CN110261320B - Method and device for fluorescence staggered differential microscopic imaging - Google Patents
Method and device for fluorescence staggered differential microscopic imaging Download PDFInfo
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/21—Polarisation-affecting properties
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- G—PHYSICS
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- 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
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
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- G—PHYSICS
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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
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/21—Polarisation-affecting properties
- G01N2021/216—Polarisation-affecting properties using circular polarised light
Abstract
Disclosure of the inventionA method of fluorescence interleaved differential microscopy imaging comprising the steps of: 1) the laser beam is collimated and converted into linearly polarized light, and the linearly polarized light is projected on a sample to be detected after phase modulation for scanning imaging; the phase modulation pattern is an all-zero phase diagram; 2) collecting fluorescence signals I excited by the sample to be detected in the two-dimensional scanning processs(x, y), wherein x, y are two-dimensional coordinates of a scanning point on a sample to be detected; 3) switching the phase modulation pattern in the step 1) into a vortex phase diagram, and 4) collecting a fluorescence signal I excited by the sample to be detected in the two-dimensional scanning processh(x, y); 5) repeating the steps 1) to 4) to obtain fluorescent signals excited by 2n samples to be detected, and calculating the light intensity of the fluorescent signals obtained by two adjacent scans in a staggered difference manner to obtain the light intensity I (x, y) of the fluorescent staggered difference signals of a time sequence, wherein the calculation formula is I (x, y) ═ Is(x，y)–βIh(x, y), β are empirical parameters. The invention also discloses a fluorescence staggered differential microscopic imaging device.
Description
Technical Field
The invention belongs to the field of super-resolution microscopic imaging, and particularly relates to a fluorescence staggered differential microscopic imaging method and device.
Background
The optical microscope has played an important role in the fields of chemistry, biology, materials and the like as a tool for scientific research and observation, and provides great convenience for people to observe microstructures and motions thereof. However, the presence of the Abbe diffraction limit makes the spatial resolution of the imaging result to be at most half the wavelength of the excitation light, which is an obstacle to the observation of finer structures. Therefore, various super-resolution methods are proposed to break through the diffraction limit, the laser confocal scanning microscopic imaging technology is most widely applied in the super-resolution method, the transverse resolution of the super-resolution method can theoretically reach 1.4 times of the diffraction limit, and meanwhile, the super-resolution method also has excellent optical slicing capability and can filter fluorescence out of a focal plane, so that the signal-to-noise ratio of an imaging result is improved.
The invention patent with publication number CN102735617B provides a super-resolution microscopic method and device, and provides a fluorescence emission differential microscopic imaging technology with higher spatial resolution on the basis of a laser confocal scanning microscopic imaging technology. The method comprises the steps of firstly scanning a sample by using a solid light spot and collecting an excited fluorescent signal, then modulating the solid light spot into a hollow light spot, then scanning the sample and collecting the excited fluorescent signal, and finally carrying out difference on the two fluorescent signals so as to obtain an imaging result with the spatial resolution far higher than that of a laser confocal scanning microscopic imaging technology. However, this technique is limited by the imaging speed, because it needs two original images to obtain a super-resolution image, i.e. the imaging speed is half of that of the laser confocal scanning micro-imaging technique.
Therefore, there is a need for a method for improving the spatial resolution of confocal laser scanning microscopy without reducing the imaging speed during microscopic imaging.
Disclosure of Invention
The invention aims to provide a fluorescence staggered differential microscopic imaging method which has the advantages of high time resolution of a laser confocal scanning microscopic imaging technology and high space resolution of a fluorescence emission differential microscopic imaging technology. The invention improves the defects of the fluorescence emission differential microscopic imaging method, and adopts a staggered differential mode to reconstruct a super-resolution image, for example, a first super-resolution image is obtained by differentiating a first original image and a second original image, a second super-resolution image is obtained by differentiating a third original image and the second original image (but not a fourth original image), and then the rest super-resolution image is reconstructed by the same method. By using the method, for n original images, n-1 fluorescence staggered differential microscopic imaging results with greatly improved spatial resolution are finally obtained, and the results are basically the same as the frames of the original images, namely the time resolution is twice of that of common fluorescence differential microscopic imaging.
