CN103543135A - Nanometer-accuracy light spot aligning method and device based on fluorescence lifetime distribution - Google Patents
Nanometer-accuracy light spot aligning method and device based on fluorescence lifetime distribution Download PDFInfo
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Abstract
The invention discloses a nanometer-accuracy light spot aligning method based on a fluorescence lifetime distribution. The nanometer-accuracy light spot aligning method is applicable to an STED (Stimulated Emission Depletion) super-resolution microscopic system with pulse excitation light and continuous loss light. The nanometer-accuracy light spot aligning method comprises the following steps: firstly, performing transverse two-dimensional scanning on samples, then performing transverse alignment according an obtained fluorescence lifetime distribution and a fluorescent light spot, subsequently, performing axial scanning imaging on a single fluorescent particle, analyzing an axial two-dimensional image with a fluorescent strength and a lifetime distribution, adjusting an divergence of the continuous loss light, and finishing axial alignment of the light spot. The invention also discloses a nanometer-accuracy light spot aligning device based on the fluorescence lifetime distribution. The device, disclosed by the invention, is simple in structure, convenient, quick and highly accurate to adjust, and high in adjustment accuracy without the need of additionally arranging an extra detection light path due to the adoption of nanogold particles; the light spot alignment accuracy can reach a nanometer order.
Description
Technical field
The invention belongs to the micro-field of optical ultra-discrimination, particularly nano-precision hot spot alignment methods and the device based on Fluorescence lifetime distribution in a kind of stimulated emission loss microscopy.
Background technology
Confocal fluorescent microscopy has the advantages such as Noninvasive, high specific and high sensitivity, is the important research method in life science always.But due to the diffraction effect of light wave, its imaging resolution is limited to half wavelength left and right, cannot meet the Research Requirements of current life science.
In order to break the restriction of the Diffraction of light wave limit, further explore the essence of life and the mechanism of disease, scientists is devoted to the research of super-resolution optical microscopy, from the nineties in 20th century, multiple optical ultra-discrimination microscopic method has been proposed: stimulated emission loss microscopy (Stimulated emission depletion microscopy, STED), photoactivation location microscopy (photoactivated localization microscopy, PALM), random optics is rebuild microscopy (stochastic optical reconstruction microscopy, STORM), saturated structures optical illumination microscopy (saturated structured illumination microscopy, SSIM), etc..Among these methods, STED super-resolution microscopy has the application prospect of the fastest writing speed and tool future at present.
First STED super-resolution microscopy is proposed in 1994 by S.W.Hell.In the STED of standard super-resolution microscopic system, the red shift loss light of a branch of phase encoding is introduced into confocal system, and the focal plane that focuses on by high-NA microcobjective produces an annular hollow hot spot (donut spot).Loss light consumes the excitation electron of the solid focal beam spot of exciting light periphery by stimulated radiation effect, suppress the autofluorescence effect of hot spot periphery and then obtain less effective point spread function (Point Spread Function, PSF).Therefore, best STED imaging effect needs the solid focal beam spot center of exciting light and the annular hollow focal beam spot center inregister of loss light.In traditional method, two focal beam spot centers aim at be by two-beam respectively scanning nano gold grain carry out imaging, then by contrast two width pictures, adjust the relative position of two-beam focal beam spots, repeatedly repeat until spot center overlaps.There is following shortcoming in this alignment methods: 1. needs additionally to add scattered light imaging optical path: because STED microscopic system itself is for wavelength of fluorescence, collection unit is divided the optical filter that filtering exciting light and loss light are housed, nanogold particle is to utilize exciting light and loss scattering of light light to carry out imaging, if therefore will adopt nanogold particle to aim at, need to add extra light path; 2. exciting light and loss light need independent scanning imagery, sample drift therebetween can produce error to be needed to proofread and correct: common acquisition system adopts avalanche diode (APD) to carry out writing scan intensity more, but APD cannot differentiate the wavelength of collecting signal, therefore exciting light and loss light need independent scanning imagery, contrast twice sweep result, will certainly introduce sample drift error.2013, there is external seminar to propose to adopt fluorescent grain to aim at (Auto-aligning stimulated emission depletion microscope using adaptive optics.Optics letters to the focal beam spot center of two-beam in STED, 2013), but its method does not overcome Time share scanning introduces the problems such as sample drift error, the method is distributed and is determined focal beam spot center by fluorescence intensity in addition, and fluorescence intensity distributes and depends on exciting light and loss light intensity ratio.
