent grain, matching spot center and record hot spot centre coordinate, extract the life distribution that described fluorescence intensity hot spot is corresponding simultaneously, the longest point in matching life-span also records the longest point coordinate, calculates spot center and the distance of life-span the longest point;
4) according to step 3) distance calculated, change the angle of the incident microcobjective of described continuous loss light, spot center and life-span the longest point is overlapped completely, the transverse direction completing hot spot is directed at;
5) to step 3) middle single the fluorescent grain selected, repeating step 1) in operation, choose the axial slices through particle center to scan, and the fluorescent samples described in moving completes axial two-dimensional scan, obtain the corresponding fluorescence intensity information of each point and the oval fluorescence hot spot of fluorescence lifetime information and single fluorescent grain;
6) according to step 5) in the oval fluorescence hot spot of single fluorescent grain and life distribution corresponding to fluorescence hot spot, regulate the divergency of described continuous loss light, make long lifetime region run through the region intermediate of fluorescence hot spot and distribute axisymmetricly along oval fluorescence hot spot major axis and minor axis, complete axially aligning of hot spot.
2. as claimed in claim 1 based on the nano-precision hot spot alignment methods of Fluorescence lifetime distribution, it is characterised in that, using the particle sparse region of fluorescent samples as described step 2) in scanning area.
3. as claimed in claim 2 based on the nano-precision hot spot alignment methods of Fluorescence lifetime distribution, it is characterised in that, described particle sparse region defining method is:
It is used alone pulse excitation light, and be rotatory polarization by pulse excitation optical modulation, then sample surfaces is focused on by microcobjective, sample surfaces is carried out two-dimensional scan, collect the fluorescence that fluorescent grain sends, obtain corresponding scan image, according to the distribution of fluorescent grain on described scan image, choose described particle sparse region.
4. as claimed in claim 1 based on the nano-precision hot spot alignment methods of Fluorescence lifetime distribution, it is characterised in that, described pulse excitation light is converted to the parallel light after phase modulated with continuous loss light.
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| GB2528976B (en) * | 2014-08-08 | 2016-12-28 | Servomex Group Ltd | Alignment device and transmitter/receiver system with two angular degrees of freedom |
| CA3021017C (en) * | 2016-06-21 | 2022-12-13 | Illumina, Inc. | Super-resolution microscopy |
| CN107015353B (en) * | 2017-03-07 | 2019-12-13 | 中国科学院化学研究所 | multicolor stimulated radiation depletion super-resolution imaging device and method and optical microscope |
| CN110274895A (en) * | 2018-03-17 | 2019-09-24 | 杨佳苗 | The discrete fluorescence spectrum of multi-detector and fluorescence lifetime detection method and device |
| CN110487756A (en) * | 2018-05-14 | 2019-11-22 | 杨佳苗 | It is divided the discrete fluorescence spectrum of pupil and fluorescence lifetime detection method and device |
| CN109324026A (en) * | 2018-10-16 | 2019-02-12 | 浙江大学 | It is copolymerized two area's fluorescence lifetime micro imaging system of burnt near-infrared |
| WO2020140283A1 (en) * | 2019-01-04 | 2020-07-09 | 深圳大学 | Imaging system of stimulated emission loss microscope |
| CN109633858B (en) * | 2019-02-19 | 2020-07-07 | 浙江大学 | Device and method for aligning focal points of correlation light beams in optical tweezers |
| CN110068554B (en) * | 2019-04-24 | 2021-12-17 | 暨南大学 | Super-resolution microscope system for detecting small-size plasma nanoparticles |
| CN114623762B (en) * | 2020-12-11 | 2023-02-10 | 中国科学院上海光学精密机械研究所 | Method for three-dimensional registration alignment of double beams and multiple beams |
| CN114324156A (en) * | 2021-11-18 | 2022-04-12 | 中国科学院化学研究所 | Stimulated radiation depletion microscope and microscopic imaging system thereof |
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