In order to achieve the above object, the fluorescence staggered differential microscopy imaging method provided by the invention comprises the following steps:
1) collimating a laser beam emitted by a laser and converting the laser beam into linearly polarized light;
2) performing phase modulation on the linearly polarized light, wherein the modulation pattern is a full-zero phase diagram, and the modulation function is f1(r, θ) ═ 0, where r is the distance to the optical axis and θ is the angle between the polar coordinate vector and the polar axis;
3) converting the linear polarized light after the phase modulation into circular polarized light;
4) the circularly polarized light is projected on a sample to be detected, and the sample is scanned and imaged under the control of a two-dimensional scanning galvanometer system;
5) collecting fluorescence signals I excited by the sample to be detected in the two-dimensional scanning process by using a detectors1(x, y), wherein x, y are two-dimensional coordinates of a scanning point on a sample to be detected;
6) switching the modulation pattern in the step 2) into a vortex phase diagram, wherein the modulation function is f1(r, θ) ═ θ, where r is the distance to the optical axis and θ is the angle between the polar coordinate vector and the polar axis;
7) repeating the step 3) and the step 4), and collecting the fluorescence signal I excited by the sample to be detected in the two-dimensional scanning process again by using the detectorh1(x, y), wherein x, y are two-dimensional coordinates of a scanning point on a sample to be detected;
8) continuously repeating the steps 2) to 7), and finally obtaining fluorescence signals excited by 2n samples to be detected, wherein Is1(x, y) to Isn(x, y) is the fluorescence signal obtained under modulation of the all-zero phase diagram, Ih1(x, y) to Ihn(x, y) is the fluorescence signal obtained under vortex phase map modulation.
9) Calculating the light intensity staggered difference of the fluorescence signals obtained by two adjacent scans to obtain the required timeThe light intensity of the fluorescence staggered differential signal of the sequence is I (x, y), and the calculation formula is I (x, y) Is(x，y)–βIh(x, y) wherein Is(x, y) is the fluorescence signal obtained under modulation of the all-zero phase diagram, Ih(x, y) is the fluorescence signal obtained under vortex phase map modulation, β is an empirical parameter, typically set to 0.7;
wherein, when the light intensity I (x, y) of the fluorescence staggered differential signal is negative, the light intensity I (x, y) is equal to zero.
The staggered difference calculation method comprises the following steps: first fluorescence staggered differential signal I1(x，y)＝Is1(x，y)–βIh1(x, y), second fluorescence staggered differential signal I2(x，y)＝Is2(x，y)–βIh1(x, y), third fluorescence staggered differential signal I3(x，y)＝Is2(x，y)–βIh2(x, y), and so on, 2n-1 fluorescence staggered differential signals I are obtained1(x, y) to I2n-1(x，y)；
The linear polarization light is p-polarized light, and the spatial light modulator is only sensitive to the p-polarized light, so that the modulation efficiency can be maximized by modulating the spatial light modulator into the p-polarized light.
Wherein the scanning by converting p-polarized light into circularly polarized light is performed in order to make the spot projected onto the sample more uniform.
The scanning step length of the two-dimensional scanning galvanometer system is set according to the required pixel size, and the scanning range of the two-dimensional scanning galvanometer system is set according to the field of view.
Wherein, the solid excitation beam modulated by the spatial light modulator in the step 2) is a Gaussian beam.
Wherein, the hollow excitation light beam modulated by the spatial light modulator in the step 6) is a ring light beam (similar to the shape of a donut).
The principle of the invention is as follows:
converting a laser beam emitted by a collimation laser into linearly polarized light; performing phase modulation on the linearly polarized light, wherein a modulation pattern is a full-zero phase diagram, converting the linearly polarized light into circularly polarized light, and projecting the circularly polarized light on a sample to be measured for two-dimensional scanning; collecting a fluorescence signal emitted by the sample to be detected by using a detector to obtain the light intensity of the fluorescence signal; switching the modulation pattern into a vortex phase diagram, carrying out phase modulation on the linearly polarized light again, and repeating the steps to obtain the light intensity of the fluorescence signal again; continuously switching the all-zero phase and the vortex; phase modulating the pattern until obtaining n fluorescence signal intensities; reconstructing a super-resolution image by adopting a staggered difference mode, for example, performing difference on the light intensity of a first fluorescence signal and the light intensity of a second fluorescence signal to obtain the light intensity of a first effective fluorescence signal, performing difference on the light intensity of a third fluorescence signal and the light intensity of the second fluorescence signal to obtain the light intensity of a second effective fluorescence signal, and reconstructing the light intensity of the remaining effective fluorescence signals by using the same method; and finally obtaining n-1 fluorescence staggered differential microscopic imaging result images with greatly improved spatial resolution.
