CN104764729A - Up-conversion-nanocrystal-based stimulated depletion super-resolution optical microscopic method and up-conversion-nanocrystal-based stimulated depletion super-resolution optical microscopic system - Google Patents
Up-conversion-nanocrystal-based stimulated depletion super-resolution optical microscopic method and up-conversion-nanocrystal-based stimulated depletion super-resolution optical microscopic system Download PDFInfo
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Abstract
The invention discloses an up-conversion-nanocrystal-based stimulated depletion super-resolution optical microscopic method and an up-conversion-nanocrystal-based stimulated depletion super-resolution optical microscopic system. An up-conversion nanocrystal and the stimulated emission depletion super-resolution fluorescence micro-imaging technique are combined, a super-resolution multiphoton fluorescence signal generated by stimulated emission depletion of an up-conversion luminescent nanometer material labeled sample is detected, a continuous low-power steady-state stimulated depletion process of multiphotons excited by a laser is realized and the simple and effective three-dimensional super-resolution imaging effect having low cost, low complexity and high resolution can be achieved. The stimulated emission depletion super-resolution optical micro-imaging system composed of a depletion light generating module, an exciting light generating module, a plurality of dichroscopes, a multiphoton micro-scanning module and a photoelectric detection module is built based on the method, and a simple and effective real-time dynamic three-dimensional image having low cost, low complexity and high resolution can be acquired.
Description
Technical field
The present invention relates to optical microscopy and optical nano Material Field, be excited loss super-resolution optical microscopic method and system particularly based on upper conversion nano crystalline substance.
Background technology
According to Ruili/Abbe criterion, the radius of the corresponding Airy disk of distance between the brightness such as two grades that optical system can be differentiated, the size of Airy disk can use full width at half maximum (full-width at half-maximum, FWHM) to be expressed as:
Wherein λ is lambda1-wavelength, and NA is lens numerical aperture, and so, the limiting resolution size of conventional optical system is in half-wavelength scope.
Stimulated emission depletion microscopy (STimulated Emission Depletion Microscopy, STED) is a kind of super-resolution imaging method utilizing the saturated nonlinear relationship of being excited loss with excited state fluorescence of fluorescence.STED microscopy comprises two bundle laser, and a branch of is exciting light, and another bundle is loss light; Excitation fluorescent material sends fluorescence, loss light is the hollow beam obtained after phase-modulation, by by excite beyond spot center the fluorescence molecule of part by stimulated radiation principle by force cancellation return ground state, thus directly reduce the halfwidth of autofluorescence point spread function, break through diffraction limit and realize super-resolution.Exciting light in above-mentioned statement is generally femtosecond pulse or continuous (CW) laser, loss light is generally high-power laser (such as, femtosecond pulse or high-power CW laser), due to the components and parts using pulsed laser to need series of complex costliness, and exciting light, loss light, detection wavelength of fluorescence interval are very little, therefore such device has cost and drops into high shortcoming.
Upper conversion nano particle (UCNPs) be a kind of nanocrystalline (as NaYF
4and Y
2o
3deng) in doping with rare-earth ions (as Er
3+, Tm
3+, Ho
3+, Nd
3+and Yb
3+deng) composite nano material, there is following advantage: (1) can use near infrared light to excite instead of ultraviolet light, be conducive to reducing biological damage, increase exciting light penetration depth; (2) multi-photon up-conversion luminescence process is based on real electron level, higher than traditional double, multi-photon dyestuff luminescence efficiency, and low-power can be used to irradiate (as: CW laser instrument) and obtain multiphoton excitation light intensity, saturation shot power is low; (3) without photobleaching, without optical flare, good stability, and fluorescence lifetime is long, emission spectrum spectral line is narrow; (4) excitation spectrum and luminescent spectrum wavelength interval greatly, do not have overlap.
According to above statement, bond material advantage, utilizes lower powered continuous wave laser to mouse out a kind of lower-cost super-resolution imaging method based on multi-photon STED, has important meaning to the application in the fields such as life science, medical research, material characterization test.
Summary of the invention
One object of the present invention is that the shortcoming overcoming prior art is with not enough, a kind of stimulated emission depletion super-resolution optical microscopic method based on upper conversion nano crystalline substance is provided, the method is excited loss method relative to tradition, and be multi-photon imaging process, result has more high resolving power.