Summary of the invention
The invention provides a kind of nano-precision hot spot alignment methods and device based on Fluorescence lifetime distribution, by surveying the horizontal and vertical Fluorescence lifetime distribution of fluorescence hot spot causing due to loss light in STED microscopy, change, adjust the relative position at life-span point spread function center and corresponding fluorescence focal beam spot center, thereby three-dimensional aligning is carried out in the center of two focal beam spots.The present invention is simple in structure, and without adopting nanogold particle, without adding extra light path, without Time share scanning imaging, alignment precision is in nanometer scale, and being specially adapted to exciting light is the STED super-resolution microscopic system that pulsed light, loss light are continuous light.
A nano-precision hot spot alignment methods for Fluorescence lifetime distribution, is applicable to have pulse excitation light and the STED super-resolution microscopic system of loss light continuously, comprises following step:
1) enable pulse excitation light and continuous loss light simultaneously, described pulse excitation light and continuously loss light are converted to rotatory polarization and focus on fluorescent samples surface by microcobjective, collect the fluorescence that fluorescent grain sends, obtain fluorescence intensity and the fluorescence lifetime of focus point;
2) fluorescent samples described in transverse shifting, repeating step 1), obtain fluorescence intensity information and the fluorescence lifetime information of each analyzing spot in corresponding scanning area;
3) to step 2) the fluorescence intensity information and the fluorescence lifetime information that obtain analyzes, select fluorescence intensity hot spot, the matching spot center of single fluorescent grain and record hot spot centre coordinate, extract the life-span distribution that described fluorescence intensity hot spot is corresponding simultaneously, the longest point in matching life-span also records the longest point coordinate, calculates the distance of spot center and longest-lived point;
4) according to step 3) calculate distance, change the angle of described continuous loss light incident microcobjective, spot center is overlapped completely with longest-lived point, complete the horizontal aligning of hot spot;
5) to single the fluorescent grain of selecting in step 3), repeating step 1) operation in, the axial slices of choosing through particle center scans, and mobile described fluorescent samples completes axial two-dimensional scan, obtains fluorescence intensity information and the fluorescence lifetime information of corresponding each point;
6) according to the oval fluorescence hot spot of single fluorescent grain in step 5) and life-span corresponding to described fluorescence hot spot, distribute, regulate the divergence of described continuous loss light, make long-life region run through the zone line 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 determines that method is: use separately pulse excitation light, and be rotatory polarization by pulse excitation optical modulation, then by microcobjective, focus on sample surfaces, sample surfaces is carried out to 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.
Wherein, pulse excitation light modulated directional light that is made as before process pulse excitation optical shutter; Continuously loss light is through the modulated directional light that is made as before continuous loss optical shutter, and passed through phase-modulation, the effect of phase-modulation be for after under focussing force at microcobjective focal beam spot become hollow bread cast hot spot.
Wherein, pulse excitation light is through the focal beam spot of microcobjective and the final multimode optical fiber end face of collecting signal conjugate points each other in system.
Wherein, described fluorescent samples is 100 nano-fluorescent grains, because 100 nano-fluorescent grains have good bleach-resistant ability, also can adopt the fluorescent grain of 40 nanometer～80 nano-scales.
In step 2) in, can also adopt galvanometer scanning system to realize the transversal scanning of sample.
