阅读说明：本技术 基于偏振结构光调制的多维层析荧光显微成像系统及方法 (Multi-dimensional chromatography fluorescence microscopic imaging system and method based on polarization structure light modulation ) 是由 席鹏 张昊 李美琪 刘文辉 戴琼海 于 2020-06-28 设计创作，主要内容包括：本发明公开了一种基于偏振结构光调制的多维层析荧光显微成像系统及方法。本发明对每个偏振调制方向的照明,都采集两张相位互补的正弦结构光照明图案,通过这两张正弦结构光照明图案的平均来获取均匀光照明图案,再将该均匀光照明图案与其中一张正弦结构光照明图案结合并利用HiLo算法即可求解出光学层析图像；只需要对这三张结构光图案平均同样可以得到均匀光照明图案,再与其中一张结构光照明图案结合即可利用HiLo算法求解出光学层析图案；本发明采取了探测端分光同时采集的方法,不需要增加额外的采集时间；本发明成像速度、较少光漂白等方面具有较大的优势。(The invention discloses a polarization structure light modulation-based multi-dimensional chromatography fluorescence microscopic imaging system and method. The method comprises the steps of collecting two sinusoidal structure light illumination patterns with complementary phases for illumination in each polarization modulation direction, obtaining a uniform light illumination pattern through averaging the two sinusoidal structure light illumination patterns, combining the uniform light illumination pattern with one of the sinusoidal structure light illumination patterns, and solving an optical tomography image by using a HiLo algorithm; uniform light illumination patterns can be obtained by averaging the three structured light patterns, and the optical tomography patterns can be solved by using a HiLo algorithm by combining the uniform light illumination patterns with one structured light illumination pattern; the invention adopts a method of simultaneously collecting light split at the detection end without adding extra collection time; the invention has great advantages in the aspects of imaging speed, less photobleaching and the like.)

1. A multi-dimensional tomography fluorescence microscopic imaging system based on polarized structure light modulation is characterized in that the multi-dimensional tomography fluorescence microscopic imaging system comprises: the device comprises a plurality of lasers, a beam combining device, an acousto-optic tunable filter, a beam expanding device, a polarization beam splitter, a half-wave plate, a spatial light modulator, a condensing lens, a spatial light filter, a vortex half-wave plate, a first dichroic mirror, a first 4f system, a second dichroic mirror, an objective lens, an emission light filter, a tube lens, a second 4f system, a diaphragm, a waveband beam splitting device and a camera; each laser emits linearly polarized laser with one waveband, and the plurality of lasers respectively emit linearly polarized laser with different wavebands; multiple beams of line polarized lasers with different wave bands are combined through a beam combining device and transmitted to an acousto-optic tunable filter on the same optical axis; the wave band of the passing linearly polarized laser is quickly selected through an acousto-optic tunable filter; the linear polarization laser with the selected wave band is expanded by a beam expanding device and then is transmitted to a spatial light modulator through a polarization beam splitter and a half-wave plate; loading periodic black-white binary periodic stripes on a spatial light modulator, reflecting laser to form multi-level linearly polarized diffracted light, and enabling the plane of the spatial light modulator to be perpendicular to an optical axis; the multi-stage linear polarization diffraction light is emitted through the half-wave plate and the polarization beam splitter, and then passes through the polarization beam splitter and the half-wave plate twice, so that the polarization direction of the multi-stage linear polarization diffraction light emitted from the polarization beam splitter is consistent with the polarization direction of the incident linear polarization laser; the multi-level linearly polarized diffraction light is focused by a condensing lens and then reaches a spatial filter; the focal plane of the condenser lens is positioned on the Fourier surface of the spatial light modulator, and the spatial filter is positioned at the focal plane of the condenser lens; the multi-order linearly polarized diffracted light passes through the spatial filter, and only +/-1 order linearly polarized diffracted light passes through the spatial filter; then the polarization direction of the plus or minus 1-level linear polarization diffraction light is adjusted to be consistent with the direction of the binary periodic stripes on the spatial light modulator through a vortex half-wave plate which is arranged close to the spatial light filter; the +/-1-order linearly polarized diffraction light passes through the first dichroic mirror, then passes through the first 4f system, then passes through the second dichroic mirror, and is converged to a sample positioned on the rear focal plane of the objective lens by the objective lens, the Fourier plane of the spatial light modulator is delayed to the rear focal plane of the objective lens by the first 4f system, namely the rear focal plane of the objective lens is positioned on the Fourier plane of the spatial light modulator by the first 4f system; the plus or minus 1-order linear polarization diffraction light interferes on the back focal plane of the objective lens to form sinusoidal stripe illumination, the sample is excited to generate fluorescence, the fluorescence is collected by the objective lens after returning, filtered by the emission optical filter after passing through the second dichroic mirror, and focused by the lens barrel lens; the first dichroic mirror and the second dichroic mirror are completely the same, but the spatial arrangement directions are mutually vertical, so that the polarization distortion introduced by independently using the second dichroic mirror is eliminated; a second 4f system is arranged between the tube lens and the camera, so that the focal plane of the tube lens is delayed to the plane of the camera; a diaphragm is firstly arranged in the middle of the second 4f system and used for controlling the size of an imaging field of view; in the middle of the second 4f system, a waveband light splitting device is arranged behind a diaphragm and spatially separates the fluorescence according to wavebands, so that the fluorescence with different wavebands is projected to different positions of the camera, and each waveband of the fluorescence corresponds to one position on the camera and serves as a spectrum detection channel; acquiring fluorescence of corresponding wave bands through different positions by a camera to obtain an original image; keeping the angle of the binary periodic stripes unchanged, changing the binary periodic stripes loaded on the spatial light modulator into complementary binary periodic stripes, and then obtaining an original image, thereby obtaining two original images of the binary periodic stripes at an angle to form a group of original images; the binary periodic stripes rotate around a horizontal optical axis, a group of original images are obtained every time the binary periodic stripes rotate pi/n, and n is larger than or equal to 3, so that n groups of original images are obtained; carrying out chromatographic image reconstruction on each group of original images according to different spectral detection channels to obtain n chromatographic images, averaging the n chromatographic images to obtain a wide-field optical chromatographic image corresponding to each spectral detection channel, and further obtaining spectral related information; and carrying out polarization demodulation on the n tomographic images to obtain polarization-related information.

