CN201716464U - Super-resolution fluorescent microscopic apparatus based on tangential polarization - Google Patents
Super-resolution fluorescent microscopic apparatus based on tangential polarization Download PDFInfo
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- CN201716464U CN201716464U CN2010202571885U CN201020257188U CN201716464U CN 201716464 U CN201716464 U CN 201716464U CN 2010202571885 U CN2010202571885 U CN 2010202571885U CN 201020257188 U CN201020257188 U CN 201020257188U CN 201716464 U CN201716464 U CN 201716464U
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Abstract
The utility model discloses a super-resolution fluorescent microscopic apparatus based on tangential polarization, comprising: a light source used for emitting an exciting light and a STED exciting light, a tangential polarization converter for converting the exciting light and the STED exciting light to tangential polarization lights, a 0-2 Pi phase plate in a vortex distribution for performing a 0-2 Pi vortex phase coding of the tangential polarization excitation lights, a spectroscope for performing a 90 degree changing of the phase coding tangential polarization exciting light and the tangential polarization STED exciting light, and an apochromatic lens for focusing the bended and converged coaxial light on the fluorescent sample to obtain a center fluorescent point; a detecting and imaging system used for collecting and detecting fluorescence emitted at the luminescent point and processing the fluorescence to obtain a super-resolution microscopic image; and a nanometer parallel moving bench used for fixing the fluorescent sample. The super-resolution fluorescent microscopic apparatus in the utility model, in the condition of guaranteeing a high resolution, greatly reduces the work power of STED, thereby reducing sample bleaching, and preventing damage upon sample.
Description
Technical field
The utility model relates to the fluorescent microscope field, is specifically related to the super-resolution fluorescence microscope equipment.
Background technology
Since Abbe in 1873 found that for the first time the far-field optics imaging has the diffraction-limited phenomenon, community of physicists just generally acknowledged that the microscopical resolution of far-field optics is subject to optical diffraction limit, and this limit is relevant with the wavelength and the numerical aperture of light source.Because the restriction of optical diffraction, the ideal point thing can not obtain a desirable picture point through optical system imaging, but becomes a Fraunhofer diffraction picture.General optical system all is circular, and it is exactly so-called Airy disk that Fraunhofer diffraction looks like.At the picture as each object point of plane all is a disc of confusion, and two discs of confusion have limited the resolution of system like this near with regard to bad differentiation, and disc of confusion is big more, and resolution is low more.Therefore, since the century, the microscopical resolution of traditional far-field optics (～200nm) be the obstacle that can't go beyond always.How to break through optical diffraction limit and further improve microscopical resolution, can observing more under optical microscope, the object of small scale is the topic of a hot topic.Potential meaning is to observe the more real-time living body biological phenomenon of small scale under optical microscope, explores the life secret.
In recent years, appearance along with new theory and technology such as the burnt microtechnic of 4 π copolymerization, stimulated emission loss microtechnic, photoactivation location microtechnic, saturated structures illumination microtechnic, the resolution of far-field optics micro-imaging is improved greatly, can see the protein of nanoscale on living cells.These technical progress certainly will greatly promote development of life science.And one of these technology, be that the stimulated emission loss microscope (Stimulated Emission Depletion (STED) microscopy) of the U.S. Patent Publication of US5731588 is described as the method that application prospect is arranged most by the patent No..It is from physically breaking the far field fluorescence microscopy of the diffraction optics limit, to the very big challenge of conventional physical viewpoint.Its concrete principle is: use a kind of circularly polarized laser, only excite the fluorophor of a point that it is fluoresced, and then suppress fluorescence intensity around that point with the circular polarization light source of a loaf of bread circle sample, so just have only the point in centre luminous and be observed less than diffraction limit.Two bundle laser (that is: exciting light and stimulated emission loss laser) are through being radiated at same position on the sample simultaneously after the ovennodulation, wherein, exciting light makes fluorescent material luminous, stimulated emission loss laser (being STED laser) is beam of laser that be close to exciting light, ring-like, that wavelength is long, make to excite in the hot spot most fluorescent material to get back to ground state by force, thereby suppress fluorescence radiation to reduce fluorescent emission hot spot area by the optical nonlinearity effect.The exciting light hot spot is after the modulation of STED laser, the spot size of emitting fluorescence molecule has reduced greatly, and increase along with the STED laser intensity, energy emitting fluorescence hot spot is more little, its full width at half maximum can reach below the diffraction limit, resolution is limited by diffraction of light no longer, thereby breaks diffraction limit.From the proposition of STED theory in 1994, after experiment for many years, developed the ultrahigh resolution microtechnic up to Hell in 2000, can obtain ultrahigh resolution 3-D view below the 100nm by 3-D scanning.