The present invention also provides a kind of nano-precision hot spot alignment device based on Fluorescence lifetime distribution, comprising:
Along the pulse the luminous light path of impulse is arranged successively pulse excitation photogenerated device and pulse excitation optical shutter;
Along the continuous loss photogenerated device that loss light light path is arranged successively continuously, continuous loss optical shutter and dichroscope;
Quarter-wave plate, for by described pulse excitation light and continuously loss optical modulation be rotatory polarization;
Microcobjective, for focusing to described rotatory polarization fluorescent samples and collect fluorescence;
Information collecting device, strength information and the life information of for gathering described microcobjective, collecting fluorescence;
And the computing machine 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 collimation lens, first polarizer, the first quarter-wave plate and the 1/1st wave plate of arranging successively along light path.
Wherein, described continuous loss photogenerated device comprises continuous wave laser, the second single-mode polarization maintaining fiber, the second collimation lens, second polarizer, the second quarter-wave plate, the 1/2nd wave plate and 0～2 π vortex phase plate of arranging successively along light path.
Wherein, described information collecting device comprises optical filter, lens, multimode optical fiber, avalanche diode and the Single Photon Counting system of arranging successively along the light path of collecting fluorescence, and Single Photon Counting system is connected with pulse excitation photogenerated device with described computing machine respectively.
Optical filter is for the light of filtering other any wavelength except spontaneous radiation wavelength of fluorescence, lens are for focusing on multimode optical fiber end face by the fluorescence of collecting, multimode optical fiber is connected with avalanche diode (APD), avalanche diode (APD) Single Photon Counting (TCSPC) system is connected, time correlation single photon technological system is connected with computing machine, Single Photon Counting system be responsible for by the photon number of avalanche diode record and photon lifetime information offer computing machine.
Principle of work of the present invention is as follows:
Through the pulse excitation light of phase-modulation with laterally forming respectively solid circles focal beam spot and annular hollow focal beam spot under the focussing force of loss light at microcobjective continuously, axially forming respectively Filled Ellipse shape focal beam spot (transverse is along optical axis direction) and bottleneck hollow hot spot.
Fluorescence lifetime refers to that fluorescence molecule decays 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 open exciting light, within the scope of exciting light focal beam spot, fluorescence molecule all will be excited to energy level spontaneous transition emitting fluorescence, not consider that under other environmental factors, the whole expression formula of any point fluorescence lifetime within the scope of hot spot that excites can be write as
τ(r)=1/k
f
Wherein, k
fthe speed of fluorescence molecule spontaneous transition emitting fluorescence, k for given fluorescence molecule kind
fvalue determines, if only open exciting light, last single fluorescence lifetime corresponding to fluorescent grain is homogeneous, as shown in Figure 3.
If open exciting light and loss light simultaneously, within the scope of exciting light focal beam spot, fluorescence molecule all will be excited to energy level, and exciting light is different from only opening, and excite the expression formula of fluorescence lifetime within the scope of hot spot to revise to be
τ(r)=1/(k
f+σI
STED(r))
Wherein, k
fthe speed of fluorescence molecule spontaneous transition emitting fluorescence, k for given fluorescence molecule kind
fvalue determines, σ is the absorption cross section of fluorescence molecule stimulated radiation, I
sTED(r) be the loss light intensity at r place in focal beam spot, exist the region fluorescence lifetime of loss light to shorten, and shortening degree is relevant with loss light intensity, if when annular hollow loss light focal beam spot overlaps with solid exciting light focal circle spot, coincidence spot center longest-lived, as shown in Figure 4.
According to the corresponding coincidence hot spot of 100 nano-fluorescent grains Fluorescence lifetime distribution, carry out nano-precision aligning, as shown in the table:
Laterally, 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 while only opening exciting light, the corresponding life-span is distributed as the round spot of homogeneous; If two-beam is opened simultaneously,, when the horizontal misalignment of two-beam, longest-lived point is not in fluorescent grain spot center, and when the lateral registration of two-beam focal beam spot, the Fluorescence lifetime distribution center of circle that fluorescent grain hot spot is corresponding is the highest.
