CN109632756A - A kind of real-time fluorescence radiation differential super-resolution microscopic method and device based on parallel beam spot scans - Google Patents
A kind of real-time fluorescence radiation differential super-resolution microscopic method and device based on parallel beam spot scans Download PDFInfo
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Abstract
The invention discloses a kind of real-time fluorescence radiation differential super-resolution microscopic method and device based on parallel beam spot scans, laser beam is divided into S polarized light and P-polarized light by this method, S polarized light is modulated to the solid hot spot of circular polarization, P-polarized light is first modulated into vortex polarised light, re-modulation is at circular polarization hollow light spot；Solid hot spot exciting light and hollow light spot exciting light are staggered at least 200nm or more on object plane；Two-dimensional scanning is carried out simultaneously to fluorescent samples using solid hot spot exciting light and hollow light spot exciting light, the negative confocal fluorescent intensity map for obtaining the positive confocal fluorescent intensity map modulated by solid hot spot and being modulated by hollow light spot；Two width fluorescence intensity figures are subjected to displacement matching.Due to being scanned simultaneously using two hot spots, compared to the way that traditional fluorescent emission difference microscopic system toggles modulation hot spot, sample rate is greater than tradition twice, realizes the micro- effect of super-resolution dynamic under cofocus scanning speed, can significantly improve image taking speed.
Description
Technical field
The invention belongs to the micro- field of super-resolution more particularly to it is a kind of can far field simultaneously obtain a width by solid hot spot tune
The negative confocal images that the positive confocal images and hollow light spot being made are modulated, and super diffraction limit is realized using difference method
The super-resolution microscopic method and device of resolution ratio.
Background technique
The development of fluorescence microscope is greatly promoted the research in the fields such as biocytology, however due to optical diffraction,
There is a resolution limit in conventional far-field optics microscopic method, according to Abbe diffraction limit theory, diffraction limit can be with
It is indicated with the full width at half maximum of the focal beam spot of object lens, i.e. Δ r=0.61 λ/NA, wherein λ is optical wavelength, and NA is objective lens numerical
Aperture.In recent ten years, various scientific research personnel achieves one and another great on breaking through optical diffraction limit
It breaks through, such as super-resolution microtechnic (Stimulation emission depletion is lost in stimulated radiation
Microscopy, STED), fluorescent radiation differential super-resolution microtechnic (Fluorescence emission difference
Microscopy, FED), Structured Illumination super-resolution microtechnic (Structure illumination microscopy,
SIM), photoactivation positioning super-resolution microtechnic (Photoactivated localization microscopy, PLAM) with
And random optical reconstruct super-resolution microtechnic (Stochastic optical reconstruction microscopy,
STORM) etc..
Fluorescent radiation differential super-resolution microscopy is a kind of novel super-resolution microscopic method just proposed recently, this method
Fluorescent image is generated using the excitation hot spot excitation of two kinds of different modes on the basis of confocal, i.e., one is modulated by solid hot spot
Obtained positive confocal microscopy view picture, another kind are the negative confocal microscopy view pictures modulated by bread-like hollow light spot,
The center of middle hollow light spot is the blackening that a size is less than diffraction limit, eliminates edge using the strength difference of this two images
The signal of excitation realizes super-resolution, is a kind of differential imaging.
Compared with other super-resolution microscopic methods, lower fluorescent bleach characteristic is may be implemented in FED, faster imaging speed
Degree, and there is certain optical chromatography ability.However, due to the principle of FED, needed in imaging a positive confocal images and
One negative confocal images needs to scan twice if this just causes to obtain a width super resolution image, so that image taking speed drops
It is low.
Summary of the invention
The present invention provides a kind of real-time fluorescence radiation differential super-resolution microscopic method and dress based on parallel beam spot scans
It sets, compared with traditional FED method, image taking speed improves twice the method for the present invention, realizes super under confocal imaging speed
Resolution imaging.
