CN106970055B - A kind of three-dimensional fluorescence difference super-resolution microscopic method and device - Google Patents
A kind of three-dimensional fluorescence difference super-resolution microscopic method and device Download PDFInfo
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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Abstract
The present invention discloses a kind of three-dimensional fluorescence difference super-resolution microscope equipment, including laser, carries the electronic sample stage of sample to be tested and light is projected to the microcobjective of the electronic sample stage；Be successively arranged between the laser and microcobjective: the light beam for issuing the laser changes into the polarizer of linearly polarized light；For modulating the first half wave plate of the linearly polarized light polarization direction；For being sequentially modulated the space optical modulator module of beam level component and vertical component；For carrying out the scanning galvanometer system of optical path-deflecting to circularly polarized light, projected on sample to be tested by the circularly polarized light of the scanning galvanometer system exit through microcobjective；It further include acquiring the detection system for the signal light that sample to be tested issues, and control the computer of the space optical modulator module and scanning galvanometer system.Invention additionally discloses the microscopic methods realized based on above-mentioned three-dimensional fluorescence difference super-resolution microscope equipment.
Description
Technical field
The invention belongs to the micro- field of optical ultra-discrimination, in particular to a kind of three-dimensional fluorescence difference super-resolution microscopic method and
Device.
Background technique
1873, Germany scientist Abbe proposed " diffraction limit " of optical imaging system, and any optical microscopy is all deposited
In a resolution limit, determined by the numerical aperture of optical wavelength and lens.Optical microscopy imaging system is due to " diffraction pole
The presence of limit ", cannot achieve 200 nanometers of high-resolution imagings below in visible light wave range.People are continually striving to thus, research hair
Open up super-resolution imaging technology, it is desirable to break through diffraction limit, obtain higher resolution ratio.It is aobvious to be engaged within 2014 fluorescence super-resolution optical
Three scientists of micro- art obtain Nobel chemistry Prize, they open human use's fluorescence labeling method and realize that super-resolution is aobvious
Micro- gate.From this, the optical microphotograph of the mankind has entered the super-resolution epoch.
Present mainstream super-resolution micro-imaging technique can be roughly divided into two classes: one kind is based on classical confocal system, such as
Stimulated radiation is quenched microscopy (STED), fluorescent emission differential microscopy (FED)；Another kind of is based on wide field imaging system, such as
Random optical recombinates microscopy (STORM), Structured Illumination microscopy (SIM), photon activation positioning microscopy (PALM) etc..
In recent years, super resolution technology is grown rapidly, developing direction not in the raising for being only confined to lateral resolution,
But develop towards dimensional resolution, raising image taking speed, system integration densification is improved.Above-mentioned super-resolution is micro-
In art, some develops to three-dimensional, provides more microscopic informations for medicine or biological study personnel.
With the promotion of resolution ratio, optical ultra-discrimination microscopic method and device are increasingly ground by medicine and field of biology
Study carefully the favor of personnel.Lossless specific of its quicklook, makes it obtain more applications, therefore, highly integrated, easy use,
High-resolution optical ultra-discrimination microscope equipment also becomes the emphasis of researchers' concern.
Summary of the invention
The present invention provides a kind of three-dimensional fluorescence difference super-resolution microscopic method and devices, may be implemented to surmount diffraction limit
Dimensional resolution.System structure is compact, single excitation light path, and adjustment is convenient, and party's subtraction unit is simple, and image taking speed is fast, to sample
Product do not have special fluorescent dye requirement.Can be applied to biology, in medical research to diffraction limit once microstructure details three
Dimension imaging.
The specific technical solution of the present invention is as follows: a kind of three-dimensional fluorescence difference super-resolution microscope equipment, including laser, holds
Carry the electronic sample stage of sample to be tested and light projected to the microcobjective of the electronic sample stage, the laser with it is micro-
It is successively arranged between object lens:
Laser for issuing the laser is converted to the collimator of directional light；
Light beam for issuing the laser changes into the polarizer of linearly polarized light；
For modulating the first half wave plate in the light beam polarization direction；
For being sequentially modulated the space optical modulator module of beam level component and vertical component；
For carrying out the scanning galvanometer system of optical path-deflecting to the light beam after the phase-modulation；The circularly polarized light passes through
The microcobjective projects on the sample to be tested；
The scanning for being respectively used to that the light beam of the scanning galvanometer system exit is focused and is collimated being sequentially arranged is saturating
Mirror and field lens；
And it is equipped with the controller for controlling the spatial light modulator and scanning galvanometer system and collects described to test sample
The detection system for the signal light that product issue.