Another object of the present invention is to provide a fluorescence staggered differential microscopy imaging apparatus for implementing the above method, which can be used to implement the above method, and uses solid light spots and hollow light spots to scan a sample alternately to excite fluorescence, thereby acquiring a series of sample images taken at different times.
An apparatus for fluorescence interleaved differential microscopy, comprising:
a laser for generating a laser beam;
a collimating objective lens for collimating the laser beam;
the polarizer is used for converting the collimated laser beam into linearly polarized light;
the half wave plate is used for converting the linearly polarized light into p-polarized light;
a spatial light modulator for phase modulating the p-polarized light;
the quarter-wave plate is used for converting the modulated p-polarized light into circularly polarized light;
the dichroic mirror is used for reflecting the circular polarization laser beam and the fluorescent beam transmitted by the sample;
the two-dimensional scanning galvanometer system is used for changing the azimuth angle deflection light path of the laser beam passing through the dichroic mirror to realize two-dimensional scanning on the sample and changing the azimuth angle deflection light path of the fluorescent beam passing through the scanning lens to realize de-scanning;
the scanning lens is used for eliminating the distortion of the laser beam passing through the two-dimensional scanning galvanometer system and collimating and beam-shrinking the fluorescent beam passing through the field lens;
the field lens is used for collimating and expanding the laser beam passing through the scanning lens and focusing the fluorescent beam passing through the microscope objective;
the microscope objective is used for focusing the laser beam collimated by the field lens to a sample stage and collecting a fluorescent signal emitted by a sample on the sample stage;
the optical filter is used for filtering stray light transmitted by the dichroic mirror;
the focusing lens is used for focusing the fluorescent light beam passing through the optical filter to the end face of the multimode optical fiber;
the pinhole is used for carrying out spatial filtering on the fluorescent light beam passing through the focusing lens;
a multimode optical fiber for coupling the spatially filtered fluorescence signal to a detector;
and the detector is used for acquiring the fluorescence signal.
The system is also provided with a controller for controlling the spatial light modulator and the scanning galvanometer system, a data acquisition card and a computer for processing the fluorescence signal;
the circularly polarized light means circularly polarized light when projected on a sample.
The two-dimensional scanning galvanometer system is conjugated with the entrance pupil surface of the microscope objective.
The pinhole and the end face of the multimode fiber are positioned at the focal plane of the focusing lens;
the multimode fiber end face diameter should be less than one airy disk diameter.
The angle between the incident and outgoing light of the spatial light modulator should be as small as possible to reduce the crosstalk effect due to light passing through more than one pixel area and to bring the phase travel close to the design value.
Preferably, the laser beam emitted by the laser device enters and exits the spatial light modulator at an included angle of 5 degrees.
The spatial light modulator can switch an all-zero phase pattern and a vortex phase pattern, and the modulation functions of the all-zero phase pattern and the vortex phase pattern are respectively f1(r, θ) ═ 0 and f1(r, θ) ═ θ, where r is the distance to the optical axis and θ is the angle between the polar coordinate vector and the polar axis;
preferably, the detector is an Avalanche Photodiode (APD);
preferably, the Numerical Aperture (NA) of the microscope objective is 1.4;
preferably, the pattern switching of the spatial light modulator and the two-dimensional scanning galvanometer system are triggered synchronously, that is, the two-dimensional scanning galvanometer system is triggered to start two-dimensional scanning of the sample after the pattern switching of the spatial light modulator, and the two-dimensional scanning galvanometer system triggers the spatial light modulator to switch the pattern after the two-dimensional scanning of the sample is completed.
Compared with the prior art, the invention has the following advantages:
(1) the method has high time resolution of a laser confocal scanning microscopic imaging technology and high space resolution of a fluorescence emission differential microscopic imaging technology;
(2) the device is simple and convenient to operate, and can be formed by modifying a traditional confocal microscope system;
(3) high resolution images can be acquired with low excitation light intensity.