The purpose of the present invention is achieved through the following technical solutions: a kind of real-time fluorescence based on parallel beam spot scans
Radiate differential super-resolution microscopic method, comprising the following steps:
(1) it is divided into S polarized light and P polarization using polarization spectroscope (PBS) after collimating the laser beam that laser issues
Light；
(2) S polarized light is modulated to the solid hot spot of circular polarization using quarter-wave plate；
(3) phase-modulation is carried out to P-polarized light, is modulated into vortex polarised light；
(4) modulated P-polarized light is further modulated to circular polarization hollow light spot using quarter-wave plate；
(5) light beam is utilized to guarantee that solid hot spot and hollow light spot will not interfere with each other according to diffraction from circular aperture limit formula
Arrangement for deflecting makes solid hot spot exciting light and hollow light spot exciting light be staggered on object plane at least 200nm or more；
(6) two-dimensional scanning is carried out simultaneously to fluorescent samples using solid hot spot exciting light and hollow light spot exciting light, filtered off
Stray light and exciting light collect fluorescence signal, obtain the positive confocal fluorescent intensity map I modulated by solid hot spot1(x,y)
With the negative confocal fluorescent intensity map I modulated by hollow light spot2(x,y)；
(7) two width fluorescence intensity figures are subjected to displacement matching, according to formula I (x, y)=I1(x,y)-γI2(x, y) is obtained
Super resolution image I (x, y), wherein For the first signal light intensity I1The maximum value of (x, y),For second signal
Light intensity I2The maximum value of (x, y)；When I (x, y) is negative, it is arranged I (x, y)=0.
Further, in the step (1), the splitting ratio of two beam polarised lights is adjusted using half wave plate, so that S is inclined
The light intensity of vibration light is weaker than P-polarized light.
Further, in the step (3), phase-modulation is carried out to P-polarized light using vortex phase plate, modulation function isWherein ρ is that certain is put at a distance from optical axis on light beam,For position polar coordinate vectors in beam orthogonal optical axis section
With the angle of X-axis.
Further, in the step (5), beam deflecting device can be using plane mirror, dichroic mirror etc..
Further, it in the step (6), is collected using avalanche photodide (APD) or photomultiplier tube (PMT) glimmering
Optical signal.
The present invention provides two kinds, and the real-time fluorescence based on parallel beam spot scans radiates differential super-resolution microscope equipment, wraps
Include objective table, the throw light to the micro- of objective table of laser, excitation photoabsorption modulation light way module, carrying fluorescent samples to be measured
Mirror holder and detection optical path submodule.
The excitation photoabsorption modulation light way module includes:
The beam expanding lens that the light of point light source for issuing laser expands as directional light；
For modulating the half wave plate of beam expanding lens outgoing light polarization direction；
For half wave plate emergent light to be divided into the polarizing beam splitter of P-polarized light and S polarized light；
For P-polarized light to be carried out to the vortex phase plate of 0~2 π phase-modulation；
For P-polarized light and S polarized light to be modulated to the quarter-wave plate of circularly polarized light；
For by the light beam splitter of P-polarized light and S-polarization combiner；
For reflecting the dichroic mirror of S polarized light and transmission fluorescence；
For two beam circularly polarized lights to be staggered the first plane mirror of at least 200nm or more on object plane；
Scanning lens for light beam splitter emergent light to be focused.
The microscope stand includes:
For scanning lens emergent light to be carried out to the second plane mirror of light beam deflection；
Guan Jing for collimating second plane mirror emergent light；
For pipe mirror emergent light to be converged to the microcobjective of objective table；
Further, the numerical aperture NA=1.49 of microcobjective, enlargement ratio are 100 times, and the focal length of Guan Jing is
200mm, the focal length of scanning lens are 50mm.
The detection optical path submodule includes two schemes:
Scheme one, specifically includes:
For filtering off the band pass filter of the stray light in dichroic mirror emergent light；
Cemented doublet for converging band pass filter emergent light；
For the two-way emergent light of cemented doublet to be carried out to the semicircular planform reflecting mirror of branch；
The first 4F lens group that two paths of signals light for semicircular planform reflecting mirror to be emitted amplifies respectively；
For the first 4F lens group outgoing beam to be carried out to the spatial filter of space filtering；Needle can be selected in spatial filter
Hole or multimode fibre realize that size should be less than an Airy spot diameter；
The first detector for space exploration filter outgoing beam；The optional Photoelectric multiplier tube of first detector
(PMT) or avalanche photodide (APD).
Scheme two, specifically includes:
For filtering off the band pass filter of the stray light in dichroic mirror emergent light；
Cemented doublet for converging band pass filter emergent light；
The 2nd 4F lens group for amplifying the two-way emergent light of cemented doublet；
Two-way light for the 2nd 4F lens group to be emitted carries out the parallel filtering optical fiber of space filtering respectively；Parallel filtering
The spacing of optical fiber is 375um, and in order to guarantee to be collected into the energy of Airy 60%, the enlargement ratio of whole system is set as 800
Times, therefore two hot spots should be 470nm ± 50nm in the spacing of object plane.