Preferably, the space optical modulator module includes:
Spatial light modulator, by the computer control load black background or simultaneously load 0~π phase modulation pattern and
0~2 π vortex phase is changed the line map case；
Reflecting mirror, the light beam for reflecting spatial light modulator are reflected into spatial light modulator again；
The first quarter-wave plate between the spatial light modulator and reflecting mirror, the light for that will pass through twice
The polarization direction of beam turns over 90 degree.
It is further preferred that being equipped between the space optical modulator module and scanning galvanometer system for converting polarised light
For the second half wave plate of circularly polarized light and the second quarter-wave plate.
In the present invention, detection system includes:
The beam splitter being arranged between the second quarter-wave plate and scanning galvanometer system.The beam splitter is in sample to be tested
For dichroic mirror should be selected when fluorescent samples.
The band pass filter of the stray light in signal light for filtering off beam splitter outgoing, the band pass filter is to be measured
Sample can be omitted when being non-fluorescence sample；
The detector of light intensity signal for detectable signal light beam, the detector are selected photomultiplier tube (PMT) or are avenged
Avalanche photo diode (APD)；
For the signal beams after filtering to be focused on the condenser lens on detector；For being carried out to the signal beams
The spatial filter of space filtering, is located at the focal plane of the condenser lens, and the spatial filter can use pin hole
Or multimode fibre, according to pin hole, the diameter of pin hole used should be less than an Airy spot diameter.
The single mode optical fiber for being filtered to the laser beam is successively arranged between the laser and the polarizer.
The spatial light modulator LCD screen loads 0~π phase modulation pattern simultaneously in the left and right sides and 0~2 π is vortexed
Phase is changed the line map case；
Load-modulate pattern and the switching frequency of black background and the space of scanning galvanometer system are swept in spatial light modulator
It is identical to retouch frequency, to realize that scanning galvanometer system cooperates the electronic too every run-down three-dimensional space of sample, spatial light modulator
Modulation function switching it is primary.
Preferably, the numerical aperture NA=1.49 of the microcobjective.
According to above-mentioned three-dimensional fluorescence difference super-resolution microscope equipment, microscopic method of the invention the following steps are included:
1) laser beam that laser issues is converted to linearly polarized light after collimation；
2) adjust the first half wave plate, make light beam polarization direction and spatial light modulator adjustable polarization direction at
The angle α；
3) polarised light is incident to the screen side of spatial light modulator, the 0~π phase modulation pattern loaded using the side
Phase-modulation is carried out to polarised light；
4) light beam after the reflection of control spatial light modulator is turned back again is incident to the screen other side of spatial light modulator,
Using the side load 0~2 π vortex phase change the line map case carry out phase-modulation；
5) modulated laser beam to the rear focuses on sample through scanning galvanometer system and microcobjective being converted into circle twice
On product and it is scanned；
6) real-time collecting sample is respectively excited the signal light issued during the scanning process, obtains single pass signal
Light intensity I1(x,y,z)；
7) black background will be only loaded in the spatial light modulator in step 3) and step 4), repeat step 3)~6), it is right
Identical three-dimensional space carries out second and scans, and obtains rescan signal light intensity I2(x,y,z)；
8) according to formula I (x, y, z)=I2(x,y,z)-r×I1(x, y, z) calculates final signal light intensity I (x, y, z), and
Super resolution image is obtained using I (x, y, z)；Wherein r=I2 max/2×I1 max, I2 maxFor I2The maximum value of (x, y, z), I1 maxFor I1
Maximum value in (x, y, z).
In the present invention, when sample to be tested is fluorescent samples, the signal light is that the circularly polarized light is thrown through microcobjective
The fluorescence inspired on sample after penetrating；When sample to be tested is non-fluorescence sample, the signal light is circularly polarized light warp
The reflected beams through sample surfaces after microcobjective projection.