Axially, the focal beam spot of exciting light is Filled Ellipse shape focal beam spot (transverse is along optical axis direction), and loss light focal beam spot is bottleneck hollow hot spot, if while only opening exciting light, the corresponding life-span is distributed as the oval spot of homogeneous; If two-beam is opened simultaneously, when two-beam axial misalignment, the long-life does not run through whole ellipse light spot and distributes and do not distribute axisymmetricly along life-span of oval spot major axis and minor axis; When two-beam focal beam spot axially overlaps, long-life 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 mixes up (guaranteeing that pulse excitation light is through the focal beam spot of microcobjective and the final multimode optical fiber end face of collecting signal conjugate points each other in system), the lateral registration of two-beam can realize by the incident angle that regulates the second dichroiscopic angle to realize loss light; The axial coincidence of two-beam can realize according to the converging and diverging degree of axial hot spot life-span profile adjustment loss light.By setting the scanning resolution (nano-precision) of nanometer translation stage, can reach the aligning of nano-precision.
The present invention is simple in structure, and alignment precision is in nanometer scale, degree of registration that can three-dimensional regulation two light beams, and being specially adapted to exciting light is the STED super-resolution microscopic system that pulsed light, loss light are continuous light.
Compared with prior art, the present invention has following useful technique effect:
(1) systematic error is little, without causing sample drift error because adopting nanogold particle to introduce Time share scanning;
(2) degree of regulation is high, and hot spot alignment precision can reach nanometer scale;
(3) apparatus structure is succinct, and fast and easy high precision is adjusted, without adding extra detection light path because of employing nanogold particle.
Accompanying drawing explanation
Fig. 1 is the device schematic diagram of the nano-precision hot spot alignment methods based on life-span distribution;
Fig. 2 be one group for the generating apparatus schematic diagram of the twin-beam of life-span distribution nano-precision hot spot alignment methods;
Fig. 3 horizontal exciting light point spread function intensity distributions and corresponding Fluorescence lifetime distribution sectional view when only opening exciting light;
Fig. 4 is for open exciting light and loss light time lateral loss luminous point spread function intensity distributions and corresponding Fluorescence lifetime distribution sectional view simultaneously;
(a～j) figure in Fig. 5 represents that different light beams focus on the light spot image that sample surfaces forms.
Embodiment
Below in conjunction with embodiment and accompanying drawing, describe the present invention in detail, but the present invention is not limited to this.
As shown in Figure 1, a kind of device of the nano-precision hot spot alignment methods based on life-span distribution, comprising: pulse excitation optical shutter 1, the first dichroscope 2, the second dichroscopes 3, quarter-wave plate 4, microcobjective 5, nanometer translation stage 6, the first optical filters 8, first lens 9, multimode optical fiber 10, avalanche diode (APD) 11, Single Photon Counting (TCSPC) system 12, continuous loss optical shutter 13, computing machine 14.
Wherein, pulse excitation optical module A transponder pulse exciting light, on the optical axis of pulse excitation light, set gradually pulse excitation optical shutter 1 and the first dichroscope 2, pulse excitation optical shutter 1 is for the switch of gating pulse exciting light, the first dichroscope 2 is negative 45 ° with the angle of pulse excitation light optical axis, and the effect of the first dichroscope 2 is for reflected impulse exciting light and transmission fluorescence, pulse excitation light becomes reflected impulse exciting light through the first dichroscope 2 reflections, reflected impulse exciting light optical axis and pulse excitation light optical axis are in 90 °, on the optical axis of reflected impulse exciting light, place successively the second dichroscope 3, quarter-wave plate 4, microcobjective 5 and nanometer translation stage 6, the second dichroscope 3 is positive 45 ° with the angle of reflected impulse exciting light, the effect of the second dichroscope 3 is Transflective pulse