For detecting the second detector of parallel filtering fiber exit beam；The optional Photoelectric multiplier tube of second detector
(PMT) or avalanche photodide (APD).
The principle of the invention is as follows: it is innovated on the basis of original fluorescent radiation differential super-resolution microscopic system,
Double light combination spot is staggered greater than the distance of an Airy, and improves detecting module, is reflected using a semicircular planform
Mirror will wherein be staggered signal reflex into independent detection system all the way, and another way then direct detection, or the parallel filter of utilization
The fluorescence signal that wave fiber-optic probe is staggered.
According to diffraction from circular aperture limit formulaWherein k is wave vector, and a is circle hole radius, and θ is
Angular aperture, I0For center intensity maxima, Airy spot diameter can be by formulaIt obtains, secondary big is relatively strong
Degree is I2≈0.00175I0, therefore when two hot spots are alternate be greater than an Airy apart from when, the influence of secondary lobe is then less than thousand points
Two, therefore two hot spots need be spaced be greater than an Airy, do not influenced with guaranteeing hot spot each other.
Compared with prior art, the invention has the following beneficial technical effects: due to being scanned simultaneously using two hot spots, phase
Than the way for toggling modulation hot spot in traditional fluorescent emission difference microscopic system, sample rate is greater than tradition twice,
It realizes the micro- effect of super-resolution dynamic under cofocus scanning speed, image taking speed can be significantly improved.
Detailed description of the invention
Fig. 1 is one schematic diagram of microscope equipment scheme of the present invention；
Fig. 2 is two schematic diagram of microscope equipment scheme of the present invention；
Fig. 3 is parallel beam spot scans proposed by the invention in object plane interval location schematic diagram；
In Fig. 4, (a) is semicircular planform reflecting mirror schematic diagram in Fig. 1 dotted line frame, is (b) parallel filtering in Fig. 2 dotted line frame
Optical fiber schematic diagram；
(a), (b) in Fig. 5 are respectively solid hot spot and hollow light spot enlarged diagram；
Fig. 6 is that solid hot spot subtracts hollow light spot differential super-resolution schematic diagram.
Specific embodiment
Below with reference to example and attached drawing, the present invention will be described in detail, however, the present invention is not limited thereto.
Embodiment 1
A kind of real-time fluorescence based on parallel beam spot scans provided in this embodiment radiates differential super-resolution microscopic method, packet
Include following steps:
(1) it is divided into S polarized light and P polarization using polarization spectroscope (PBS) after collimating the laser beam that laser issues
Light；
(2) S polarized light is modulated to the solid hot spot of circular polarization using quarter-wave plate；
(3) phase-modulation is carried out to P-polarized light, is modulated into vortex polarised light；
(4) modulated P-polarized light is further modulated to circular polarization hollow light spot using quarter-wave plate；
(5) light beam is utilized to guarantee that solid hot spot and hollow light spot will not interfere with each other according to diffraction from circular aperture limit formula
Arrangement for deflecting makes solid hot spot exciting light and hollow light spot exciting light be staggered on object plane at least 200nm or more；
(6) two-dimensional scanning is carried out simultaneously to fluorescent samples using solid hot spot exciting light and hollow light spot exciting light, filtered off
Stray light and exciting light collect fluorescence signal, obtain the positive confocal fluorescent intensity map I modulated by solid hot spot1(x,y)
With the negative confocal fluorescent intensity map I modulated by hollow light spot2(x,y)；
(7) two width fluorescence intensity figures are subjected to displacement matching, according to formula I (x, y)=I1(x,y)-γI2(x, y) is obtained
Super resolution image I (x, y), wherein For the first signal light intensity I1The maximum value of (x, y),For second signal
Light intensity I2The maximum value of (x, y)；When I (x, y) is negative, it is arranged I (x, y)=0.
Further, in the step (1), the splitting ratio of two beam polarised lights is adjusted using half wave plate, so that S is inclined
The light intensity of vibration light is weaker than P-polarized light.
Further, in the step (3), phase-modulation is carried out to P-polarized light using vortex phase plate, modulation function isWherein ρ is that certain is put at a distance from optical axis on light beam,For position polar coordinate vectors in beam orthogonal optical axis section
With the angle of X-axis.