Although the STED microtechnic has obtained certain development, yet up to the present STED principle and method also are not widely used, be in particular in: (1) existing pulse STED measuring system light path complexity, the optical component that uses is many, comprises the electronic control system of expensive pulsed laser, complexity etc.(2) very high to the stability requirement of system, in order to guarantee resolution, 2～3 hours needs of general work are recalibrated.(3) in order to reach the STED effect, the optical intensity density of STED laser exceeds 1000-10000 doubly than exciting light, even higher, and so high light intensity is a fatal damage to most of biological sample.(4) resolution of theory unlimited in fact is limited, because also increased fluorescent bleach in the high-resolution while of pursuit, thereby reduces imaging resolution and image quality.Afterwards, in order to enlarge the depth of focus of STED system, the patent No. is the mode that the United States Patent (USP) of US7709809 has proposed the combination bit phase-plate.In order to improve system stability, application number: 201010177761.6 Chinese patent has proposed a kind of method of real-Time Compensation drift.And for the above-mentioned too high problem of STED operating power, never be well solved.Thereby the operating power that how to reduce STED laser reduces the bleaching of sample, avoids the damage to sample, is a difficult problem of STED microtechnic always.Be to utilize a kind of circularly polarized light to assemble in the existing STED system as exciting light, with the circularly polarized light (STED light) of another wavelength through the vortex phase board generation bagel fluorophore emitting fluorescence that disinthibites.Its characteristics: the one, the circular polarization exciting light is difficult to focus on below the diffraction limit; The 2nd, utilize circularly polarized light to disinthibite and excite hot spot fluorescence on every side by vortex phase board formation bagel, it suppresses effect has only by increasing the STED laser power.
The utility model content
The utility model provides a kind of super-resolution fluorescence microscope equipment based on tangential polarization, and at the power that guarantees to reduce greatly under the high-resolution condition STED laser, thereby the damage to sample is avoided in the bleaching of reduction sample.
A kind of super-resolution fluorescence microscope equipment based on tangential polarization comprises:
Be used to send first light source that the fluorescence excitation sample produces the exciting light of fluorescence; Be used for described exciting light is converted to the first tangential polarization converter of tangential polarization exciting light; Be used for the tangential polarization exciting light is carried out 0～2 π phase board (Vortex 0～2 π PhasePlate) of the vortex distribution of 0～2 π vortex phase coding; Be used for the tangential polarization exciting light of phase coding is carried out 90 ° of first spectroscopes of turning back;
Be used to send the secondary light source that the inhibition fluorescent samples sends the STED laser of fluorescence; Be used for described STED laser is converted to the second tangential polarization converter of tangential polarization STED laser; Be used for tangential polarization STED laser is carried out 90 ° of second spectroscopes of turning back;
The axis light that is used for converging after first and second spectroscopes are turned back focuses on the secondary color aplanat that disappears that obtains the center luminous point on the fluorescent samples, because two bundle axis light focus on the same point on the fluorescent samples, the solid fluorescent emission hot spot that the excitation light generation diffraction limit is following, the hollow fluorescence that STED laser produces a loaf of bread loop-shaped suppresses hot spot, 2 spot center points overlap fully, the fluorescent emission that solid fluorescent emission hot spot and hollow fluorescence suppress the hot spot intersection is suppressed, and therefore solid fluorescent emission hot spot has only the central point emitting fluorescence to become luminous point;
Be used to collect and survey fluorescence that luminous point sends and obtain the detection imaging system of the micro-image of ultrahigh resolution according to the fluorescence light intensity that detects, described detection imaging system comprises: be used to collect the secondary color aplanat that disappears of the fluorescence that luminous point sends, the wave filter that is used for elimination ground unrest and background stray light, the detector that is used for filtered fluorescence signal is surveyed, be used for the fluorescence light intensity of surveying is handled and the image device of imaging.