Wherein, x on sample, y, z-axis direction are determined by 3-D scanning mode.
Preferably, enabling I (x, y, z)=0 when final signal light intensity I (x, y, z) is negative value.
The principle of the invention is as follows:
According to classical diffraction theory, actual optical system is to the focusing effect of directional light, and nonideal point, but one
The shuttle shape spatial distribution of its bulk can be calculated, long axis prolongs optical axis direction, is diffraction spot or Airy on focal plane.Airy
Sample in range can all be excited to issue signal light, so that the Sample details within the scope of Airy can not be resolved.
Therefore, the resolution ratio of microscopic system is limited by diffraction limit.So breaking through the limitation of diffraction limit, microscopic system is improved
Resolution ratio, it is crucial for reducing Airy area.Theoretically Airy can not be reduced by optical device, but can be led to
It crosses other means and reduces the final equivalent excitation area of system, propose high-resolution purpose to reach.It is similarly super for three-dimensional
For resolution, the resolution of microscope is improved, it is crucial for reducing the volume of space-focusing hot spot.
In the methods of the invention, 0~2 π of load is vortexed on the right side of load 0~π phase modulation pattern on the left of spatial light modulator
Phase modulation pattern.After light beam is by 0~π phase-modulation, according to vectorial field diffraction theory, integral calculation is visitd by Di it is found that
Light field of the light beam after microcobjective focuses at this time is the extremely weak hollow cylinder of an intensity, cylinder both ends in focal plane spatial neighborhood
For the stronger thin solid cylinder of intensity.It after light beam is modulated by 0~2 π vortex phase, can similarly be calculated, light beam passes through at this time
Micro objective is distributed after focusing in focal plane spatial neighborhood for a hollow cylinder, is the hollow light of baked donut shape on focal plane
Spot
Light beam is incident on the left side of spatial light modulator first, the 0~π phase diagram loaded on the left of spatial light modulator at this time
Case is only modulated the component in beam level direction, and vertical component is unmodulated.When light beam through polarization be rotated by 90 ° after, again
When being incident on spatial light modulator upper left side, horizontal component before becomes vertical component, will not be modulated again, before
Vertical component becomes horizontal component, 0~2 π vortex phase pattern modulates loaded on the right side of spatial light modulator.In this way, two
The component in direction is modulated by different modulation patterns, when light beam focuses on focal plane through microcobjective at this time, above two sky
Between optical field distribution be superimposed, approximate hollow ellipsoids optical field distribution is obtained near focal plane, long axis prolongs optical axis direction.This is hollow ellipse
It is I that ball excites scope, which excites signal light intensity obtained by sample,1(x,y,z)。
When spatial light modulator load black background be theoretically any modulation is not done to exciting light, one can consider that
Spatial light modulator only serves the effect of plane mirror.At this point, visiing integral calculation by Di it is found that light beam is through micro-imaging object lens
It is a solid hot spot near focal plane after focusing.Signal light intensity obtained by excitation sample is I in the solid hot spot excites scope2
(x,y,z).According to formula I (x, y, z)=I2(x,y,z)-r×I1(x, y, z) calculates final signal light intensity I (x, y, z).Obvious I
The luminous volume of useful signal light at each scanning element corresponding to (x, y, z) will be less than I2Each scanning element corresponding to (x, y, z)
Locate the volume that shines.Therefore, compared with normal optical microscopic method, present invention decreases the luminous volumes of useful signal light, thus
The resolution ratio of super diffraction limit may be implemented.
Compared with the prior art, the invention has the following beneficial technical effects:
(1) it may be implemented under the premise of lower excitation light power, the resolution ratio of three-dimensional super diffraction limit be provided；
(2) due to only needing twice sweep, the system scanned compared to previous similar approach 3 times, image taking speed improves three
/ mono-.
(3) single excitation light path, so that system compact, saves the step of multichannel tune is overlapped, be easy to adjustment.
(4) single spatial light modulator loads two width modulation patterns, brief cost.