2. The multi-dimensional tomographic fluorescence microscopy imaging system of claim 1, wherein the beam combining means employs a mirror and a dichroic mirror.

3. The multi-dimensional tomographic fluorescence microscopy imaging system of claim 1, wherein the first 4f system and the second 4f system are optical plane delay-and-zoom 4f systems.

4. The multi-dimensional tomographic fluorescence microscopy imaging system of claim 1, wherein the beam expanding device is a 4f system.

5. The multi-dimensional tomographic fluorescence microscopy imaging system of claim 1, wherein the spatial filter corresponds to a rotation angle of the binary periodic fringes and comprises n pairs of clear holes, each pair of clear holes being located on a diameter of a pass circle, and an angle between each adjacent pair of clear holes being pi/n.

6. The multi-dimensional tomographic fluorescence microscopy imaging system according to claim 1, wherein the wavelength-splitting means employs m-1 dichroic mirrors to split the fluorescence into m wavelength bands, and one or more reflecting mirrors are disposed in each of the wavelength bands, so as to adjust spatial positions of optical paths of the fluorescence of the corresponding wavelength band to form m spectral detection channels.

7. A microscopic imaging method of the polarized structured light modulation based multi-dimensional tomographic fluorescence microscopic imaging system according to claim 1, wherein the microscopic imaging method comprises the steps of:

1) each laser emits linearly polarized laser with one waveband, and the plurality of lasers respectively emit linearly polarized laser with different wavebands;

2) multiple beams of line polarized lasers with different wave bands are combined through a beam combining device and transmitted to an acousto-optic tunable filter on the same optical axis; the wave band of the passing linearly polarized laser is quickly selected through an acousto-optic tunable filter;

3) the linear polarization laser with the selected wave band is expanded by a beam expanding device and then is transmitted to a spatial light modulator through a polarization beam splitter and a half-wave plate;

4) loading periodic black-white binary periodic stripes on a spatial light modulator, reflecting laser to form multi-level linearly polarized diffracted light, and enabling the plane of the spatial light modulator to be vertical to a horizontal optical axis;

5) the multi-stage linear polarization diffraction light is emitted through the half-wave plate and the polarization beam splitter, and the linear polarization laser passes through the polarization beam splitter and the half-wave plate twice, so that the polarization direction of the multi-stage linear polarization diffraction light emitted from the polarization beam splitter is consistent with the polarization direction of the incident linear polarization laser;

6) the multi-level linearly polarized diffraction light is focused by a condensing lens and then reaches a spatial filter; the focal plane of the condenser lens is positioned on the Fourier surface of the spatial light modulator, and the spatial filter is positioned at the focal plane of the condenser lens; the multi-order linearly polarized diffracted light passes through the spatial filter, and only +/-1 order linearly polarized diffracted light passes through the spatial filter;

7) the plus or minus 1-order linear polarization diffraction light passes through a vortex half-wave plate which is arranged close to the spatial filter, and the polarization direction of the plus or minus 1-order linear polarization diffraction light is adjusted to be consistent with the direction of the binary periodic fringes on the spatial light modulator;

8) the +/-1-order linearly polarized diffraction light passes through the first dichroic mirror, then passes through the first 4f system, then passes through the second dichroic mirror, and is converged to a sample positioned on the rear focal plane of the objective lens by the objective lens, the Fourier plane of the spatial light modulator is delayed to the rear focal plane of the objective lens by the first 4f system, namely the rear focal plane of the objective lens is positioned on the Fourier plane of the spatial light modulator by the first 4f system;

9) the plus or minus 1-order linear polarization diffraction light interferes on the back focal plane of the objective lens to form sinusoidal stripe illumination, the sample is excited to generate fluorescence, the fluorescence is collected by the objective lens after returning, filtered by the emission optical filter after passing through the second dichroic mirror, and focused by the lens barrel lens; the first dichroic mirror and the second dichroic mirror are completely the same, but the spatial arrangement directions are mutually vertical, so that the polarization distortion introduced by independently using the second dichroic mirror is eliminated;

10) a second 4f system is arranged between the tube lens and the camera, so that the focal plane of the tube lens is delayed to the plane of the camera;

11) a diaphragm is firstly placed in the middle of the second 4f system, so that the size of an imaging field of view is controlled;

12) in the middle of the second 4f system, a waveband light splitting device is arranged behind a diaphragm and spatially separates the fluorescence according to wavebands, so that the fluorescence with different wavebands is projected to different positions of the camera, and each waveband of the fluorescence corresponds to one position on the camera and serves as a spectrum detection channel;

13) acquiring fluorescence of corresponding wave bands through different positions by a camera to obtain an original image;

14) keeping the angle of the binary periodic stripes unchanged, changing the binary periodic stripes loaded on the spatial light modulator into complementary binary periodic stripes, and repeating the steps 1) -13) to obtain another original image, thereby obtaining two original images of the binary periodic stripes at an angle to form a group of original images;

15) the binary periodic stripes rotate around a horizontal optical axis by pi/n, the steps 1) to 14) are repeated, a group of original images are obtained at each angle, n is larger than or equal to 3, so that n groups of original images are obtained, and the binary periodic stripes at each angle correspond to one polarization modulation;

16) after n groups of original images are obtained, carrying out chromatographic image reconstruction on each group of original images according to different spectral detection channels to obtain n chromatographic images, and averaging the n chromatographic images to obtain a wide-field optical chromatographic image corresponding to each spectral detection channel so as to obtain spectral related information; polarization demodulation is performed on the n tomographic images to obtain polarization-related information.