The nanometer translation stage that is used for fixing fluorescent samples scans fluorescent samples by mobile nanometer translation stage, thereby obtains two-dimentional micro-image.By fluorescent samples is carried out 3-D scanning, can obtain three-dimensional micro-image.
In the utility model, the exciting light that is used for fluorescence excitation sample generation fluorescence that is produced by first light source is converted to the tangential polarization exciting light through the first tangential polarization converter; 0～2 π phase board (Vortex 0～2 π Phase Plate) that distributes through vortex carries out the tangential polarization exciting light that 0～2 π vortex phase coding obtains phase coding again, is undertaken 90 ° by first spectroscope again and turns back; Be used to send and suppress the STED laser that fluorescent samples sends fluorescence and be converted to tangential polarization STED laser by what secondary light source produced through the second tangential polarization converter; Being undertaken 90 ° by second spectroscope again turns back; The axis light of converging after will turning back through first and second spectroscopes, the secondary color aplanat focuses on through disappearing, phase coding tangential polarization exciting light is focusing on the solid fluorescent emission hot spot that obtains below the diffraction limit on the fluorescent samples, tangential polarization STED laser focuses on the hollow fluorescence that forms donut-like and suppresses hot spot on fluorescent samples, 2 spot center points overlap fully, the fluorescent emission that solid fluorescent emission hot spot and hollow fluorescence suppress the hot spot intersection is suppressed, therefore solid fluorescent emission hot spot has only the central point emitting fluorescence to become luminous point, obtains the center luminous point on fluorescent samples; Center luminous point emitted fluorescence is collected fluorescence signal, surveys the fluorescence light intensity and is processed into picture by surveying imaging system; Mobile nanometer translation stage scans fluorescent samples, thereby obtains the micro-image of the ultrahigh resolution (resolution that 100nm is following) of two dimension.Fluorescent samples is carried out 3-D scanning, can obtain three-dimensional micro-image.
In the utility model, described first and second light sources can preferably use laser instrument for the light source of general generation collimation laser.
In the utility model, the described first and second tangential polarization converters can be for realizing any device and the device of the conversion of tangential polarization light in the prior art, as spatial light modulator of forming by microstructure grating and interferometer etc., be preferably the polarization converter Radial-Azimuthal Polarization Converter of Sweden (ARCoptix) company, can directly other polarized light be converted to tangential polarization light.
In the utility model, 0～2 π phase board (Vortex 0～2 π PhasePlate) that described vortex distributes is the phase board of 2 π for thickness is increased to a pairing phase place of light wavelength magnitude gradually along angle.0～2 π phase board (Vortex 0～2 π Phase Plate) that described vortex distributes is determined by following formula the bit phase delay amount Δ α of incident light:
In the formula,
Angle for polar coordinates vector in position on the phase board plane and x axle.
Described 0～2 π vortex phase coding is meant for cross-section center symmetrical circular incident beam, adopt a thickness to increase gradually along angle, pairing phase place is that 0～2 π phase board (Vortex 0～2 π Phase Plate) that the vortex of 2 π distributes makes this incident beam produce bit phase delay with respect to initial phase when being increased to a light wavelength magnitude, the size of bit phase delay is by the angle decision of the radius of crossing specified point in the light beam circular cross section with+X-axis, span is put the range-independence in the center of circle with this between 0～2 π.
0～2 π phase board (Vortex 0～2 π Phase Plate) that described vortex distributes preferably adopts vortex phase board (Vortex 0～2 π Phase Plate) VPP-1A of U.S. RPC photonics company, in fact also can adopt the spatial light modulator as the operation of 0～2 π vortex to realize identical functions.