Another object of the present invention is to provide a kind of and realizes the above-mentioned micro imaging system of being excited loss super-resolution optical microscopic method based on upper conversion nano crystalline substance, this system utilizes steady laser, utilize the character advantage of up-conversion, realize multiphoton excitation process, thus realize semiconductor steady laser and excite multi-photon to be excited loss, cheap, build conveniently, be easy to promote.
Object of the present invention is realized by following technical scheme: be excited loss super-resolution optical microscopic method based on upper conversion nano crystalline substance, comprises the following steps:
(1) first continuous wave laser produces steady laser bundle, after spatial phase modulation, form hollow beam, and this hollow beam is used as loss light;
(2) second continuous wave lasers produce near infrared steady laser bundle, and this light beam is used as exciting light; Wherein, the wavelength of the second continuous wave laser is in near-infrared band, the wavelength of the first continuous wave laser is shorter than the wavelength of the second continuous wave laser, the luminescent spectrum of the centre wavelength coupling UCNPs of described first continuous wave laser, the excitation wavelength of the Wavelength matched UCNPs of described second continuous wave laser;
(3) loss light and exciting light are spatially collimated co-axial couplings, and by the laser beam focus after coupling at UCNPs itself or by the fluorescently-labeled sample of UCNPs, make sample produce super-resolution multiphoton fluorescence signal;
(4) utilize photodetector to detect above-mentioned super-resolution multiphoton fluorescence signal, carry out XYZ scanning direction, obtain fluorescence imaging picture.
Preferably, the wavelength range of described second continuous wave laser is between 700nm ~ 1800nm.
A kind ofly realize the above-mentioned micro imaging system of being excited loss super-resolution optical microscopic method based on upper conversion nano crystalline substance, comprise loss photogenerated module, exciting light generation module, high saturating low anti-dichroic mirror, the anti-dichroic mirror of low height, multi-photon microscan module and photoelectric detection module, described loss photogenerated module is for generating the hollow beam as loss light, described exciting light generation module is for generating the near infrared steady laser bundle as exciting light, and hollow beam and near infrared steady laser bundle are parallel to each other; The saturating low anti-dichroic mirror of described height and hollow beam angle at 45 ° are placed, and be placed on perpendicular to this hollow beam and pass on the optical axis of the saturating low anti-dichroic mirror of this height, hollow beam is by high saturating low anti-dichroic mirror post deflection 90 °; The anti-dichroic mirror of described low height and near infrared steady laser bundle angle at 45 ° are placed, be placed on perpendicular near infrared steady laser bundle and pass on the optical axis of the anti-dichroic mirror of this low height, near infrared steady laser bundle is by the anti-dichroic mirror post deflection of low height 90 °, the anti-dichroic mirror of low height is arranged on below high saturating low anti-dichroic mirror, and at 45 ° with optical axis; It is a branch of coupled laser bundle that hollow beam after deflection and near infrared steady laser bundle spatially collimate co-axial couplings, this laser beam focuses on the sample of up-conversion luminescent material mark on objective table by multi-photon microscan module, the super-resolution multiphoton fluorescence signal that photoelectric detection module is excited for detecting above-mentioned sample.
Concrete, described loss photogenerated module comprises the first continuous wave laser, and along the first collimator and extender mirror, polaroid, quarter-wave plate, spatial phase modulation plate that the laser beam working direction that this laser instrument is launched is placed successively, the laser beam that described first continuous wave laser sends becomes a branch of parallel beam after the first collimator and extender mirror, then be modulated into hollow beam through polaroid, quarter-wave plate, spatial phase modulation plate, the central light strength of this hollow beam is 0.
Concrete, described exciting light generation module comprises the second continuous wave laser and the second collimator and extender mirror, the laser beam that described second continuous wave laser sends is parallel with the laser beam that the first continuous wave laser sends, the excitation wavelength of the Wavelength matched UCNPs of described second continuous wave laser.
Concrete, described multi-photon microscan module comprises the scanning galvanometer and object lens placed successively along coupled laser bundle working direction, and the laser beam after coupling is focused on by object lens after scanning galvanometer, and the sample on described objective table is placed on the focal plane of object lens.