8. The microscopic imaging method according to claim 7, wherein in step 16), the reconstruction of the wide-field optical tomography image and the demodulation of the polarization information for the different spectral detection channels comprises the following steps:

a) original image:

in n groups of original images, the corresponding polarization modulation direction of each group of original images is theta_i(i 1.. n), each group comprises two sinusoidal illumination patterns, the stripe directions of the two sinusoidal illumination patterns are consistent with the polarization modulation direction, and the phase difference of the stripes is pi;

b) alignment of different spectral detection channels:

each original image comprises m spectrum detection channels which are positioned at non-overlapping spatial positions, different spectrum detection channels are cut and separated, and then patterns of different spectrum detection channels are spatially aligned by multiplying each spectrum detection channel by a corresponding affine transformation matrix;

c) polarization modulated illumination intensity correction:

for each spectral detection channel, 2n original images are obtained, respectively

d) And (3) solving optical tomography images under different polarization modulations:

averaging two sinusoidal illumination patterns of each polarization modulation to obtain an image I corresponding to the uniform illumination under the polarization modulation_U，Pi：

Binding of I_U，PiAnd the corrected image I_SI1，PiOr I_SI2，PiObtaining an optical tomography image I corresponding to the polarization modulation by adopting a high-low frequency fusion reconstruction algorithm (HiLo) algorithm_OS，Pi；

e) Obtaining a wide-field optical tomography image:

averaging different polarization tomography patterns to obtain wide-field optical tomography image I_osNamely:

f) repeating the steps b) to e) for each spectral detection channel so as to obtain wide-field chromatographic images of all spectral detection channels;

g) obtaining polarization information of the sample:

polarization information of the sample includes fluorescent dipolesA polarization direction alpha and a polarization modulation degree OUF when the polarization modulation direction is theta_iTime, fluorescence dipole excitation intensity I_OS，PiComprises the following steps:

I_OS，Pi＝I_DC+I_AC·cos(2θ_i-2α)，(i＝1，...，n)

wherein, I_DCIs a direct current component, I_ACIs an alternating component, θ_iThe polarization modulation direction is shown as alpha, the polarization direction of the fluorescent dipole is shown as alpha, the fluorescent molecules and the biomolecules form a dipole, and the expression of the dipole in a matrix form is as follows:

wherein, theta_iFor the known polarization modulation direction, alpha, I is determined by matrix inversion_DC，I_ACDegree of polarization modulation, OUF ═ 2I_AC/(I_AC+I_DC) And further obtain the related information of the spectrum.

9. A microscopic imaging method according to claim 8, wherein in step b), the alignment of the patterns of different spectral detection channels by multiplying each spectral detection channel by a respective affine transformation matrix comprises the steps of:

i. preparing a sparse single-layer fluorescent microsphere fixed sample wafer, and imaging the sample wafer to obtain n groups of original images;

ii, intercepting and separating different spectral detection channels of each original image, and solving wide-field optical chromatographic patterns corresponding to the spectral detection channels according to the steps d) and e);

and iii, positioning the position coordinates of the center of the fluorescent microsphere, and solving an affine transformation matrix corresponding to the pattern transformation of all the spectrum detection channels to the first spectrum detection channel according to the coordinates.

10. The microscopic imaging method according to claim 8, wherein in step c), the light intensity correction matrix corresponding to different polarization modulation directions is obtained by calibration, comprising the following steps:

i. preparing a dense fluorescent microsphere fixed sample wafer, imaging, and acquiring n groups of original images, wherein the fluorescent microspheres in the sample wafer should cover the whole imaging field of view as much as possible;

ii, obtaining images of each spectral detection channel without light intensity correction after processing according to the step b)

Averaging two sinusoidal illumination patterns in each polarization modulation direction to obtain an image corresponding to uniform illumination under the polarization modulation

Dividing the pattern of uniform illumination in each polarization modulation direction by the pattern of uniform illumination in the first polarization modulation direction to obtain the corresponding intensity correction matrix in each polarization modulation direction.

Technical Field

The invention relates to the field of optical microscopic imaging, in particular to a multi-dimensional chromatography fluorescence microscopic imaging system and method based on polarization structure light modulation.

Background

Fluorescence microscopy imaging can carry out non-invasive and specific observation on biological samples, and has very important significance in the fields of research on subcellular structure functions and the like. Basic physical properties of fluorescence include fluorescence intensity, polarization, spectrum, fluorescence lifetime, etc., and detecting these physical properties as much as possible can obtain more diverse information inside the cell. The method provided by the invention can be used for detecting the multi-dimensional information such as the intensity, the spectrum, the polarization and the like of the fluorescence. The invention adopts polarization modulation illumination at the illumination end, and can solve the polarization information of the sample by the modulation illumination in no less than three polarization directions; and at the detection end, the dichroic mirror is used for separating the signals of different spectral bands of the fluorescence on the light path and projecting the signals to different positions of the camera simultaneously so as to realize the simultaneous acquisition of the signals of the plurality of spectral bands.

In conventional wide-field fluorescence microscopy, fluorescence of a sample in a three-dimensional volume is excited, so that a background signal and a focal plane signal are mixed and detected, and the detection of information such as polarization, spectrum and the like deviates. One of the solutions is to use a fluorescence confocal microscopy technique, but the technique needs to realize two-dimensional imaging through point scanning, and needs hundreds of detections, thereby greatly reducing the imaging speed.

Disclosure of Invention

Aiming at the problems in the prior art, the invention provides a multi-dimensional chromatography fluorescence microscopic imaging system and method based on polarization structure light modulation; aiming at the acquisition of fluorescence multidimensional information, the invention can realize fluorescence polarization analysis by introducing polarization modulation illumination at the illumination end besides detecting intensity, and realizes fluorescence spectrum information detection by simultaneously acquiring spectrum segments at the detection end; aiming at the interference of background noise on focal plane signals, the method reduces the fluorescence background noise by introducing HiLo technology so as to improve the accuracy of information measurement such as intensity, spectrum, polarization and the like.