Further, in the step (5), beam deflecting device is using plane mirror, dichroic mirror etc..
Further, it in the step (6), is collected using avalanche photodide (APD) or photomultiplier tube (PMT) glimmering
Optical signal.
Embodiment 2
As shown in Figure 1, a kind of real-time fluorescence based on parallel beam spot scans provided in this embodiment radiates differential super-resolution
Microscope equipment, including laser 14, excitation photoabsorption modulation light way module, objective table 1, the throw light for carrying fluorescent samples to be measured
To the microscope stand and detection optical path submodule of objective table 1；
The excitation photoabsorption modulation light way module includes:
The beam expanding lens 12 that the light of point light source for issuing laser 14 expands as directional light；
The half wave plate 11 for being emitted light polarization direction for modulating beam expanding lens 12；
For 11 emergent light of half wave plate to be divided into the polarizing beam splitter 10 of P-polarized light and S polarized light；
For P-polarized light to be carried out to the vortex phase plate 9 of 0~2 π phase-modulation；
For P-polarized light and S polarized light to be modulated to the quarter-wave plate 8 of circularly polarized light；
For by the light beam splitter 6 of P-polarized light and S-polarization combiner；
For reflecting the dichroic mirror 7 of S polarized light and transmission fluorescence；
For two beam circularly polarized lights to be staggered the first plane mirror 13 of at least 200nm or more on object plane, pass through tune
The deflection angle of whole first plane mirror 13, so that closing hollow light spot and solid hot spot after beam forms certain angle.
Scanning lens 5 for 6 emergent light of light beam splitter to be focused.
The microscope stand includes:
For 5 emergent light of scanning lens to be carried out to the second plane mirror 4 of light beam deflection；
Pipe mirror 3 for collimating 4 emergent light of second plane mirror；
The microcobjective 2 of objective table 1 is converged to for 3 emergent light of pipe mirror；
Further, the numerical aperture NA=1.49 of microcobjective 2, enlargement ratio are 100 times, and the focal length of Guan Jing 3 is
200mm, the focal length of scanning lens 5 are 50mm.
The detection optical path submodule includes:
For filtering off the band pass filter 15 of the stray light in 7 emergent light of dichroic mirror；
Cemented doublet 16 for converging 15 emergent light of band pass filter；
For the two-way emergent light of cemented doublet 16 to be carried out to the semicircular planform reflecting mirror 17 of branch；
The first 4F lens group 18 that two paths of signals light for semicircular planform reflecting mirror 17 to be emitted amplifies respectively；
For 18 outgoing beam of the first 4F lens group to be carried out to the spatial filter 19 of space filtering；Spatial filter 19 can
Pin hole or multimode fibre is selected to realize that size should be less than an Airy spot diameter；
The first detector 20 for 19 outgoing beam of space exploration filter；Photomultiplier transit can be selected in first detector 20
Manage (PMT) or avalanche photodide (APD).
The device further includes the computer 24 for controlling laser 14 and the first detector 20.
Embodiment 3
As shown in Fig. 2, a kind of real-time fluorescence based on parallel beam spot scans provided in this embodiment radiates differential super-resolution
Microscope equipment, including laser 14, excitation photoabsorption modulation light way module, objective table 1, the throw light for carrying fluorescent samples to be measured
To the microscope stand and detection optical path submodule of objective table 1；
The excitation photoabsorption modulation light way module includes:
The beam expanding lens 12 that the light of point light source for issuing laser 14 expands as directional light；
The half wave plate 11 for being emitted light polarization direction for modulating beam expanding lens 12；
For 11 emergent light of half wave plate to be divided into the polarizing beam splitter 10 of P-polarized light and S polarized light；
For P-polarized light to be carried out to the vortex phase plate 9 of 0~2 π phase-modulation；
For P-polarized light and S polarized light to be modulated to the quarter-wave plate 8 of circularly polarized light；
For by the light beam splitter 6 of P-polarized light and S-polarization combiner；
For reflecting the dichroic mirror 7 of S polarized light and transmission fluorescence；
For two beam circularly polarized lights to be staggered the first plane mirror 13 of at least 200nm or more on object plane；For inciting somebody to action
The scanning lens 5 that 6 emergent light of light beam splitter is focused.