Concrete, described photoelectric detection module comprises coaxial bandpass filter, condenser lens and the photodetector placed successively, described bandpass filter, condenser lens are arranged on along in the opposite direction of coupled laser bundle working direction, and described photodetector is connected with outer computer.Up-conversion luminescent material mark sample be excited loss laser exciting under transmitting along the high-resolution fluorescence in all directions, part fluorescence signal is collected by object lens, after scanning galvanometer, the anti-dichroic mirror of low height, high saturating low anti-dichroic mirror, by photoelectric detector after bandpass filter and condenser lens.
Preferably, described scanning galvanometer is arranged on a whirligig, and whirligig is controlled by a computing machine, and this computing machine is also connected with photodetector.Therefore after the signal of the complete one-time detection of photoelectric detector, just transmit a signal to computing machine, then this computing machine is rotated by whirligig gated sweep galvanometer, utilizes the mode of focal beam spot scanning samples to obtain a width two dimensional image.
Further, described objective table side is provided with for the motor of driving objective table along Z-direction movement.By this motor, 3-D view can be obtained in conjunction with whirligig.
Preferably, the saturating low anti-dichroic mirror reflects wavelength of described height is less than the wavelength of loss light, is greater than the light of the wavelength of loss light through wavelength.
Preferably, the anti-dichroic mirror reflects wavelength of described low height is greater than the wavelength of exciting light, is less than the light of the wavelength of exciting light through wavelength.
Compared with prior art, tool has the following advantages and beneficial effect in the present invention:
1, micro imaging system of the present invention is to UCNPs itself or by its fluorescently-labeled imaging of samples, namely being combined of stimulated emission depletion microscopy (STimulated Emission Depletion Microscopy, STED) and up-conversion (UCNPs) is realized.The character advantage of up-conversion is utilized to realize above-mentioned two, multiphoton excitation process, thus realize semiconductor steady laser and excite multi-photon to be excited loss, be excited with existing multi-photon the femtosecond pulse exciting light that loss technology adopts and compare, cheap, build conveniently, be easy to promote.
2, the process of fluorescence excitation of the present invention is multiphoton excitation process, and its fluorescence intensity is proportional to the high-order power of incident light light intensity, and it is higher that the imaging facula resolution that therefore the present invention produces is excited loss relative to tradition.Meanwhile, exciting light and radiant light have relatively large anti-Stokes displacement, are convenient to the autofluorescence interference eliminating sample.It is lower that this anti-Stokes displacement also requires to the selection of the components and parts such as dichroscope, optical filter.
3, the present invention utilizes the low saturation shot power benefits of UCNPs material, ultra low power visible ray or near infrared light is used to be excited to be worn to picture, the loss light beam be used as with traditional high power CW laser or femtosecond pulse is compared, and system is simple, cheap.
4, the present invention relies on technology and material character, effectively reduce the photothermal injury of sample, improve imaging depth, avoid the defects such as the common flicker of conventional fluorescent microtechnic, photobleaching and photochemical degradation simultaneously, therefore, the present invention is a kind of super-resolution imaging technology can observed biological sample long-time continuous.
5, the present invention second steady laser (exciting light) wavelength coverage is 700-1800nm, is in near-infrared band, and scattering is little has larger attenuation length in tissue the inside for these wavelength, is conducive to the imaging depth improving STED microscopy.
Accompanying drawing explanation
Fig. 1 is the structural representation of micro imaging system of the present invention.
Fig. 2 is upper conversion nano particle NaYF
4: Yb
3+/ Er
3+fluorescence spectrum figure.
Fig. 3 is upper conversion nano particle NaYF
4: Yb
3+/ Tm
3+fluorescence spectrum figure.
Fig. 4 is for adopting NaYF
4: Yb
3+/ Er
3+as the transmission electron microscope picture of up-conversion.
Fig. 5 is NaYF
4: Er
3+level structure and between them by energy trasfer realize up-conversion luminescence process energy level transition mechanism.
In Fig. 1: the 1-the first continuous wave laser, 2-the first collimator and extender mirror, 3-polaroid, 4-quarter-wave plate, 5-spatial phase modulation plate, 6-high saturating low anti-dichroic mirror, 7-the second continuous wave laser, 8-the second collimator and extender mirror, the anti-dichroic mirror of 9-low height, 10-the first catoptron, 11-scanning galvanometer, 12-object lens, 13-UCNPs or UCNPs marks sample, 14-the second catoptron, 15-bandpass filter, 16-condenser lens, 17-photodetector.