The invention aims to provide a multi-dimensional tomography fluorescence microscopic imaging system based on polarization structure light modulation.

The invention relates to a multi-dimensional chromatography fluorescence microscopic imaging system based on polarization structure light modulation, which comprises: the device comprises a plurality of lasers, a beam combining device, an acousto-optic tunable filter, a beam expanding device, a polarization beam splitter, a half-wave plate, a spatial light modulator, a condensing lens, a spatial light filter, a vortex half-wave plate, a first dichroic mirror, a first 4f system, a second dichroic mirror, an objective lens, an emission light filter, a tube lens, a second 4f system, a diaphragm, a waveband beam splitting device and a camera; each laser emits linearly polarized laser with one waveband, and the plurality of lasers respectively emit linearly polarized laser with different wavebands; multiple beams of line polarized lasers with different wave bands are combined through a beam combining device and transmitted to an acousto-optic tunable filter on the same optical axis; the wave band of the passing linearly polarized laser is quickly selected through an acousto-optic tunable filter; the linear polarization laser with the selected wave band is expanded by a beam expanding device and then is transmitted to a spatial light modulator through a polarization beam splitter and a half-wave plate; loading periodic black-white binary periodic stripes on a spatial light modulator, reflecting laser to form multi-level linearly polarized diffracted light, and enabling the plane of the spatial light modulator to be perpendicular to an optical axis; the multi-stage linear polarization diffraction light is emitted through the half-wave plate and the polarization beam splitter, and then passes through the polarization beam splitter and the half-wave plate twice, so that the polarization direction of the multi-stage linear polarization diffraction light emitted from the polarization beam splitter is consistent with the polarization direction of the incident linear polarization laser; the multi-level linearly polarized diffraction light is focused by a condensing lens and then reaches a spatial filter; the focal plane of the condenser lens is positioned on the Fourier surface of the spatial light modulator, and the spatial filter is positioned at the focal plane of the condenser lens; the multi-order linearly polarized diffracted light passes through the spatial filter, and only +/-1 order linearly polarized diffracted light passes through the spatial filter; then the polarization direction of the plus or minus 1-level linear polarization diffraction light is adjusted to be consistent with the direction of the binary periodic stripes on the spatial light modulator through a vortex half-wave plate which is arranged close to the spatial light filter; the +/-1-order linearly polarized diffraction light passes through the first dichroic mirror, then passes through the first 4f system, then passes through the second dichroic mirror, and is converged to a sample positioned on the rear focal plane of the objective lens by the objective lens, the Fourier plane of the spatial light modulator is delayed to the rear focal plane of the objective lens by the first 4f system, namely the rear focal plane of the objective lens is positioned on the Fourier plane of the spatial light modulator by the first 4f system; the plus or minus 1-order linear polarization diffraction light interferes on the back focal plane of the objective lens to form sinusoidal stripe illumination, the sample is excited to generate fluorescence, the fluorescence is collected by the objective lens after returning, filtered by the emission optical filter after passing through the second dichroic mirror, and focused by the lens barrel lens; the first dichroic mirror and the second dichroic mirror are completely the same, but the spatial arrangement directions are mutually vertical, so that the polarization distortion introduced by independently using the second dichroic mirror is eliminated; a second 4f system is arranged between the tube lens and the camera, so that the focal plane of the tube lens is delayed to the plane of the camera; a diaphragm is firstly arranged in the middle of the second 4f system and used for controlling the size of an imaging field of view; in the middle of the second 4f system, a waveband light splitting device is arranged behind a diaphragm and spatially separates the fluorescence according to wavebands, so that the fluorescence with different wavebands is projected to different positions of the camera, and each waveband of the fluorescence corresponds to one position on the camera and serves as a spectrum detection channel; acquiring fluorescence of corresponding wave bands through different positions by a camera to obtain an original image; keeping the angle of the binary periodic stripes unchanged, changing the binary periodic stripes loaded on the spatial light modulator into complementary binary periodic stripes, and then obtaining an original image, thereby obtaining two original images of the binary periodic stripes at an angle to form a group of original images; the binary periodic stripes rotate around a horizontal optical axis, a group of original images are obtained every time the binary periodic stripes rotate pi/n, and n is larger than or equal to 3, so that n groups of original images are obtained; carrying out chromatographic image reconstruction on each group of original images according to different spectral detection channels to obtain n chromatographic images, averaging the n chromatographic images to obtain a wide-field optical chromatographic image corresponding to each spectral detection channel, and further obtaining spectral related information; and carrying out polarization demodulation on the n tomographic images to obtain polarization-related information.

The beam combining device adopts a reflecting mirror and a dichroic mirror.

The first 4f system and the second 4f system are optical plane delay scaling 4f systems, namely, one optical plane is transferred to another position and is scaled.

The beam expanding device adopts a 4f system.

The spatial filter corresponds to the rotation angle of the binary periodic stripes and comprises n pairs of light through holes, each pair of light through holes is positioned on the diameter of one passing circle, and the included angle between each adjacent pair of light through holes is pi/n.

The wave band light splitting device adopts m-1 dichroic mirrors to split the fluorescence into m wave bands, and one or more reflectors are arranged on each wave band, so that the spatial position of the light path of the fluorescence of the corresponding wave band is adjusted, and m spectrum detection channels are formed.

The invention also aims to provide a multi-dimensional tomography fluorescence microscopic imaging method based on polarization structure light modulation.