excitation light, fluorescence and the continuous loss light of reflection, the effect of quarter-wave plate 4 is that to guarantee to incide the light of microcobjective 5 be rotatory polarization, microcobjective 5 is for focusing on exciting light, loss light and collection fluorescence, nanometer translation stage 6 is for the axially scanning of two dimension of horizontal two peacekeepings of complete paired samples, on the reverse extending line of reflected impulse exciting light, place successively the first optical filter 8, first lens 9 and multimode optical fiber 10, the effect of the first optical filter 8 is only to allow the fluorescence of sample spontaneous transition to pass through, and the end face of multimode optical fiber 10 is placed on the focal plane of first lens 9, multimode optical fiber 10 is connected with avalanche diode (APD) 11, and the effect of avalanche diode (APD) 11 is to survey the fluorescent photon of being collected by microcobjective 5, avalanche diode (APD) 11 is connected with Single Photon Counting (TCSPC) system 12 with pulse excitation light generation module A, and Single Photon Counting (TCSPC) system is for being converted into strength information and life information by the photon signal coming from APD conduction, computing machine 14 is connected with nanometer translation stage 6 with Single Photon Counting (TCSPC) system 12, and computing machine is by control time be correlated with single photon counting (TCSPC) system 12 and the horizontal and vertical two-dimentional intensity of the complete paired samples of nanometer translation stage 6 and the imaging in life-span, loss optical module B launches continuous loss light continuously, on the optical axis of continuous loss light, set gradually continuous loss optical shutter 13 and the first dichroscope 3, loss light overlaps with reflected impulse exciting light optical axis through the second dichroscope 3 reflections continuously, and loss optical shutter 13 is for controlling the switch of continuous loss light continuously.
The present embodiment gives the generating apparatus of set of pulses exciting light and continuous loss light, but generation method is not limited in this.
As Fig. 2 A, a kind of generating apparatus of the pulse excitation light for life-span distribution nano-precision hot spot alignment methods, comprises pulsed laser 15, the first single-mode polarization maintaining fiber 16, the first collimation lens 17, the first polarizer 18, the first quarter-wave plates 19 and the 1/1st wave plates 20.
Wherein, pulsed laser 15 Emission Lasers, outgoing after the first single-mode polarization maintaining fiber 16 couplings, successively through the first collimation lens 17, first polarizer 18, the 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 collimation lens 17, the effect of the first collimation lens 17 is for collimated light beam, the effect of first polarizer 18 is used for guaranteeing that collimated light is line polarisation, the first quarter-wave plate 19 and the 1/1st wave plate 20 are for the phase differential of compensated pulse exciting light light path and then guarantee that final focal beam spot is rotatory polarization.
As Fig. 2 B, a kind of generating apparatus of the continuous loss light for life-span distribution nano-precision hot spot alignment methods, comprise continuous wave laser 21, the second single-mode polarization maintaining fiber 22, the second collimation lens 23, the second polarizer 24, the second quarter-wave plate 25, the 2 1/ 2nd wave plates 26 and 0～2 π vortex phase plates 27.
Wherein, continuous wave laser 21 Emission Lasers, outgoing after the second single-mode polarization maintaining fiber 22 couplings, successively through the second collimation lens 23, the second polarizer 24, the second quarter-wave plate 25, the 2 1/ 2nd wave plates 26 and 0～2 π vortex phase plates 27.The outgoing end face of the second single-mode polarization maintaining fiber 22 is at the front focus place of the second collimation lens 23, the effect of the second collimation lens 23 is for collimated light beam, the effect of second polarizer 24 is used for guaranteeing that collimated light is line polarisation, the second quarter-wave plate 25 and the 1/2nd wave plate 26 be for compensating the phase differential of continuous loss light light path and then guaranteeing that final focal beam spot is rotatory polarization, and 0～2 π vortex phase plate 27 is used to form laterally as annular hollow, be axially bottleneck hollow hot spot.