Embodiment
Below in conjunction with embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited thereto.
Embodiment 1
As shown in Figure 1, be excited loss super-resolution optical microscopic system based on upper conversion nano crystalline substance, comprise loss photogenerated module, exciting light generation module, the collimation co-axial couplings module, multi-photon microscan module and the photoelectric detection module that are made up of two dichroic mirrors.
The present embodiment adopts NaYF
4: Yb
3+/ Er
3+as up-conversion fluorescent marker, see Fig. 2, its photoluminescence fluorescence spectrum has two emission peaks at 532nm and 655nm place, therefore the present embodiment employing centre wavelength is first continuous wave laser 1 of 532nm, its laser sent forms hollow beam as being excited loss light after spatial phase modulation plate phase-modulation, employing centre wavelength is the exciting light of the second continuous wave laser 7 as fluorescent marker of 975nm, detect and collimate by above-mentioned two laser the super-resolution two-photon fluorescence signal produced after co-axial couplings focusing scanning irradiates, obtain fluorescence imaging.Fig. 4 is the Electronic Speculum figure of upper conversion nano particle UCNPs.
Concrete, the course of work of the present embodiment is as follows: loss photogenerated module comprises the first continuous wave laser 1, and along the first collimator and extender mirror 2, polaroid 3, quarter-wave plate 4, spatial phase modulation plate 5 that the laser beam working direction that this laser instrument 1 is launched is placed successively, the wavelength that described first continuous wave laser sends is that the laser beam of 532nm becomes a branch of collimated laser beam after the first collimator and extender mirror shaping, then become hollow beam through polaroid, quarter-wave plate, spatial phase modulation plate phase-modulation, the central light strength of this hollow beam is zero.Exciting light generation module comprises the second continuous wave laser 7 and the second collimator and extender mirror 8, and the centre wavelength that described second continuous wave laser sends is that the laser beam of 975nm is parallel with the laser beam that the first continuous wave laser sends.
Described collimation co-axial couplings module comprises high saturating low anti-dichroic mirror 6 and the anti-dichroic mirror 9 of low height, high saturating low anti-dichroic mirror and hollow beam angle at 45 ° are placed, be placed on perpendicular to this hollow beam and pass on the optical axis of the saturating low anti-dichroic mirror of this height, hollow beam is by high saturating low anti-dichroic mirror post deflection 90 °.The anti-dichroic mirror of low height and near infrared steady laser bundle angle at 45 ° are placed, be placed on perpendicular near infrared steady laser bundle and pass on the optical axis of the anti-dichroic mirror of this low height, near infrared steady laser bundle is by the anti-dichroic mirror post deflection of low height 90 °, the anti-dichroic mirror of low height is arranged on below high saturating low anti-dichroic mirror, and at 45 ° with optical axis.It is a branch of coupled laser bundle that hollow beam after deflection and near infrared steady laser bundle spatially collimate co-axial couplings, and in the present embodiment, the critical wavelength of high saturating low anti-dichroic mirror and the anti-dichroic mirror of low height lays respectively in the wavelength coverage of 532nm ~ 655nm and 655nm ~ 975nm.
Coupled laser bundle after collimation co-axial couplings focuses on the NaYF on objective table by multi-photon microscan module
4: Yb
3+/ Er
3+on the sample of up-conversion luminescent material mark, produce super-resolution two-photon fluorescence signal, multi-photon microscan module comprises the first catoptron 10, scanning galvanometer 11, the object lens 12 placed successively along coupled laser bundle working direction, above-mentioned super-resolution two-photon fluorescence signal wavelength is 655nm fluorescence signal, described scanning galvanometer is arranged on a whirligig, whirligig is controlled by an outer computer, and this computing machine is also connected with aftermentioned photodetector.655nm super-resolution two-photon fluorescence signal after object lens, scanning galvanometer, the first catoptron, the anti-dichroic mirror of low height, high saturating low anti-dichroic mirror return, detects this 655nm fluorescence signal by photoelectric detection module successively.