In the utility model, the described secondary color aplanat that disappears is preferably the high-NA secondary color aplanat that disappears, described high-NA NA=1～1.4.The high-NA secondary color aplanat that disappears is preferably 1.4NA, and 100 times of amplifications have the flat field correcting function;
In the utility model, described nanometer translation stage adopts high precision nanometer translation stage, is preferably the high precision nanometer translation stage of German PI company;
In the utility model, described wave filter comprises narrowband light filter and spatial filter, and described narrowband light filter is used for narrow-band filtering and removes ground unrest, can choose according to fluorescigenic scope; Described spatial filter can adopt optical fiber or pin hole, preferably adopts pin hole, the fluorescence of collecting is focused on the back remove background stray light by spatial filter, and described aperture size can be determined according to confocal principle.
In the utility model, described detector is a single-photon detector, is preferably the avalanche diode single-photon detector of high-quantum efficiency.
In the utility model, described first and second spectroscopes preferably adopt dichroscope, exciting light and STED laser vertical are turned back, and fluorescence is directly seen through, do not need to add in addition optical element like this, disappear in only need the light path through the fluorescence excitation emission reception of secondary color aplanat of the fluorescence that luminous point sends sees through two dichroscopes more successively and can collect and finish.
In the utility model, utilize a branch of tangential polarization exciting light under the operation of vortex phase board, focus on and obtain the following focal beam spot of diffraction limit; Utilize another bundle tangential polarization STED laser on the fluorescent samples same position, to be focused to bagel, the luminous of exciting light spot edge of disinthibiting.Because the distinctive focus characteristics of tangential polarization STED laser, it is faster than the bagel convergence that circularly polarized light forms, and therefore under relatively low STED operating power, can reach the resolution identical with prior art.Comprehensive above-mentioned 2 advantages under the situation that guarantees resolution, can reduce the STED operating power greatly, overcome prior art in the deficiency of STED operating power aspect too high.
Principle of work of the present utility model is as follows:
Will be from the exciting light and the STED laser of two laser instruments difference outgoing, after being converted to tangential polarization light by two tangential polarization converters respectively, further the tangential polarization exciting light is carried out 0～2 π vortex phase coding, break original interference field, make that converging hot spot the hollow phenomenon do not occur, obtains solid fluorescent emission hot spot; After phase coding tangential polarization exciting light and tangential polarization STED Laser Modulation be coaxial emergent light, the secondary color aplanat that disappears by high-NA focuses on the measured fluorescent samples, because exciting light is the following solid hot spot of a diffraction limit, STED laser is the hollow hot spot of a loaf of bread round, 2 spot center points overlap fully, and the hollow hot spot of donut-like suppresses the luminous of exciting light spot edge, therefore two hot spot laps are suppressed, have only central point to launch fluorescence, can realize reducing of fluorescence hot spot, the raising of resolution; As seen, the utility model adopts tangential polarization and carries out the exciting light of 0～2 π vortex phase coding, itself just can obtain the following focal beam spot of diffraction limit, realized high resolving power, and the available technology adopting circularly polarized laser directly focuses on (modulating mutually without any the position) as exciting light, exciting light can not be focused on below the diffraction limit; And the utility model adopts tangential polarization STED light not need to carry out the vortex phase coding, directly focus on and just can obtain bagel hot spot (hollow hot spot), and other STED polarized light just has the bagel hot spot after need focusing on through the vortex phase coding.Because the distinctive focus characteristics of tangential polarization STED laser, it is faster than the bagel convergence that circularly polarized light forms, and therefore under relatively low STED operating power, can reach the resolution identical with prior art.So, under the situation that guarantees resolution, can reduce the STED operating power greatly.The utility model is assembled theory and combining with high-NA and is got up light beam polarization is theoretical, overcomes prior art in the deficiency of STED operating power aspect too high.
With respect to prior art, the utlity model has following beneficial technical effects:
Guaranteeing to greatly reduce the operating power of STED under the high-resolution situation, thereby reducing the bleaching of sample, avoiding damage, overcoming prior art in the deficiency of STED operating power aspect too high to sample.
In addition, because exciting light and STED light all are tangential polarization light, under high numerical aperture lens focuses on, has only radially (x, y direction) component, all there is not z to (depth of focus direction) component, and other polarized light all can have three durection components (x, y, z) in the prior art under high-NA focuses on, and is difficult to distinguish, therefore, the utility model provides better means to the fluorescence polarization The Characteristic Study.