The microscope stand includes:
For 5 emergent light of scanning lens to be carried out to the second plane mirror 4 of light beam deflection；
Pipe mirror 3 for collimating 4 emergent light of second plane mirror；
The microcobjective 2 of objective table 1 is converged to for 3 emergent light of pipe mirror；
Further, the numerical aperture NA=1.49 of microcobjective 2, enlargement ratio are 100 times, and the focal length of Guan Jing 3 is
200mm, the focal length of scanning lens 5 are 50mm.
The detection optical path submodule includes:
For filtering off the band pass filter 15 of the stray light in 7 emergent light of dichroic mirror；
Cemented doublet 16 for converging 15 emergent light of band pass filter；
The 2nd 4F lens group 21 for amplifying the two-way emergent light of cemented doublet 16；
Two-way light for the 2nd 4F lens group 21 to be emitted carries out the parallel filtering optical fiber 22 of space filtering respectively；Parallel
The spacing for filtering optical fiber 22 is 375um, in order to guarantee to be collected into the energy of Airy 60%, the enlargement ratio setting of whole system
It is 800 times, therefore two hot spots should be 470nm ± 50nm in the spacing of object plane.
For detecting the second detector 23 of 22 outgoing beam of parallel filtering optical fiber；Photoelectricity times can be selected in second detector 23
Increase pipe (PMT) or avalanche photodide (APD).
The device further includes the computer 24 for controlling laser 14 and the second detector 23.
Above-mentioned specific embodiment is used to illustrate the present invention, rather than limits the invention, of the invention
In spirit and scope of protection of the claims, to any modifications and changes that the present invention makes, protection model of the invention is both fallen within
It encloses.
Claims (10)
1. a kind of real-time fluorescence based on parallel beam spot scans radiates differential super-resolution microscopic method, which is characterized in that this method
The following steps are included:
(1) it is divided into S polarized light and P-polarized light using polarization spectroscope (PBS) after collimating the laser beam that laser issues；
(2) S polarized light is modulated to the solid hot spot of circular polarization using quarter-wave plate；
(3) phase-modulation is carried out to P-polarized light, is modulated into vortex polarised light；
(4) modulated P-polarized light is further modulated to circular polarization hollow light spot using quarter-wave plate；
(5) it is deflected to guarantee that solid hot spot and hollow light spot will not be interfered with each other using light beam according to diffraction from circular aperture limit formula
Device makes solid hot spot exciting light and hollow light spot exciting light be staggered on object plane at least 200nm or more；
(6) two-dimensional scanning is carried out to fluorescent samples using solid hot spot exciting light and hollow light spot exciting light simultaneously, is filtered off spuious
Light and exciting light collect fluorescence signal, obtain the positive confocal fluorescent intensity map I modulated by solid hot spot1(x, y) and by
The negative confocal fluorescent intensity map I that hollow light spot is modulated2(x,y)；
(7) two width fluorescence intensity figures are subjected to displacement matching, according to formula I (x, y)=I1(x,y)-γI2(x, y) obtains oversubscription
Distinguish image I (x, y), wherein For the first signal light intensity I1The maximum value of (x, y),For second signal light intensity
I2The maximum value of (x, y)；When I (x, y) is negative, it is arranged I (x, y)=0.
2. a kind of real-time fluorescence based on parallel beam spot scans according to claim 1 radiates the micro- side of differential super-resolution
Method, which is characterized in that in the step (1), the splitting ratio of two beam polarised lights is adjusted using half wave plate, so that S-polarization
The light intensity of light is weaker than P-polarized light.
3. a kind of real-time fluorescence based on parallel beam spot scans according to claim 1 radiates the micro- side of differential super-resolution
Method, which is characterized in that in the step (3), phase-modulation is carried out to P-polarized light using vortex phase plate, modulation function isWherein ρ is that certain is put at a distance from optical axis on light beam,For position polar coordinate vectors in beam orthogonal optical axis section
With the angle of X-axis.
4. a kind of real-time fluorescence based on parallel beam spot scans according to claim 1 radiates the micro- side of differential super-resolution
Method, which is characterized in that in the step (5), beam deflecting device can be using plane mirror, dichroic mirror etc..
5. a kind of real-time fluorescence based on parallel beam spot scans according to claim 1 radiates the micro- side of differential super-resolution
Method, which is characterized in that in the step (6), collect fluorescence using avalanche photodide (APD) or photomultiplier tube (PMT)
Signal.