In the present embodiment, photoelectric detection module comprises coaxial the second catoptron 14, bandpass filter 15, condenser lens 16 and the photodetector 17 placed successively, described condenser lens is arranged on the opposite direction of coupled laser bundle working direction, and described photodetector is connected with outer computer.After the complete one-time detection signal of photoelectric detector, just transmit a signal to computing machine, then this computing machine is rotated by whirligig gated sweep galvanometer, the mode of focal beam spot scanning samples is utilized to obtain a width two dimensional image, described objective table side is provided with for the motor of driving objective table along Z-direction movement, by this motor, 3-D view can be obtained in conjunction with whirligig.
Embodiment 2
The present embodiment except following characteristics other structures with embodiment 1:
As shown in Figure 3, the present embodiment adopts NaYF
4: Yb
3+/ Tm
3+as up-conversion fluorescent marker, its photoluminescence fluorescence spectrum has two emission peaks at 480nm and 800nm place, therefore the first continuous wave laser that employing centre wavelength is 800nm forms hollow beam as being excited loss light after spatial phase modulation plate phase-modulation, employing centre wavelength is the exciting light of the second continuous wave laser as fluorescent marker of 975nm, photoelectric detection module detection is collimated the 480nm super-resolution two-photon fluorescence signal produced after co-axial couplings focusing scanning irradiates by above-mentioned two laser, obtain fluorescence imaging.
In the present embodiment, the critical wavelength of high saturating low anti-dichroic mirror and the anti-dichroic mirror of low height lays respectively in the wavelength coverage of 480nm ~ 800nm and 800nm ~ 975nm.
Embodiment 3
The present embodiment except following characteristics other structures with embodiment 1:
As shown in Figure 5, the present embodiment adopts NaYF
4: Er
3+as up-conversion fluorescent marker, Er
3+simultaneously as sensitized ions and activating ion, its photoluminescence fluorescence spectrum level structure has two emission peaks at 655nm and 545nm place, exciting light energy level difference as shown in Figure 5, therefore the first continuous wave laser that employing centre wavelength is 655nm forms hollow beam as being excited loss light after spatial phase modulation plate phase-modulation, employing centre wavelength is the exciting light of the second continuous wave laser as fluorescent marker of 1490nm, photoelectric detection module detection is collimated the 545nm super-resolution three-photon fluorescent signal produced after co-axial couplings focusing scanning irradiates by above-mentioned two laser, obtain fluorescence imaging.
In the present embodiment, the critical wavelength of high saturating low anti-dichroic mirror and the anti-dichroic mirror of low height lays respectively in the wavelength coverage of 540nm ~ 655nm and 655nm ~ 1490nm.
Above-described embodiment is the present invention's preferably embodiment; but embodiments of the present invention are not restricted to the described embodiments; change, the modification done under other any does not deviate from Spirit Essence of the present invention and principle, substitute, combine, simplify; all should be the substitute mode of equivalence, be included within protection scope of the present invention.
Claims (10)
1. be excited loss super-resolution optical microscopic method based on upper conversion nano crystalline substance, it is characterized in that, comprise the following steps:
(1) first continuous wave laser produces steady laser bundle, after spatial phase modulation, form hollow beam, and this hollow beam is used as stimulated emission depletion light;
(2) second continuous wave lasers produce near infrared steady laser bundle, and this light beam is used as exciting light; Wherein, the wavelength of the second continuous wave laser is in near-infrared band, the wavelength of the first continuous wave laser is shorter than the wavelength of the second continuous wave laser, the centre wavelength of described first continuous wave laser matches the luminescent spectrum of conversion nano particle, the excitation spectrum of the Wavelength matched UCNPs of described second continuous wave laser;
(3) loss light and exciting light are spatially collimated co-axial couplings, and by the laser beam focus after coupling at UCNPs itself or by the fluorescently-labeled sample of UCNPs, make sample produce super-resolution multiphoton fluorescence signal;
(4) utilize photodetector to detect above-mentioned super-resolution multiphoton fluorescence signal, and carry out XYZ scanning direction, obtain fluorescence imaging picture.
2. be according to claim 1ly excited loss super-resolution optical microscopic method based on upper conversion nano crystalline substance, it is characterized in that, the wavelength range of described second continuous wave laser is between 700nm ~ 1800nm.