Description of drawings
Fig. 1 is the super-resolution fluorescence microscope equipment synoptic diagram based on tangential polarization of the present utility model;
Fig. 2 is a tangential polarization light synoptic diagram in the utility model;
Fig. 3 is the synoptic diagram of 0～2 π phase board (Vortex 0～2 π PhasePlate) of the utility model mesoscale eddies distribution; Wherein, a is the horizontal synoptic diagram of 0～2 π phase board of vortex distribution, and b is the schematic perspective view of 0～2 π phase board of vortex distribution;
Fig. 4 is the light intensity section comparison diagram of other polarized light in phase coding tangential polarization exciting light and the prior art on high-NA disappears secondary color aplanat focusing back focal plane;
Fig. 5 is the light intensity section comparison diagram of other phase coding polarized light in tangential polarization STED laser and the prior art on high-NA disappears secondary color aplanat focusing back focal plane;
Among the figure:
Embodiment
Describe the utility model in detail below in conjunction with drawings and Examples, but the utility model is not limited in this.
As shown in Figure 1 a kind of based on tangential polarization super-resolution fluorescence microscope equipment comprises:
0～2 π phase board 4, first dichroscope 5, second dichroscope 7, the high-NA that first laser instrument 1, second laser instrument 2, the first tangential polarization converter 3, the second tangential polarization converter 6, vortex distribute disappear secondary color aplanat 8, nanometer translation stage 10, narrow band pass filter 11, achromatism condenser lens 12, pin hole 13 and detector 14.
The first tangential polarization converter 3 and the second tangential polarization converter 6 are the polarization converter Radial-Azimuthal Polarization Converter of Sweden (ARCoptix) company, and 0～2 π phase board 4 that vortex distributes is vortex phase board (Vortex 0～2 π PhasePlate) VPP-1A of U.S. RPC photonics company.
In the formula,
Angle for position polar coordinates vector and X-axis in the light beam vertical Z axle section.
With 0～2 π phase board 4 of tangential polarization exciting light by the vortex distribution, light is modulated to 0～2 π vortex position phase again, light becomes phase coding tangential polarization exciting light R1.The cardinal principle of 0～2 π phase board 4 that vortex distributes is to make the light beam by it produce different phase delay to reach the purpose of phase coding at diverse location.Be the horizontal synoptic diagram and the schematic perspective view of 0～2 π phase board 4 of vortex distribution as shown in Figure 3.The bit phase delay amount Δ α of 4 pairs of incident lights of 0～2 π phase board that vortex distributes is determined by following formula:
In the formula,
Angle for position polar coordinates vector and X-axis on the phase board plane.
Phase coding tangential polarization exciting light R1 turns back 90 ° through first dichroscope 5, and light is R3.
On-axis rays R23 is through the high-NA secondary color aplanat 8 that disappears, wherein, exciting light forms the super-resolution focus hot spot on high-NA disappears the focal plane, picture side of secondary color aplanat 8, STED light forms the focal beam spot of bagel on high-NA disappears the focal plane, picture side of secondary color aplanat 8.2 spot center points overlap fully, disappear secondary color aplanat 8 of high-NA adopts the microcobjective that 100 of NA=1.4 * the secondary color difference that disappears has flat field correction herein, and use the immersion oil of refractive index n=1.518 in the picture side of object lens (object lens focusing hot spot one side), purpose is in order to make object lens reach 1.4 numerical aperture, can to reach better focusing effect.In this case, to form with the desirable focus of geometrical optics on high-NA disappears the focal plane, picture side of secondary color aplanat 8 be the focal beam spot at center for R3 and R2 light beam.Near the focal beam spot light beam Electric Field Distribution can be calculated by following formula:
In the formula,
Be to be the cylindrical coordinate of initial point with desirable focal position, C is a normaliztion constant, and A1 is light beam R2 or R3 light distribution parameter, and A2 is the disappear structural parameters of secondary color aplanat 8 of numerical aperture.Δ α is the bit phase delay amount,
Be the angle of polar coordinates vector in position on the phase board plane and x axle, i is an imaginary unit, and k=2 π/λ, n are medium refraction index, and θ is disappear the focused ray behind the secondary color aplanat and the angle of optical axis.Adopt above-mentioned formula can calculate the size of the hot spot on the focal plane respectively.