Drawings
FIG. 1 is a schematic diagram of an apparatus for fluorescence cross-over differential microscopy according to the present invention;
FIG. 2 is a normalized intensity profile of a solid laser beam in the present invention;
FIG. 3 is a normalized intensity profile of a hollow laser beam in accordance with the present invention;
FIG. 4 is a graph comparing the normalized distribution of the effective signal intensity of the present invention and the conventional confocal signal intensity.
FIG. 5 shows the result of processing the data set obtained by scanning the same longitudinal drift sample four times by the differential fluorescence emission microscopy imaging technique of the present invention and the conventional differential fluorescence emission microscopy imaging technique.
Detailed Description
The present invention will be described in detail with reference to the following examples and drawings, but the present invention is not limited thereto.
The fluorescence staggered differential microscopy imaging device shown in fig. 1 comprises: the device comprises a laser 1, a single-mode optical fiber 2, a collimating lens 3, a polarizer 4, a half-wave plate 5, a first reflector 6, a spatial light modulator 7, a quarter-wave plate 8, a dichroic mirror 9, a two-dimensional scanning galvanometer system 10, a scanning lens 11, a field lens 12, a second reflector 13, a microscope objective 14, a sample stage 15, an optical filter 16, a focusing lens 17, a pinhole 18, a multimode optical fiber 19, a detector 20, a computer and data acquisition card 21 and a display 22.
The optical fiber scanning device comprises a single-mode optical fiber 2, a collimating lens 3, a polarizer 4, a half-wave plate 5, a first reflecting mirror 6, a spatial light modulator 7, a quarter-wave plate 8, a dichroic mirror 9, a two-dimensional scanning mirror vibrating system 10, a scanning lens 11, a field lens 12, a second reflecting mirror 13, a microscope objective 14 and a sample stage 15 which are sequentially arranged on an optical axis of a laser beam emitted by a laser 1. The laser beam is collimated by a collimating objective lens 3, the laser beam is modulated into p-polarized light by a polarizer 4 and a half-wave plate 5, the laser beam is subjected to phase modulation by a spatial light modulator 7, the laser beam projected on a sample is circularly polarized light by a quarter-wave plate 8, the laser beam is reflected by a dichroic mirror 9 and penetrates through a fluorescent beam emitted by the sample, a two-dimensional scanning galvanometer system 10, a scanning lens 11 and a field lens 12 realize two-dimensional scanning of the sample, and the laser beam is focused to a sample stage 15 by a microscope objective lens 14 and a fluorescent signal emitted by the sample is collected;
the optical filter 16, the focusing lens 17, the pinhole 18, the multimode fiber 19 and the detector 20 are sequentially arranged on an optical axis of the fluorescent beam transmitted by the dichroic mirror 9, stray light is filtered by the optical filter 16, the fluorescent beam is focused on the end face of the pinhole 18 multimode fiber 19 by the focusing lens 17, spatial filtering is performed on the pinhole 18, the fluorescent beam is coupled to the detector 20 by the multimode fiber 19, and a fluorescent signal is acquired by the detector 20.
Wherein, the computer and data acquisition card 21 is used for scanning of the two-dimensional scanning galvanometer system 13, signal acquisition of the detector array 22 and final staggered differential calculation;
the method using fluorescence interleaved differential microscopy using the apparatus shown in figure 1 is as follows:
1) an excitation light beam (in this embodiment, 635nm wavelength red light is used as the excitation light) emitted by the laser 1 is coupled into the single-mode optical fiber 2, collimated by the collimating lens 3, then made into linearly polarized light by the polarizer 4, then converted into p-polarized light by the half-wave plate 5, and then reflected by the first reflecting mirror 6 onto the spatial light modulator 7.