Detailed description of the invention
Fig. 1 is the three-dimensional super-resolution schematic device based on fluorescence stimulated emission differential in the present invention；
Fig. 2 is 0~π phase modulation pattern in the present invention；
Fig. 3 is in the present invention by the light field in the direction focal plane xy and the direction xz that after 0~π phase-modulation, light beam is focused
Distribution；
Fig. 4 is 0~2 π vortex phase modulation pattern in the present invention；
Fig. 5 is in the present invention by after 0~2 π phase-modulation, the light field in the direction focal plane xy and the direction xz that light beam focuses
Distribution；
The optical field distribution in the direction focal plane xy and the direction xz after light beam focuses after modulating twice in Fig. 6 present invention；
Fig. 7 is the direction xy optical field distribution and the direction xy light field at the method for the present invention focal plane at common Laser Scanning Confocal Microscope focal plane
Distribution, i.e., common confocal microscope transverse direction efficient lighting area and present system transverse direction efficient lighting area；
Fig. 8 is the direction xz optical field distribution and the direction xz light field at the method for the present invention focal plane at common Laser Scanning Confocal Microscope focal plane
Distribution, i.e., common confocal microscope longitudinal direction efficient lighting area and present system longitudinal direction efficient lighting area.
Specific embodiment
Below with reference to embodiment and attached drawing, the present invention will be described in detail, but the present invention is not limited to this.
Three-dimensional fluorescence difference super-resolution as shown in Figure 1 is micro-, comprising: laser 1, single mode optical fiber 2a, collimator 3 are polarized
Device 4, reflecting mirror 5a, 1/2 wave plate 6a, D-shaped reflecting mirror 7, spatial light modulator 8, quarter wave plate 9a, lens 10, reflecting mirror 5b, instead
Penetrate mirror 5c, 1/2 wave plate 6b, quarter wave plate 9b, four band logical dichroic mirrors 11, galvanometer scanning system 12, scanning mirror 13, field lens 14 is micro-
Object lens 15, sample too 16, four band pass filters 17, electronic aperture 18, single mode optical fiber 2b, detector 19, control system and PC machine
20。
Wherein, thin optical fiber 2a, collimator 3, the polarizer 4 and reflecting mirror 5a be sequentially located at laser 1 outgoing optical axis it
On, the light transmission axis direction of the polarizer 4 should make the light intensity after transmission maximum.
Wherein, D-shaped reflecting mirror is located on 1 optical axis of laser after turning back, and light beam is turned back for the first time and is incident to sky
Between the left side of optical modulator 8.
Wherein, quarter wave plate 9a, lens 10, reflecting mirror 5b are located on the beam optical axis after spatial light modulator 8 is turned back,
Reflecting mirror 5b also is located on the focal plane of lens 10.
Light beam is reflected by reflecting mirror 5b, and again pass by quarter wave plate 9a, lens 10 are incident on the right side of spatial light modulator,
Reflecting mirror 5c is reflexed to through spatial light modulator for the second time, is transferred by 5c to light beam.Wherein 1/2 wave plate 6b, quarter wave plate 9b
And four band logical dichroic mirror 11 be located on optical axis after reflecting mirror 5c turns back.
Light beam is reflected into galvanometer scanning system 12 by dichroic mirror 11, wherein scanning mirror 13, field lens 14,15 and of microcobjective
Electronic sample stage 16 is sequentially located on the optical axis of scanning galvanometer system exit light beam.Electronic sample stage 16 is located at 15 focal plane of object lens
Place.
Four band pass filters 17, electronic aperture 18 and detector 19 are located on signal light optical axis.
Control system and PC machine 20 are connected with spatial light modulator 8, detector 19 and scanning galvanometer system, for controlling
The switching of pattern in spatial light modulator 8.Spatial light modulator is under the control of upper PC machine and control system 20, in phase tune
Switch between pattern and black background.
In above-mentioned apparatus, the numerical aperture NA=1.49 of microcobjective 15；Aperture used is an electronic small aperture apparatus, from
A series of apertures with condenser lens and changeable diameter use the aperture of 0.7 Airy in the present invention in device；Detection
Device 19 is photomultiplier tube (PMT).