The invention discloses a polarization structure light modulation-based multi-dimensional chromatography fluorescence microscopic imaging method, which comprises the following steps of:

1) each laser emits linearly polarized laser with one waveband, and the plurality of lasers respectively emit linearly polarized laser with different wavebands;

2) multiple beams of line polarized lasers with different wave bands are combined through a beam combining device and transmitted to an acousto-optic tunable filter on the same optical axis; the wave band of the passing linearly polarized laser is quickly selected through an acousto-optic tunable filter;

3) the linear polarization laser with the selected wave band is expanded by a beam expanding device and then is transmitted to a spatial light modulator through a polarization beam splitter and a half-wave plate;

4) loading periodic black-white binary periodic stripes on a spatial light modulator, reflecting laser to form multi-level linearly polarized diffracted light, and enabling the plane of the spatial light modulator to be vertical to a horizontal optical axis;

5) the multi-stage linear polarization diffraction light is emitted through the half-wave plate and the polarization beam splitter, and the linear polarization laser passes through the polarization beam splitter and the half-wave plate twice, so that the polarization direction of the multi-stage linear polarization diffraction light emitted from the polarization beam splitter is consistent with the polarization direction of the incident linear polarization laser;

6) the multi-level linearly polarized diffraction light is focused by a condensing lens and then reaches a spatial filter; the focal plane of the condenser lens is positioned on the Fourier surface of the spatial light modulator, and the spatial filter is positioned at the focal plane of the condenser lens; the multi-order linearly polarized diffracted light passes through the spatial filter, and only +/-1 order linearly polarized diffracted light passes through the spatial filter;

7) the plus or minus 1-order linear polarization diffraction light passes through a vortex half-wave plate which is arranged close to the spatial filter, and the polarization direction of the plus or minus 1-order linear polarization diffraction light is adjusted to be consistent with the direction of the binary periodic fringes on the spatial light modulator;

8) the +/-1-order linearly polarized diffraction light passes through the first dichroic mirror, then passes through the first 4f system, then passes through the second dichroic mirror, and is converged to a sample positioned on the rear focal plane of the objective lens by the objective lens, the Fourier plane of the spatial light modulator is delayed to the rear focal plane of the objective lens by the first 4f system, namely the rear focal plane of the objective lens is positioned on the Fourier plane of the spatial light modulator by the first 4f system;

9) the plus or minus 1-order linear polarization diffraction light interferes on the back focal plane of the objective lens to form sinusoidal stripe illumination, the sample is excited to generate fluorescence, the fluorescence is collected by the objective lens after returning, filtered by the emission optical filter after passing through the second dichroic mirror, and focused by the lens barrel lens; the first dichroic mirror and the second dichroic mirror are completely the same, but the spatial arrangement directions are mutually vertical, so that the polarization distortion introduced by independently using the second dichroic mirror is eliminated;

10) a second 4f system is arranged between the tube lens and the camera, so that the focal plane of the tube lens is delayed to the plane of the camera;

11) a diaphragm is firstly placed in the middle of the second 4f system, so that the size of an imaging field of view is controlled;

12) in the middle of the second 4f system, a waveband light splitting device is arranged behind a diaphragm and spatially separates the fluorescence according to wavebands, so that the fluorescence with different wavebands is projected to different positions of the camera, and each waveband of the fluorescence corresponds to one position on the camera and serves as a spectrum detection channel;

13) acquiring fluorescence of corresponding wave bands through different positions by a camera to obtain an original image;

14) keeping the angle of the binary periodic stripes unchanged, changing the binary periodic stripes loaded on the spatial light modulator into complementary binary periodic stripes, and repeating the steps 1) -13) to obtain another original image, thereby obtaining two original images of the binary periodic stripes at an angle to form a group of original images;

15) the binary periodic stripes rotate around a horizontal optical axis by pi/n, the steps 1) to 14) are repeated, a group of original images are obtained at each angle, n is larger than or equal to 3, so that n groups of original images are obtained, and the binary periodic stripes at each angle correspond to one polarization modulation;

16) after n groups of original images are obtained, carrying out chromatographic image reconstruction on each group of original images according to different spectral detection channels to obtain n chromatographic images, and averaging the n chromatographic images to obtain a wide-field optical chromatographic image corresponding to each spectral detection channel so as to obtain spectral related information; polarization demodulation is performed on the n tomographic images to obtain polarization-related information.

In step 16), reconstructing the wide-field optical tomography image and demodulating the polarization information for different spectral detection channels includes the following steps:

a) original image:

in n groups of original images, the corresponding polarization modulation direction of each group of original images is theta_i(i 1.. n), each group comprises two sinusoidal illumination patterns, the stripe directions of the two sinusoidal illumination patterns are consistent with the polarization modulation direction, and the phase difference of the stripes is pi;

b) alignment of different spectral detection channels:

each original image comprises m spectrum detection channels which are positioned at non-overlapping spatial positions, different spectrum detection channels are cut and separated, and then patterns of different spectrum detection channels are spatially aligned by multiplying each spectrum detection channel by a corresponding affine transformation matrix;

c) polarization modulated illumination intensity correction:

for each spectral detection channel, 2n original images are obtained, respectivelyAnd

where subscript Pi (i ═ 1.,. n) represents different polarization modulations, subscript SIj (j ═ 1, 2) represents two sinusoidal illumination patterns of different phases, i.e., binary when the polarization modulation direction is in the order of twoWhen the angle of the periodic stripes changes, the intensity of +/-1-order linear polarization diffraction light fluctuates, so that the illumination light intensity under different polarization modulation needs to be corrected, and the image corresponding to each polarization modulation direction is divided by the respective correction matrix to obtain a corrected image I_SI1，PiAnd I_SI2，Pi；

d) And (3) solving optical tomography images under different polarization modulations:

averaging two sinusoidal illumination patterns of each polarization modulation to obtain an image I corresponding to the uniform illumination under the polarization modulation_U，Pi：

Binding of I_U，PiAnd the corrected image I_SI1，PiOr I_SI2，PiObtaining an optical tomography image I corresponding to the polarization modulation by adopting a high-low frequency fusion reconstruction algorithm (HiLo) algorithm_OS，Pi；

e) Obtaining a wide-field optical tomography image:

averaging different polarization tomography patterns to obtain wide-field optical tomography image I_osNamely:

f) repeating the steps b) to e) for each spectral detection channel so as to obtain wide-field chromatographic images of all spectral detection channels;

g) obtaining polarization information of the sample:

the polarization information of the sample comprises a fluorescence dipole polarization direction alpha and a polarization modulation degree OUF, and when the polarization modulation direction is theta_iTime, fluorescence dipole excitation intensity I_OS，PiComprises the following steps:

I_OS，Pi＝I_DC+I_AC·cos(2θ_i-2α)，(i＝1，...，n)

wherein, I_DCIs a direct current component, I_ACIs an alternating component, θ_iIs the polarization modulation direction, alpha is the fluorescence coupleThe polarization direction of the polaron, the fluorescent molecules and the biomolecules form a dipole and are expressed in a matrix form as follows:

wherein, theta_iFor the known polarization modulation direction, alpha, I is determined by matrix inversion_DC,I_ACDegree of polarization modulation, OUF ═ 2I_AC/(I_AC+I_DC) And further obtain the related information of the spectrum.