3. one kind realizes the micro imaging system of being excited loss super-resolution optical microscopic method based on upper conversion nano crystalline substance described in any one of claim 1-2, it is characterized in that, comprise loss photogenerated module, exciting light generation module, high saturating low anti-dichroic mirror, the anti-dichroic mirror of low height, multi-photon microscan module and photoelectric detection module, described loss photogenerated module is for generating the hollow beam as loss light, described exciting light generation module is for generating the near infrared steady laser bundle as exciting light, and hollow beam and near infrared steady laser bundle are parallel to each other; The saturating low anti-dichroic mirror of described height and hollow beam angle at 45 ° are placed, and be placed on perpendicular to this hollow beam and pass on the optical axis of the saturating low anti-dichroic mirror of this height, hollow beam is by high saturating low anti-dichroic mirror post deflection 90 °; The anti-dichroic mirror of described low height and near infrared steady laser bundle angle at 45 ° are placed, be placed on perpendicular near infrared steady laser bundle and pass on the optical axis of the anti-dichroic mirror of this low height, near infrared steady laser bundle is by the anti-dichroic mirror post deflection of low height 90 °, the anti-dichroic mirror of low height is arranged on below high saturating low anti-dichroic mirror, and at 45 ° with optical axis; It is a branch of coupled laser bundle that hollow beam after deflection and near infrared steady laser bundle spatially collimate co-axial couplings, this laser beam focuses on the sample of up-conversion luminescent material mark on objective table by multi-photon microscan module, the super-resolution multiphoton fluorescence signal that photoelectric detection module is excited for detecting above-mentioned sample.
4. micro imaging system according to claim 3, it is characterized in that, described loss photogenerated module comprises the first continuous wave laser, and along the first collimator and extender mirror, polaroid, quarter-wave plate, spatial phase modulation plate that the laser beam working direction that this laser instrument is launched is placed successively, the laser beam that described first continuous wave laser sends becomes a branch of parallel beam after the first collimator and extender mirror, then be modulated into hollow beam through polaroid, quarter-wave plate, spatial phase modulation plate, the central light strength of this hollow beam is 0.
5. micro imaging system according to claim 4, it is characterized in that, described exciting light generation module comprises the second continuous wave laser and the second collimator and extender mirror, the laser beam that described second continuous wave laser sends is parallel with the laser beam that the first continuous wave laser sends, the excitation wavelength of the Wavelength matched UCNPs of described second continuous wave laser.
6. micro imaging system according to claim 3, it is characterized in that, described multi-photon microscan module comprises the scanning galvanometer and object lens placed successively along coupled laser bundle working direction, laser beam after coupling is focused on by object lens after scanning galvanometer, and the sample on described objective table is placed on the focal plane of object lens.
7. micro imaging system according to claim 3, it is characterized in that, described photoelectric detection module comprises coaxial bandpass filter, condenser lens and the photodetector placed successively, described bandpass filter, condenser lens are arranged on along in the opposite direction of coupled laser bundle working direction, and described photodetector is connected with outer computer.
8. micro imaging system according to claim 6, is characterized in that, described scanning galvanometer is arranged on a whirligig, and whirligig is controlled by a computing machine, and this computing machine is also connected with photodetector.
9. micro imaging system according to claim 8, is characterized in that, described objective table side is provided with for the motor of driving objective table along Z-direction movement.
10. micro imaging system according to claim 3, is characterized in that, the saturating low anti-dichroic mirror reflects wavelength of described height is less than the wavelength of loss light, is greater than the light of the wavelength of loss light through wavelength;
The anti-dichroic mirror reflects wavelength of described low height is greater than the wavelength of exciting light, is less than the light of the wavelength of exciting light through wavelength.
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| CN110836876A (en) * | 2018-08-15 | 2020-02-25 | 浙江大学 | Super-resolution microscopy method and system based on saturated pumping-stimulated radiation detection |
| CN110836876B (en) * | 2018-08-15 | 2021-05-14 | 浙江大学 | Super-resolution microscopy method and system based on saturated pumping-stimulated radiation detection |
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| CN110646385A (en) * | 2019-10-10 | 2020-01-03 | 安徽师范大学 | Single-particle imaging device and method based on up-conversion luminescence total internal reflection |
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| CN113933317A (en) * | 2021-06-16 | 2022-01-14 | 北京工业大学 | Double-beam combining method and device for laser microscopic detection |
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