2) The spatial light modulator 7 performs phase modulation on the linearly polarized light, the modulation pattern is a full-zero phase diagram, and the modulation function is f1(r, θ) ═ 0, where r is the distance to the optical axis and θ is the angle between the polar coordinate vector and the polar axis;
3) the quarter-wave plate 8 converts the excitation light beam projected on the sample to be measured placed on the sample stage 14 into circularly polarized light;
4) the polarized and modulated excitation light beam is reflected to a two-dimensional scanning galvanometer system 10 by a dichroic mirror 9, the two-dimensional scanning galvanometer system 10 deflects a light path by changing the azimuth angle of the excitation light beam, distortion of an emergent excitation light beam is eliminated after a lens 11 is scanned, a field lens 12 performs collimation and beam expansion, and finally a microscope objective 14 focuses the excitation light beam on a sample to be measured on a sample table 15;
5) the sample to be measured emits fluorescence signals, the fluorescence signals are collected by a microscope objective 14, the fluorescence signals are contracted through a field lens 12 and a scanning lens 11, the fluorescence signals are de-scanned through a two-dimensional scanning galvanometer system 10, the fluorescence signals are transmitted out of a dichroic mirror 9, and the transmitted light enters a multimode optical fiber 19 after being filtered by an optical filter 17 and spatially filtered by a pinhole 18 and is coupled into a detector 20. Collecting fluorescence signals I excited by the sample to be detected by using a detector 20s1(x, y), wherein x, y are two-dimensional coordinates of the scanning point on the sample to be detected, and transmit the data to the computer and data acquisition card 21. Fluorescent Signal Is1The Point Spread Function (PSF) curve of (x, y) is shown in fig. 2;
6) switching the modulation pattern in the step 2) into a vortex phase diagram, wherein the modulation function is f1(r, θ) ═ θ, where r is the distance to the optical axis and θ is the sandwich of the polar coordinate vector and the polar axisAn angle;
7) repeating the step 3), the step 4), and collecting the fluorescence signal I excited by the sample to be detected again by using the detector 20h1(x, y), wherein x, y are two-dimensional coordinates of the scanning point on the sample to be detected and transmit the data to the computer and data acquisition card 21. Fluorescent Signal Ih1The point spread function curve of (x, y) is shown in FIG. 3;
8) continuously repeating the steps 2) to 7), and finally obtaining fluorescence signals excited by 2n samples to be detected, wherein Is1(x, y) to Isn(x, y) is the fluorescence signal obtained under modulation of the all-zero phase diagram, Ih1(x, y) to Ihn(x, y) is the fluorescence signal obtained under vortex phase map modulation.
9) Using a computer 21 to calculate the light intensity of the fluorescence signal obtained by two adjacent scans in a staggered difference manner to obtain the light intensity I (x, y) of 2n-1 fluorescence staggered difference signals required by us, wherein the calculation formula is that I (x, y) is equal to Is(x，y)–βIh(x, y) wherein Is(x, y) is the fluorescence signal obtained under modulation of the all-zero phase diagram, Ih(x, y) is the fluorescence signal obtained under vortex phase map modulation, β is an empirical parameter, typically set to 0.7;
the comparison curve of the point spread function of the effective fluorescence signal I (x, y) of the method of the present invention and the light intensity of the conventional confocal signal is shown in FIG. 3. As can be seen from FIG. 4, the size of the point spread function of the effective signal light intensity I (x, y) of the invention is obviously reduced compared with the common laser confocal scanning microscopic imaging technology, so that the imaging result of the method of the invention has higher spatial resolution and can realize super-resolution imaging.
The results of processing the data set obtained by scanning the same longitudinal drift sample four times by the method of the present invention and the common fluorescence emission differential microscopic imaging technique are shown in fig. 5. The raw data are four fluorescence signal plots obtained using the above method for a 200 nm particle sample that drifts longitudinally, two of which are the imaging results in the all-zero phase pattern and the other two are the imaging results in the vortex phase pattern. The two images on the upper part of FIG. 5 are the processing results of the common fluorescence emission differential microscopic imaging method, and the three images on the lower part of FIG. 5 are the processing results of the method of the present invention, wherein the second image obtained by the method of the present invention is the middle process of the two images of the common fluorescence emission differential microscopic imaging method. Therefore, when the number of the acquired images is larger, the frame number can be increased to 2 times, namely the method has higher time resolution and can realize rapid super-resolution imaging.