Wherein, in step b), the alignment of the patterns of the different spectral detection channels is performed by multiplying each spectral detection channel by a respective corresponding affine transformation matrix, comprising the steps of:

i. preparing a sparse single-layer fluorescent microsphere fixed sample wafer, and imaging the sample wafer by using the polarization structure light modulation-based multi-dimensional chromatography fluorescence microscopic imaging system to obtain n groups of original images;

ii, intercepting and separating different spectral detection channels of each original image, and solving wide-field optical chromatographic patterns corresponding to the spectral detection channels according to the steps d) and e);

and iii, positioning the position coordinates of the center of the fluorescent microsphere, and solving an affine transformation matrix corresponding to the pattern transformation of all the spectrum detection channels to the first spectrum detection channel according to the coordinates.

In step c), the light intensity correction matrix corresponding to different polarization modulation directions is obtained through calibration, and the method comprises the following steps:

i. preparing a dense fluorescent microsphere fixed sample wafer and adopting the polarization structure light modulation-based multi-dimensional chromatography fluorescence microscopic imaging system to image and obtain n groups of original images, wherein the fluorescent microspheres in the sample wafer should cover the whole imaging field of view as much as possible;

ii, obtaining images of each spectral detection channel without light intensity correction after processing according to the step b)

Averaging two sinusoidal illumination patterns in each polarization modulation direction to obtain an image corresponding to uniform illumination under the polarization modulation

Dividing the pattern of uniform illumination in each polarization modulation direction by the pattern of uniform illumination in the first polarization modulation direction to obtain the corresponding intensity correction matrix in each polarization modulation direction.

The invention has the advantages that:

compared with the traditional wide-field fluorescence microscopy, the method has the advantages that two sinusoidal structure light illumination patterns with complementary phases are collected for illumination in each polarization modulation direction, the uniform light illumination pattern is obtained through averaging the two sinusoidal structure light illumination patterns, the uniform light illumination pattern is combined with one sinusoidal structure light illumination pattern, and the optical tomography image can be solved by utilizing the HiLo algorithm. Since at least 3 polarization modulation directions of illumination are required to solve polarization information, the invention requires at least 6 detections for one reconstruction. For the acquisition of the spectral information, the method adopts a method of simultaneously acquiring the light split at the detection end without increasing extra acquisition time. Compared with fluorescence confocal microscopy, which requires hundreds of detections, the method has great advantages in terms of imaging speed, less photobleaching, and the like.

Meanwhile, the method provided by the invention can also be applied to data acquired by a commercial structured light microscope (SIM), and the super-resolution resolving capability can be obtained while the multi-dimensional chromatographic signal is obtained. The data collected by the SIM and the method of the invention mainly have the following two differences: firstly, the 2D-SIM collects three sinusoidal structure light illumination patterns with the phase difference of 2 pi/3 for each polarization modulation illumination, uniform light illumination patterns can be obtained by averaging the three structure light patterns, the uniform light illumination patterns can be solved by using a HiLo algorithm by combining with one of the structure light illumination patterns, and the 3D-SIM collects 5 images for each polarization modulation direction and can obtain uniform light illumination patterns by averaging the images in the same way; second, commercial SIMs cannot simultaneously collect fluorescence signals in multiple spectral bands, but only sequentially collect signals in different spectral bands.

Drawings

FIG. 1 is a schematic diagram of one embodiment of a polarization structure light modulation based multi-dimensional tomographic fluorescence microscopy imaging system of the present invention;

fig. 2 is a flowchart of reconstruction of wide-field optical tomographic images and demodulation of polarization information for different spectral detection channels according to the polarization structure light modulation-based multi-dimensional tomographic fluorescence microscopy imaging method of the present invention.

Detailed Description

The invention will be further elucidated by means of specific embodiments in the following with reference to the drawing.