Adopt a kind of nano-precision hot spot alignment methods distributing based on the life-span shown in Fig. 1, its process is as follows:
(1) choose suitable transversal scanning region;
Pulse excitation optical shutter 1 is opened, and loss optical shutter 13 is closed continuously, and pulse excitation light becomes reflected impulse exciting light, reflected impulse exciting light light path and pulse excitation light light path quadrature through the first dichroscope 2 reflections, reflected impulse exciting light, after the second dichroscope 3 transmissions, is modulated to rotatory polarization through quarter-wave plate 4, finally by microcobjective 5, focuses on sample surfaces, 100 nano-fluorescent grain samples 7 are fixed on nanometer translation stage 6, and fluorescent grain ground state electronics is reflected the supreme energy level of pulse excitation optical excitation, spontaneous transition emitting fluorescence, fluorescence is collected through same microcobjective 5, successively successively by quarter-wave plate 4, the second dichroscope 3, the first dichroscope 2 and the first optical filter 8, by first lens 9, focus on multimode optical fiber 10 end faces, and avalanche diode (APD) 11 records that connected by multimode optical fiber 10 other ends, avalanche diode 11 is connected with Single Photon Counting (TCSPC) system 12 with pulse excitation light generation module A, Single Photon Counting system 12 be responsible for by the photon number of avalanche diode 11 records and photon lifetime information offer computing machine 14, computing machine 14 can calculate fluorescence intensity and the fluorescence lifetime of this analyzing spot, complete after this some record, enter down any scanning and record, computing machine completes horizontal two-dimensional scan by controlling nanometer translation stage 6, by single pass, obtain fluorescence intensity information and the fluorescence lifetime information of each point, by single pass, obtain the horizontal two-dimentional intensity image forming with life information simultaneously, after image obtains, close pulse excitation optical shutter 1, on scan image, choose sample particle sparse region and carry out hot spot calibration.
(2) obtain horizontal two-dimentional fluorescence intensity figure and the life-span distributed image of opening after loss light;
Scanning imagery is carried out in the region that step (1) is chosen, open pulse excitation optical shutter 1 and continuous loss optical shutter 13 simultaneously, continuously loss light becomes the continuous loss light of reflection through the second dichroscope 3 reflections, reflects continuous loss light path with continuous loss light light path quadrature, originally overlap with reflected impulse excitation light roadbed, reflect continuous loss light and be modulated to rotatory polarization through quarter-wave plate 4, finally through microcobjective 5, focus on sample surfaces, reflecting continuous loss light focal beam spot overlaps substantially with reflected impulse exciting light focal beam spot, wherein the focal beam spot of reflected impulse exciting light is filled circles spot, and the focal beam spot that reflects continuous loss light is annular hollow circle spot, fluorescent grain ground state electronics is reflected the supreme energy level of pulse excitation optical excitation, harmless, deplete in region, upper energy level electronics spontaneous transition emitting fluorescence, deplete region diminishing, the upper energy level electronics stimulated radiation transition transmitting excited radiation light identical with loss optical wavelength, the fluorescence producing and excited radiation light are collected through same microcobjective 5, successively successively by quarter-wave plate 4, the second dichroscope 3, the first dichroscope 2, fluorescence focuses on multimode optical fiber 10 end faces through the first optical filter 8 by first lens 9, and avalanche diode (APD) 11 records that connected by multimode optical fiber 10 other ends, excited radiation light is by the second dichroscope 3 reflection and the first optical filter 8 filterings, avalanche diode 11 is connected with Single Photon Counting (TCSPC) system 12 with pulse excitation light generation module A, Single Photon Counting system 12 be responsible for by the photon number of avalanche diode record and photon lifetime information offer computing machine 14, computing machine 14 can calculate fluorescence intensity and the fluorescence lifetime of this analyzing spot, complete after this some record, enter down any scanning and record, computing machine 14 completes two-dimensional scan by controlling nanometer translation stage 6, by single pass, obtain fluorescence intensity information and the fluorescence lifetime information of each point, by single pass, obtain the horizontal two-dimentional intensity image forming with life information, after image obtains, close pulse excitation optical shutter 1 and continuous loss optical shutter 13.
(3) according to Fluorescence lifetime distribution and fluorescence hot spot, laterally aim at;
Fluorescence intensity figure and Fluorescence lifetime distribution figure that step (2) is obtained analyze.Select fluorescence intensity hot spot, the matching spot center of single fluorescent grain and record corresponding coordinate, extract the life-span distribution that this fluorescence hot spot is corresponding simultaneously, the longest point in matching life-span also records corresponding coordinate, calculate spot center and longest-lived point distance, the angle that need to rotate according to distance calculating the second dichroscope is also rotated the second dichroscope 3 until spot center overlaps completely with longest-lived point.