The above description is only exemplary of the preferred embodiments of the present invention, and is not intended to limit the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A method of fluorescence interleaved differential microscopy, comprising the steps of:
1) the laser beam is collimated and converted into linearly polarized light, and the linearly polarized light is projected on a sample to be detected after phase modulation for scanning imaging;
the phase modulation pattern is an all-zero phase diagram, and the modulation function is f1(r, θ) ═ 0, where r is the distance to the optical axis and θ is the angle between the polar coordinate vector and the polar axis;
2) collecting fluorescence signals I excited by the sample to be detected in the two-dimensional scanning processs(x, y), wherein x, y are two-dimensional coordinates of a scanning point on a sample to be detected;
3) switching the phase modulation pattern in the step 1) into a vortex phase pattern, wherein the modulation function is f1(r, θ) ═ θ, where r is the distance to the optical axis and θ is the angle between the polar coordinate vector and the polar axis;
4) collecting fluorescence signals I excited by the sample to be detected in the two-dimensional scanning processh(x, y), wherein x, y are two-dimensional coordinates of a scanning point on a sample to be detected;
5) repeating the steps 1) to 4) to obtain fluorescent signals excited by 2n samples to be detected, and calculating the light intensity of the fluorescent signals obtained by two adjacent scans in a staggered difference manner to obtain the light intensity I (x, y) of the fluorescent staggered difference signals of a time sequence, wherein the calculation formula is I (x, y) ═ Is(x，y)–βIh(x, y), β is an empirical parameter;
the staggered difference calculation method comprises the following steps: first fluorescence staggered differential signal I1(x，y)＝Is1(x，y)–βIh1(x, y), second fluorescence staggered differential signal I2(x，y)＝Is2(x，y)–βIh1(x, y), third fluorescence staggered differential signal I3(x，y)＝Is2(x，y)–βIh2(x, y), and so on, 2n-1 fluorescence staggered differential signals I are obtained1(x, y) to I2n-1(x，y)。
2. The method of fluorescence interleaved differential microscopy as defined in claim 1 wherein the fluorescence interleaved differential signal intensity I (x, y) is made equal to zero when it is negative.
3. The method of claim 1, wherein the phase-modulated linear polarization is converted to circular polarization and projected onto the sample.
4. The method of fluorescence interlaced differential microscopy according to claim 1, wherein said linearly polarized light is p-polarized light.
5. The method for fluorescence interleaved differential microscopy as defined in claim 1 wherein in step 1) the phase modulated solid excitation beam is a gaussian beam.
6. The method for fluorescence interleaved differential microscopy as defined in claim 1 wherein in step 3) the phase modulated hollow excitation beam is a ring beam.
7. A fluorescence interleaved differential microscopy imaging device based on the method as claimed in any one of claims 1 to 6, characterized in that it comprises:
the laser device, the polarizer, the spatial light modulator and the two-dimensional scanning galvanometer system are sequentially arranged, and after laser beams emitted by the laser device are converted into linearly polarized light through the polarizer and are modulated with the phase of the spatial light modulator, the two-dimensional scanning galvanometer system controls scanning imaging;
the microscope objective is used for focusing the laser beam emitted by the two-dimensional scanning galvanometer system to the sample stage and collecting a fluorescent signal emitted by a sample on the sample stage;
a detector for collecting the fluorescence signal;
a controller for controlling the spatial light modulator and the scanning galvanometer system, and a computer for processing the fluorescence signal.
8. The fluorescence interlaced differential microscopy imaging apparatus according to claim 7, wherein the controller controls the phase modulation pattern of the spatial light modulator to switch between:
the phase modulation pattern is an all-zero phase diagram, and the modulation function is f1(r, θ) ═ 0, where r is the distance to the optical axis and θ is the angle between the polar coordinate vector and the polar axis;
the phase modulation pattern is switched into a vortex phase diagram, and the modulation function of the vortex phase diagram is f1(r, θ) ═ θ, where r is the distance to the optical axis and θ is the angle of the polar coordinate vector with the polar axis.
9. The fluorescence interlaced differential microscopy imaging device according to claim 7, having located between said polarizer and spatial light modulator:
the half wave plate is used for converting the linearly polarized light into p-polarized light;
and a quarter wave plate for converting the modulated p-polarized light into circularly polarized light.
10. The fluorescence interlaced differential microscopy imaging device according to claim 7, wherein said laser beam emitted by said laser is incident on and exits said spatial light modulator at an angle of 5 degrees.
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