As shown in fig. 1, the polarization structure light modulation-based multi-dimensional tomographic fluorescence microscopy imaging system of the present embodiment includes: two lasers Ls1 and Ls2, a beam combining device, an acousto-optic tunable filter AOTF, a beam expanding device, a polarization beam splitter PBS, a half-wave plate HWP, a spatial light modulator SLM, a condenser lens L3, a spatial filter Mk, a vortex half-wave plate VHWP, a first dichroic mirror DM1, a first 4f system, a second dichroic mirror DM2, an objective lens OB, an emission filter EF, a tube lens TL, a second 4f system, a diaphragm AP, a waveband beam splitting device and a camera C; each laser emits linearly polarized laser with one waveband, and the two lasers Ls1 and Ls2 respectively emit linearly polarized laser with different wavebands; the first beam is reflected by a first reflecting mirror M1, combined with the second beam polarization laser by a third dichroic mirror DM3, and transmitted to the acousto-optic tunable filter AOTF along the same optical axis; the wave band of the passing linearly polarized laser is quickly selected through an acousto-optic tunable filter; the linear polarization laser with the selected wave band is expanded by a beam expanding device, the beam expanding device adopts a first lens, a second lens L1 and a second lens L2 with different focal lengths, and the linear polarization laser is reflected by a second reflecting mirror M2 and then sequentially passes through a polarization beam splitter PBS and a half-wave plate HWP to reach a spatial light modulator SLM; loading periodic black-white binary periodic stripes on a spatial light modulator, reflecting laser to form multi-level linearly polarized diffracted light, and enabling the plane of the spatial light modulator to be perpendicular to an optical axis; the multi-stage linear polarization diffraction light is emitted through the half-wave plate and the polarization beam splitter, and then passes through the polarization beam splitter and the half-wave plate twice, so that the polarization direction of the multi-stage linear polarization diffraction light emitted from the polarization beam splitter is consistent with the polarization direction of the incident linear polarization laser; the multi-stage linearly polarized diffraction light is focused by a condenser lens L3 and then reaches a spatial filter Mk; the focal plane of the condenser lens is positioned on the Fourier surface of the spatial light modulator, and the spatial filter is positioned at the focal plane of the condenser lens; the multi-order linearly polarized diffracted light passes through the spatial filter, and only +/-1 order linearly polarized diffracted light passes through the spatial filter; then the polarization direction of the plus or minus 1 level linear polarization diffraction light is adjusted to be consistent with the direction of the binary periodic fringes on the spatial light modulator through a vortex half-wave plate VHWP which is arranged close to the spatial light filter; the +/-1-order linearly polarized diffracted light passes through a first dichroic mirror DM1 and then passes through a first 4f system, the first 4f system adopts fourth and fifth lenses L4 and L5, a third reflector M3 is arranged between the fourth lens L4 and the fifth lens L5 and then passes through a second dichroic mirror DM2 to be converged on a sample SP positioned on a rear focal plane of the objective lens by an objective lens OB, and the Fourier plane of the spatial light modulator is delayed to the rear focal plane of the objective lens by the first 4f system, namely the rear focal plane of the objective lens is positioned on the Fourier plane of the spatial light modulator through the first 4f system; the plus or minus 1-order linear polarization diffraction light interferes on the back focal plane of the objective lens to form sinusoidal stripe illumination, the sample SP is excited to generate fluorescence, the fluorescence is collected by the objective lens after returning, filtered by an emission filter EF after passing through a second dichroic mirror, and focused by a tube lens TL; the first dichroic mirror and the second dichroic mirror are completely the same, but the spatial arrangement directions are mutually vertical, so that the polarization distortion introduced by independently using the second dichroic mirror is eliminated; a second 4f system is provided between the tube lens and the camera, the second 4f system including sixth and seventh lenses L6 and L7, thereby retarding a focal plane of the tube lens to a plane where the camera is located; firstly, placing a diaphragm AP in a second 4f system for controlling the size of an imaging field of view; in the middle of the second 4f system, a waveband splitting device is arranged behind the diaphragm AP and comprises a fourth dichroic mirror DM4, a fifth dichroic mirror DM5 and fourth to seventh reflecting mirrors M4 to M7; the fourth dichroic mirror DM4 and the fifth dichroic mirror DM5 spatially divide the fluorescence into three different wavebands according to the wavebands, and project the fluorescence of the three different wavebands to different positions of the camera C through the fourth to seventh reflecting mirrors M4 to M7 respectively, wherein each waveband of the fluorescence corresponds to one position on the camera and is used as a spectrum detection channel; the camera C acquires fluorescence of corresponding wave bands through different positions to obtain an original image; keeping the angle of the binary periodic stripes unchanged, changing the binary periodic stripes loaded on the spatial light modulator into complementary binary periodic stripes, and then obtaining an original image, thereby obtaining two original images of the binary periodic stripes at an angle to form a group of original images; the binary periodic stripes rotate around a horizontal optical axis, a group of original images are obtained every time the binary periodic stripes rotate pi/n, and n is 3, so that three groups of original images are obtained; carrying out chromatographic image reconstruction on each group of original images according to different spectral detection channels to obtain three chromatographic images, averaging the three chromatographic images to obtain a wide-field optical chromatographic image corresponding to each spectral detection channel, and further obtaining spectral related information; and carrying out polarization demodulation on the three tomographic images to obtain polarization-related information.

The multi-dimensional chromatography fluorescence microscopic imaging method based on polarization structure light modulation comprises the following steps:

1) each laser emits linearly polarized laser with one waveband, and the two lasers respectively emit two sections of linearly polarized laser with different wavebands;

2) the first beam is reflected by a first reflecting mirror M1, combined with the second beam polarization laser by a third dichroic mirror DM3, and transmitted to the acousto-optic tunable filter AOTF along the same optical axis; the wave band of the passing linearly polarized laser is quickly selected through an acousto-optic tunable filter;

3) the linear polarization laser with the selected wave band is expanded by a beam expanding device and then is transmitted to a spatial light modulator through a polarization beam splitter and a half-wave plate;

4) loading periodic black-white binary periodic stripes on a spatial light modulator, reflecting laser to form multi-level linearly polarized diffracted light, and enabling the plane of the spatial light modulator to be vertical to a horizontal optical axis;

5) the multi-stage linear polarization diffraction light is emitted through the half-wave plate and the polarization beam splitter, and the linear polarization laser passes through the polarization beam splitter and the half-wave plate twice, so that the polarization direction of the multi-stage linear polarization diffraction light emitted from the polarization beam splitter is consistent with the polarization direction of the incident linear polarization laser;

6) the multi-level linearly polarized diffraction light is focused by a condensing lens and then reaches a spatial filter; the focal plane of the condenser lens is positioned on the Fourier surface of the spatial light modulator, and the spatial filter is positioned at the focal plane of the condenser lens; the multi-order linearly polarized diffracted light passes through the spatial filter, and only +/-1 order linearly polarized diffracted light passes through the spatial filter;

7) the plus or minus 1-order linear polarization diffraction light passes through a vortex half-wave plate which is arranged close to the spatial filter, and the polarization direction of the plus or minus 1-order linear polarization diffraction light is adjusted to be consistent with the direction of the binary periodic fringes on the spatial light modulator;