Wherein light beam R3 is a small solid hot spot after focusing on, and its full width at half maximum is below diffraction limit.Fig. 4 is the light intensity section comparison diagram of other polarized light in phase coding tangential polarization exciting light and the prior art on high-NA disappears secondary color aplanat 8 focusing back focal planes.Can draw phase coding tangential polarization exciting light focus point full width at half maximum minimum of the present utility model by Fig. 4, its full width at half maximum is 0.34 optical wavelength, in 0.357 of diffraction limit (numerical aperture all is NA=1.4 here) below the optical wavelength.As adopting the aperture to block further optimization again, can reach the full width at half maximum of 0.26 optical wavelength, so, do not using STED to carry out under the condition of delustring, the following high resolving power of diffraction limit has just been arranged.Although the focusing tangential polarization light that blocks after the optimization with footpath with holes has secondary lobe, but these secondary lobes are beyond full width at half maximum is half-wavelength, utilize the influence beyond can the elimination half-wavelength of copolymerization Jiao's receive mode, therefore systemic resolution is not impacted at this secondary lobe.Therefore, utilize the tangential polarization exciting light can realize the focusing that diffraction limit is following.
And tangential polarization STED laser R2 is the hollow hot spot of a bagel after focusing on, and to carrying out further delustring in exciting light R3 hot spot marginal portion, further improves systemic resolution.The utility model unlike previous technologies be, tangential polarization STED light does not need to carry out 0～2 π vortex phase coding, directly focus on and just can obtain bagel hot spot (hollow hot spot), and other STED polarized light just has the bagel hot spot after need focusing on through the vortex phase coding again.Fig. 5 is the light intensity section comparison diagram of other phase coding polarized light in tangential polarization STED laser and the prior art on high-NA disappears secondary color aplanat 8 focusing back focal planes.As seen from Figure 5, radial polarisation and linear polarization can't be used in the STED microtechnic because the central point energy is non-vanishing, and be used in the STED microtechnic, the energy highest point of tangential polarization STED laser of the present utility model than the energy highest point of circularly polarized light more near central point.If the energy peak behind circularly polarized light and tangential polarization STED laser focusing is respectively made straight line to the central energy minimum point, the slope that can find straight line under the tangential polarization STED laser more precipitous (absolute value of slope is bigger), this explanation tangential polarization STED laser uses the convergence ratio circularly polarized light when doing the exciting light delustring to use the convergence when doing the exciting light delustring to want soon.That is to say that under identical power condition tangential polarization STED light is stronger to the extinction capability of exciting light, can obtain higher resolution.Therefore, under identical resolution situation, the operating power of the needed STED light of tangential polarization light is littler, has played the effect that reduces the STED luminous power.
Exciting light R3 is after STED light R2 modulation, the area of fluorescence excitation dwindles greatly, reach diameter below 100nm, the central point emitted fluorescence is collected through disappear secondary color aplanat 8 of above-mentioned same high-NA, see through first dichroscope 5 and second dichroscope 7, pass through narrow band pass filter 11 filtering again with the elimination ground unrest, and focus on the pin hole 13 through achromatism condenser lens 12, fluorescence signal is after the further elimination background stray light of needle passing hole 13 filtering, see through pin hole 13 and incide the fluorescence light intensity that obtains on the sample certain any fluorescence signal on the photodetector 14, handle by 10 pairs of fluorescent samples scannings of mobile nanometer translation stage with to the fluorescence intensity follow-up data again, obtain the micro-image of ultrahigh resolution.