(4) obtain the axial two dimensional image of the fluorescence intensity of opening after loss light and life-span distribution;
Single the fluorescent grain that step (3) is selected carries out axial scan imaging.Open pulse excitation optical shutter 1 and continuous loss optical shutter 13 simultaneously, the axial slices of choosing through particle center scans, computing machine completes axial two-dimensional scan by controlling nanometer translation stage, the fluorescence intensity information of the each point obtaining and fluorescence lifetime information form the axial two-dimentional intensity image with life information, close pulse excitation optical shutter 1 and continuous loss optical shutter 13 after image obtains.
(5) according to axially Fluorescence lifetime distribution and axial fluorescence hot spot axially align;
The fluorescence intensity that step (4) is obtained distributes and Fluorescence lifetime distribution is analyzed.Select the fluorescence intensity hot spot of single fluorescent grain, extract the life-span distribution that this fluorescence hot spot is corresponding simultaneously, the converging and diverging degree of regulation loss light makes long-life region run through fluorescence hot spot zone line.
Wherein, the effect of the first dichroscope 2 is the fluorescence that reflected impulse exciting light transmission spontaneous transition produce; The effect of the second dichroscope 3 is the fluorescence that transmitted pulse exciting light and spontaneous transition produce, and reflects continuous loss light simultaneously; The reason that adopts 100 nano-fluorescent grains is because 100 nano-fluorescent grains have good bleach-resistant ability, also can adopt the fluorescent grain of 40 nanometer～80 nano-scales; The effect of the first optical filter 8 is filtering pulse excitation light, continuous loss light and excited radiation light, only allows autofluorescence to pass through.
Wherein, pulse excitation light modulated directional light that is made as before process pulse excitation optical shutter; Continuously loss light is through the modulated directional light that is made as before continuous loss optical shutter, and passed through phase-modulation, the effect of phase-modulation be for after under focussing force at microcobjective focal beam spot become hollow bread cast hot spot.
Wherein, pulse excitation light is through the focal beam spot of microcobjective and the final multimode optical fiber end face of collecting signal conjugate points each other in system.
Claims (8)
1. the nano-precision hot spot alignment methods based on Fluorescence lifetime distribution, is applicable to have pulse excitation light and the STED super-resolution microscopic system of loss light continuously, it is characterized in that, comprises following step:
1) enable pulse excitation light and continuous loss light simultaneously, described pulse excitation light and continuously loss light are converted to rotatory polarization and focus on fluorescent samples surface by microcobjective, collect the fluorescence that fluorescent grain sends, obtain fluorescence intensity and the fluorescence lifetime of focus point;
2) fluorescent samples described in transverse shifting, repeating step 1), obtain fluorescence intensity information and the fluorescence lifetime information of each analyzing spot in corresponding scanning area;
3) to step 2) the fluorescence intensity information and the fluorescence lifetime information that obtain analyzes, select fluorescence intensity hot spot, the matching spot center of single fluorescent grain and record hot spot centre coordinate, extract the life-span distribution that described fluorescence intensity hot spot is corresponding simultaneously, the longest point in matching life-span also records the longest point coordinate, calculates the distance of spot center and longest-lived point;
4) according to step 3) calculate distance, change the angle of described continuous loss light incident microcobjective, spot center is overlapped completely with longest-lived point, complete the horizontal aligning of hot spot;
5) to single the fluorescent grain of selecting in step 3), repeating step 1) operation in, the axial slices of choosing through particle center scans, and mobile described fluorescent samples completes axial two-dimensional scan, obtains fluorescence intensity information and the fluorescence lifetime information of corresponding each point;
6) according to the oval fluorescence hot spot of single fluorescent grain in step 5) and life-span corresponding to fluorescence hot spot, distribute, regulate the divergence of described continuous loss light, make long-life region run through the zone line of fluorescence hot spot and distribute axisymmetricly along oval fluorescence hot spot major axis and minor axis, complete axially aligning of hot spot.