6. a kind of real-time fluorescence based on parallel beam spot scans radiates differential super-resolution microscope equipment, which is characterized in that the device
It is shown including laser, excitation photoabsorption modulation light way module, the objective tables of carrying fluorescent samples to be measured, throw light to objective table
Micro mirror frame and detection optical path submodule.
The excitation photoabsorption modulation light way module includes:
The beam expanding lens that the light of point light source for issuing laser expands as directional light；
For modulating the half wave plate of beam expanding lens outgoing light polarization direction；
For half wave plate emergent light to be divided into the polarizing beam splitter of P-polarized light and S polarized light；
For P-polarized light to be carried out to the vortex phase plate of 0~2 π phase-modulation；
For P-polarized light and S polarized light to be modulated to the quarter-wave plate of circularly polarized light；
For by the light beam splitter of P-polarized light and S-polarization combiner；
For reflecting the dichroic mirror of S polarized light and transmission fluorescence；
For two beam circularly polarized lights to be staggered the first plane mirror of at least 200nm or more on object plane；
Scanning lens for light beam splitter emergent light to be focused.
The microscope stand includes:
For scanning lens emergent light to be carried out to the second plane mirror of light beam deflection；
Guan Jing for collimating second plane mirror emergent light；
For pipe mirror emergent light to be converged to the microcobjective of objective table；
The detection optical path submodule includes:
For filtering off the band pass filter of the stray light in dichroic mirror emergent light；
Cemented doublet for converging band pass filter emergent light；
For the two-way emergent light of cemented doublet to be carried out to the semicircular planform reflecting mirror of branch；
The first 4F lens group that two paths of signals light for semicircular planform reflecting mirror to be emitted amplifies respectively；
For the first 4F lens group outgoing beam to be carried out to the spatial filter of space filtering；
The first detector for space exploration filter outgoing beam.
7. the real-time fluorescence according to claim 6 based on parallel beam spot scans radiates differential super-resolution microscope equipment,
It is characterized in that, the spatial filter selects pin hole or multimode fibre to realize that size should be less than an Airy spot diameter.
8. a kind of real-time fluorescence based on parallel beam spot scans radiates differential super-resolution microscope equipment, which is characterized in that the device
It is shown including laser, excitation photoabsorption modulation light way module, the objective tables of carrying fluorescent samples to be measured, throw light to objective table
Micro mirror frame and detection optical path submodule.
The excitation photoabsorption modulation light way module includes:
The beam expanding lens that the light of point light source for issuing laser expands as directional light；
For modulating the half wave plate of beam expanding lens outgoing light polarization direction；
For half wave plate emergent light to be divided into the polarizing beam splitter of P-polarized light and S polarized light；
For P-polarized light to be carried out to the vortex phase plate of 0~2 π phase-modulation；
For P-polarized light and S polarized light to be modulated to the quarter-wave plate of circularly polarized light；
For by the light beam splitter of P-polarized light and S-polarization combiner；
For reflecting the dichroic mirror of S polarized light and transmission fluorescence；
For two beam circularly polarized lights to be staggered the first plane mirror of at least 200nm or more on object plane；
Scanning lens for light beam splitter emergent light to be focused.
The microscope stand includes:
For scanning lens emergent light to be carried out to the second plane mirror of light beam deflection；
Guan Jing for collimating second plane mirror emergent light；
For pipe mirror emergent light to be converged to the microcobjective of objective table；
The detection optical path submodule includes:
For filtering off the band pass filter of the stray light in dichroic mirror emergent light；
Cemented doublet for converging band pass filter emergent light；
The 2nd 4F lens group for amplifying the two-way emergent light of cemented doublet；
Two-way light for the 2nd 4F lens group to be emitted carries out the parallel filtering optical fiber of space filtering respectively；
For detecting the second detector of parallel filtering fiber exit beam.
9. the real-time fluorescence according to claim 8 based on parallel beam spot scans radiates differential super-resolution microscope equipment,
It is characterized in that, the spacing of the parallel filtering optical fiber is 375um, entire to be in order to guarantee to be collected into the energy of Airy 60%
The enlargement ratio of system is set as 800 times, therefore two hot spots should be 470nm ± 50nm in the spacing of object plane.
10. the real-time fluorescence according to claim 6 or 8 based on parallel beam spot scans radiates the micro- dress of differential super-resolution
It sets, which is characterized in that the numerical aperture NA=1.49 of the microcobjective, enlargement ratio are 100 times, and the focal length of Guan Jing is
200mm, the focal length of scanning lens are 50mm.
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