Realize that the process of three-dimensional super-resolution is as follows using Fig. 1 shown device:
The light beam coupling that laser 1 issues imports collimator 3 into single mode optical fiber 2a, by single mode optical fiber 2a, light beam from
Collimator 3 is directional light after being emitted, and is converted to linearly polarized light through polarizing film 4.By reflecting mirror 5a, 1/2 wave plate 6a and D-shaped reflection
Mirror 7 is incident on 8 left side of spatial light modulator.Wherein, 1/2 wave plate fast axle is adjusted, so that the polarization direction of light beam and horizontal direction
Angle is 54.5 degree.0~π phase modulation pattern is loaded on the left of spatial light modulator 8 at this time, as shown in Figure 2.0~π phase-modulation
Modulation function can use polar coordinatesIt is expressed as,
Wherein, θmaxFor the maximum value of incident light radius；
At this point, polarization direction from the horizontal by 54.5 degree of light beam horizontal component by above-mentioned 0~π phase modulation function
Modulation.After being converted to rotatory polarization, the focal plane vicinity optical field distribution after object lens focus is as shown in Figure 3 for it.Light beam is by space
Light reflection again passes by lens 10 and quarter wave plate 9a after reflecting mirror 5b reflection by quarter wave plate 9a and lens 10, incident
On the right side of to spatial light modulator.Reflecting mirror 5b is located in the focus of lens 10, so that shadow of the face shape of reflecting mirror to Beam Wave-Front
Sound is preferably minimized.The fast axle of quarter wave plate 9a is adjusted, so that incident polarization light beam, twice after quarter wave plate 9, polarization direction turns
It crosses 90 degree and is incident on 8 right side of spatial light modulator.0~2 π vortex phase modulation pattern, such as Fig. 4 are loaded on the right side of spatial light modulator
Shown, phase modulation function can be write as:
At this point, becoming vertical component by the horizontal component of 8 left side pattern modulates of spatial light modulator before, can not be adjusted
System.Before unmodulated vertical component becomes horizontal component, i.e., 0~2 π vortex phase loaded on the right side of spatial light modulator 8
Position modulation pattern modulation.Focal plane vicinity optical field distribution such as Fig. 5 institute after the component is converted into rotatory polarization, after object lens focus
Show.
Light beam reflects after reflection on the right side of spatial light modulator, then by reflecting mirror 5c, by 1/2 wave plate 6b and quarter wave plate
9b is converted into rotatory polarization.Circularly polarized light beam is reflected through four band logical dichroic mirrors 11, into galvanometer scanning system 12, then through scanning mirror 13
Enter object lens 15 with field lens 14, focus on sample surface, optical field distribution is as shown in fig. 6, light field shown in as Fig. 3 and Fig. 5
Superposition.By the region of rotatory polarization Shu Jifa on sample, signal light is issued, through field lens 14, scanning mirror 13, galvanometer system 12, four bands
Logical dichroic mirror 11, four bandpass filters 17 enter electronic aperture 18, by the included lens focus of electronic aperture 18 to aperture, then by
Signal light is imported detector 19 by single mode optical fiber 2b, and then enters 20 memory of PC machine.
Controller and PC machine 20 and spatial light modulator 8, detector 19, galvanometer scanning system 12 and electronic sample stage phase
Even.Galvanometer scanning system 12 is controlled by controller and PC machine 20 and electronic sample stage 16 completes the point by point scanning of three-dimensional space, and
Each point signal is recorded, to obtain signal light intensity I1(x,y,z)。
By the pattern on controller adjustment space optical modulator 8, make its full frame load black background, repeats above-mentioned step
Suddenly, signal light intensity I is obtained2(x,y,z).Utilize formula I (x, y, z)=I2(x,y,z)-r×I1(x, y, z) obtains final effective
Signal light intensity I (x, y, z).The direction xy optical field distribution and xy at the method for the invention focal plane at common Laser Scanning Confocal Microscope focal plane
Direction optical field distribution is as shown in Figure 7.The direction xz optical field distribution and the method for the invention are burnt at common Laser Scanning Confocal Microscope focal plane
The direction xz optical field distribution is as shown in Figure 8 at face.