2. the nano-precision hot spot alignment methods based on Fluorescence lifetime distribution as claimed in claim 1, is characterized in that, usings the particle sparse region of fluorescent samples as described step 2) in scanning area.
3. the nano-precision hot spot alignment methods based on Fluorescence lifetime distribution as claimed in claim 2, is characterized in that, described particle sparse region determines that method is:
Use separately pulse excitation light, and be rotatory polarization by pulse excitation optical modulation, then by microcobjective, focus on sample surfaces, sample surfaces is carried out to 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. the nano-precision hot spot alignment methods based on Fluorescence lifetime distribution as claimed in claim 1, is characterized in that, described pulse excitation light and continuously loss light are converted to the directional light after phase-modulation.
5. the nano-precision hot spot alignment device based on Fluorescence lifetime distribution, is characterized in that, comprising:
Along the pulse the luminous light path of impulse is arranged successively pulse excitation photogenerated device and pulse excitation optical shutter;
Along the continuous loss photogenerated device that loss light light path is arranged successively continuously, continuous loss optical shutter and dichroscope;
Quarter-wave plate, for by described pulse excitation light and continuously loss optical modulation be rotatory polarization;
Microcobjective, for focusing to described rotatory polarization fluorescent samples and collect fluorescence;
Information collecting device, strength information and the life information of for gathering described microcobjective, collecting fluorescence;
And the computing machine being connected with described information collecting device.
6. the nano-precision hot spot alignment device based on Fluorescence lifetime distribution as claimed in claim 5, it is characterized in that, described pulse excitation photogenerated device comprises pulsed laser, the first single-mode polarization maintaining fiber, the first collimation lens, first polarizer, the first quarter-wave plate and the 1/1st wave plate of arranging successively along light path.
7. the nano-precision hot spot alignment device based on Fluorescence lifetime distribution as claimed in claim 5, it is characterized in that, described continuous loss photogenerated device comprises continuous wave laser, the second single-mode polarization maintaining fiber, the second collimation lens, second polarizer, the second quarter-wave plate, the 1/2nd wave plate and 0～2 π vortex phase plate of arranging successively along light path.
8. the nano-precision hot spot alignment device based on Fluorescence lifetime distribution as claimed in claim 5, it is characterized in that, described information collecting device comprises optical filter, lens, multimode optical fiber, avalanche diode and the Single Photon Counting system of arranging successively along the light path of collecting fluorescence, and Single Photon Counting system is connected with pulse excitation photogenerated device with described computing machine respectively.
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| CN107015353A (en) * | 2017-03-07 | 2017-08-04 | 中国科学院化学研究所 | Polychrome stimulated radiation exhausts super-resolution imaging device, method and light microscope |
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| CN105372178A (en) * | 2014-08-08 | 2016-03-02 | 仕富梅集团公司 | Alignment device and transmitter/receiver system with two angular degrees of freedom |
| CN109313328A (en) * | 2016-06-21 | 2019-02-05 | 伊鲁米那股份有限公司 | Super-resolution microscopy |
| CN107015353A (en) * | 2017-03-07 | 2017-08-04 | 中国科学院化学研究所 | Polychrome stimulated radiation exhausts super-resolution imaging device, method and light microscope |
| 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 |
| CN109633858A (en) * | 2019-02-19 | 2019-04-16 | 浙江大学 | To the device and method of irradiating light beam focus alignment in a kind of optical tweezer |
| CN110068554A (en) * | 2019-04-24 | 2019-07-30 | 暨南大学 | A kind of super-resolution microscopic system of small-size plasma nano particle detection |
| CN110068554B (en) * | 2019-04-24 | 2021-12-17 | 暨南大学 | Super-resolution microscope system for detecting small-size plasma nanoparticles |
| CN114623762A (en) * | 2020-12-11 | 2022-06-14 | 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 |
| 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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