Claims (9)
1. the super-resolution fluorescence microscope equipment based on tangential polarization is characterized in that, comprising:
Be used to send first light source that the fluorescence excitation sample produces the exciting light of fluorescence; Be used for described exciting light is converted to the first tangential polarization converter of tangential polarization exciting light; Be used for the tangential polarization exciting light is carried out 0～2 π phase board of the vortex distribution of 0～2 π vortex phase coding; Be used for phase coding tangential polarization exciting light is carried out 90 ° of first spectroscopes of turning back;
Be used to send the secondary light source that the inhibition fluorescent samples sends the STED laser of fluorescence; Be used for described STED laser is converted to the second tangential polarization converter of tangential polarization STED laser; Be used for tangential polarization STED laser is carried out 90 ° of second spectroscopes of turning back;
The axis light that is used for converging after first and second spectroscopes are turned back focuses on the secondary color aplanat that disappears that obtains the center luminous point on the fluorescent samples;
Be used to collect and survey fluorescence that luminous point sends and the detection imaging system that obtains the micro-image of ultrahigh resolution according to the fluorescence light intensity that detects;
And the nanometer translation stage that is used for fixing fluorescent samples.
2. super-resolution fluorescence microscope equipment as claimed in claim 1 is characterized in that, also comprises: be used for tangential polarization exciting light or phase coding tangential polarization exciting light are carried out the diaphragm that the central shielding optical filtering forms annular beam.
3. super-resolution fluorescence microscope equipment as claimed in claim 1 or 2, it is characterized in that described detection imaging system comprises: be used to collect the secondary color aplanat that disappears of the fluorescence that luminous point sends, the wave filter that is used for elimination ground unrest and background stray light, the detector that is used for filtered fluorescence signal is surveyed, be used for the fluorescence light intensity of surveying is handled and the image device of imaging.
4. super-resolution fluorescence microscope equipment as claimed in claim 3 is characterized in that described wave filter comprises narrowband light filter and spatial filter.
5. super-resolution fluorescence microscope equipment as claimed in claim 3 is characterized in that, described spatial filter is optical fiber or pin hole.
6. super-resolution fluorescence microscope equipment as claimed in claim 3 is characterized in that, described detector is the avalanche diode single-photon detector of high-quantum efficiency.
7. super-resolution fluorescence microscope equipment as claimed in claim 1 is characterized in that, the described secondary color aplanat that disappears is the secondary color aplanat that disappears of high-NA, described high-NA NA=1～1.4.
8. super-resolution fluorescence microscope equipment as claimed in claim 1 is characterized in that, described high-NA NA=1.4.
9. super-resolution fluorescence microscope equipment as claimed in claim 1 is characterized in that, described first and second spectroscopes all adopt dichroscope.
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| CN101907766A (en) * | 2010-07-09 | 2010-12-08 | 浙江大学 | Super-resolution fluorescence microscopy method and device based on tangential polarization |
| CN102305782A (en) * | 2011-08-10 | 2012-01-04 | 浙江大学 | Method and device for analyzing fluorescent correlation spectroscopy based on medium microsphere |
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| CN101907766A (en) * | 2010-07-09 | 2010-12-08 | 浙江大学 | Super-resolution fluorescence microscopy method and device based on tangential polarization |
| CN102305782A (en) * | 2011-08-10 | 2012-01-04 | 浙江大学 | Method and device for analyzing fluorescent correlation spectroscopy based on medium microsphere |
| CN102914525B (en) * | 2012-04-10 | 2016-06-01 | 广东工业大学 | The novel fluorescence life-span microscopic imaging device of optically-based addition heterodyne modulation and method |
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| CN103389573A (en) * | 2013-07-31 | 2013-11-13 | 北京信息科技大学 | STED (stimulated emission depletion) micro imaging method and device based on radially polarized vortex beam |
| CN103389573B (en) * | 2013-07-31 | 2015-04-08 | 北京信息科技大学 | STED (stimulated emission depletion) micro imaging method and device based on radially polarized vortex beam |
| CN104089935A (en) * | 2014-07-11 | 2014-10-08 | 中国科学院化学研究所 | Super-resolution fluorescence lifetime correlation spectroscopy system |
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| CN107014793B (en) * | 2017-04-21 | 2019-07-30 | 浙江大学 | One kind is based on double galvanometer doublet multi-mode wide fields super-resolution micro imaging system |
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