The foregoing is merely preferable implementation examples of the invention, are not intended to restrict the invention, it is all in spirit of that invention and
Within principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (9)
1. a kind of three-dimensional fluorescence difference super-resolution microscope equipment including laser, carries the electronic sample stage of sample to be tested and incites somebody to action
Light projects the microcobjective of the electronic sample stage, it is characterised in that:
It is successively arranged between the laser and microcobjective:
Light beam for issuing the laser changes into the polarizer of linearly polarized light；
For modulating the first half wave plate of the linearly polarized light polarization direction；
For being sequentially modulated the space optical modulator module of beam level component and vertical component；
For carrying out the scanning galvanometer system of optical path-deflecting to circularly polarized light, by the circularly polarized light of the scanning galvanometer system exit
It is projected on sample to be tested through microcobjective；
It further include acquiring the detection system for the signal light that sample to be tested issues, and control the space optical modulator module and scanning
The computer of galvanometer system；
The space optical modulator module includes:
Spatial light modulator controls load black background by the computer or loads 0~π phase modulation pattern and 0~2 simultaneously
π vortex phase is changed the line map case；
Reflecting mirror, the light beam for reflecting spatial light modulator are reflected into spatial light modulator again；
The first quarter-wave plate between the spatial light modulator and reflecting mirror, for by the light beam passed through twice
Polarization direction turns over 90 degree.
2. three-dimensional fluorescence difference super-resolution microscope equipment as described in claim 1, it is characterised in that: the space light modulation
The second half wave plate and the two or four for polarised light to be converted to circularly polarized light is equipped between module and scanning galvanometer system
/ mono- wave plate.
3. three-dimensional fluorescence difference super-resolution microscope equipment as claimed in claim 2, it is characterised in that: by the described 1st/
The light beam of one wave plate outgoing is incident to space optical modulator module after a D-shaped reflecting mirror.
4. three-dimensional fluorescence difference super-resolution microscope equipment as claimed in claim 2, it is characterised in that: the detection system packet
It includes:
The beam splitter being arranged between the second quarter-wave plate and scanning galvanometer system,
The detector of light intensity signal for detectable signal light beam,
For the signal beams after filtering to be focused on the condenser lens on detector,
With the spatial filter for the signal beams to be carried out with space filtering.
5. a kind of microscopic method realized based on any one of Claims 1 to 4 three-dimensional fluorescence difference super-resolution microscope equipment,
Characterized in that it comprises the following steps:
1) laser beam that laser issues is converted to linearly polarized light after collimation；
2) the first half wave plate is adjusted, keeps the polarization direction of light beam and spatial light modulator adjustable polarization direction at α angle；
3) polarised light is incident to the screen side of spatial light modulator, using 0~π phase modulation pattern of side load to inclined
The light that shakes carries out phase-modulation；
4) light beam after the reflection of control spatial light modulator is turned back again is incident to the screen other side of spatial light modulator, utilizes
The side load 0~2 π vortex phase change the line map case carry out phase-modulation；
5) modulated laser beam to the rear is focused on sample through scanning galvanometer system and microcobjective being converted into circle twice
And it is scanned；
6) real-time collecting sample is respectively excited the signal light issued during the scanning process, obtains single pass signal light intensity
I1(x,y,z)；
7) black background will be only loaded in the spatial light modulator in step 3) and step 4), repeat step 3)~6), to identical
Three-dimensional space carry out second and scan, obtain rescan signal light intensity I2(x,y,z)；
8) according to formula I (x, y, z)=I2(x,y,z)-r×I1(x, y, z) calculates final signal light intensity I (x, y, z), and utilizes I
(x, y, z) obtains super resolution image；Wherein r=I2 max/2×I1 max, I2 maxFor I2The maximum value of (x, y, z), I1 maxFor I1(x,y,
Z) maximum value in.
6. microscopic method as claimed in claim 5, which is characterized in that when sample to be tested is fluorescent samples, the signal light
The fluorescence inspired on sample after microcobjective projects for circularly polarized light；It is described when sample to be tested is non-fluorescence sample
Signal light is for circularly polarized light through the reflected beams of sample surfaces after microcobjective projects.
7. microscopic method as claimed in claim 5, which is characterized in that if final signal light intensity I (x, y, z) is negative value, enable I
(x, y, z)=0.
8. microscopic method as claimed in claim 5, which is characterized in that the numerical aperture NA=1.49 of the microcobjective.
9. microscopic method as claimed in claim 5, which is characterized in that in step 2), adjust the first half wave plate
Fast axle, so that the polarization direction of light beam and horizontal direction angle are 54.5 degree.
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