8) the +/-1-order linearly polarized diffraction light passes through the first dichroic mirror, then passes through the first 4f system, then passes through the second dichroic mirror, and is converged to a sample positioned on the rear focal plane of the objective lens by the objective lens, the Fourier plane of the spatial light modulator is delayed to the rear focal plane of the objective lens by the first 4f system, namely the rear focal plane of the objective lens is positioned on the Fourier plane of the spatial light modulator by the first 4f system;

9) the plus or minus 1-order linear polarization diffraction light interferes on the back focal plane of the objective lens to form sinusoidal stripe illumination, the sample is excited to generate fluorescence, the fluorescence is collected by the objective lens after returning, filtered by the emission optical filter after passing through the second dichroic mirror, and focused by the lens barrel lens; the first dichroic mirror and the second dichroic mirror are completely the same, but the spatial arrangement directions are mutually vertical, so that the polarization distortion introduced by independently using the second dichroic mirror is eliminated;

10) a second 4f system is arranged between the tube lens and the camera, so that the focal plane of the tube lens is delayed to the plane of the camera;

11) in the middle of the second 4f system, a diaphragm is firstly arranged for controlling the size of the imaging field of view

12) In the middle of the second 4f system, a waveband light splitting device is arranged behind a diaphragm and spatially separates the fluorescence according to wavebands, so that the fluorescence with different wavebands is projected to different positions of the camera, and each waveband of the fluorescence corresponds to one position on the camera and serves as a spectrum detection channel;

13) acquiring fluorescence of corresponding wave bands through different positions by a camera to obtain an original image;

14) keeping the angle of the binary periodic stripes unchanged, changing the binary periodic stripes loaded on the spatial light modulator into complementary binary periodic stripes, and repeating the steps 1) -13) to obtain another original image, thereby obtaining two original images of the binary periodic stripes at an angle to form a group of original images;

15) rotating the binary periodic stripes around a horizontal optical axis by pi/n, repeating the steps 1) to 14), obtaining a group of original images at each angle, wherein n is 3, thereby obtaining three groups of original images, wherein the binary periodic stripes at each angle correspond to one polarization modulation, and each group corresponds to different polarization modulation directions theta_i(i ═ 1, 2, 3) at 0 °, 60 ° and 120 °, respectively, and six original images were obtained in total;

16) after three groups of original images are obtained, carrying out chromatographic image reconstruction on each group of original images according to different spectral detection channels to obtain three chromatographic images, and averaging the three chromatographic images to obtain a wide-field optical chromatographic image corresponding to each spectral detection channel so as to obtain spectral related information; the three tomographic images are polarization-demodulated to obtain polarization-related information, as shown in fig. 2, which includes the following specific steps:

a) original image:

in three groups of original images, each group of original images corresponds to a corresponding polarization modulation direction theta_i(i 1.. 3), each group comprises two sinusoidal illumination patterns, the stripe directions of the two sinusoidal illumination patterns are consistent with the polarization modulation direction, and the phase difference of the stripes is pi;

b) alignment of different spectral detection channels:

each original image comprises three spectrum detection channels which are positioned at non-overlapping spatial positions, different spectrum detection channels are cut and separated, and then patterns of different spectrum detection channels are spatially aligned by multiplying each spectrum detection channel by a corresponding affine transformation matrix;

c) polarization modulated illumination intensity correction:

for each spectral detection channel, six original images are obtained, namely

Subscript Pi (I ═ 1.,. 3) represents different polarization modulations, subscript SIj (j ═ 1, 2) represents two sinusoidal illumination patterns with different phases, when the polarization modulation direction, that is, the angle of the binary periodic fringes changes, the intensity of the + -1 order linear polarization diffracted light fluctuates, so the illumination intensity under different polarization modulations needs to be corrected, and the image corresponding to each polarization modulation direction is divided by the respective correction matrix to obtain the corrected image I_SI1，P1,I_SI2，P1,I_SI1，P2,I_SI2，P2,I_SI1，P3,I_SI2，P3；

d) And (3) solving optical tomography images under different polarization modulations:

averaging two sinusoidal illumination patterns of each polarization modulation to obtain an image I corresponding to the uniform illumination under the polarization modulation_U，Pi：

Binding of I_U，PiAnd the corrected image I_SI1，PiOr I_SI2，PiObtaining an optical tomography image I corresponding to the polarization modulation by adopting a high-low frequency fusion reconstruction algorithm (HiLo) algorithm_OS，Pi；

e) Obtaining a wide-field optical tomography image:

averaging different polarization tomography patterns to obtain wide-field optical tomography image I_osNamely:

I_os＝(I_OS，P1+I_OS，P2+I_OS，P3)/3.

f) repeating the steps b) to e) for each spectral detection channel so as to obtain wide-field chromatographic images of all spectral detection channels;

g) obtaining polarization information of the sample:

the polarization information of the sample comprises a fluorescence dipole polarization direction alpha and a polarization modulation degree OUF, and when the polarization modulation direction is theta_iTime, fluorescence dipole excitation intensity I_OS，PiComprises the following steps:

I_OS，Pi＝I_DC+I_AC·cos(2θ_i-2α)，i＝1，2，3

wherein, IDC is direct current component, IAC is alternating current component, and theta i is polarization modulation direction, and alpha is the dipole direction, and fluorescence molecule and biomolecule form the dipole, and the expression is matrix form:

wherein, theta_iFor the known polarization modulation direction, alpha, I is determined by matrix inversion_DC,I_ACDegree of polarization modulation, OUF ═ 2I_AC/(I_AC+I_DC) And further obtain the related information of the spectrum.

Wherein, in step b), the alignment of the patterns of the different spectral detection channels is performed by multiplying each spectral detection channel by a respective corresponding affine transformation matrix, comprising the steps of:

i. preparing a sparse single-layer fluorescent microsphere fixed sample wafer, and acquiring n (n is 3) groups of original images of the sample wafer by adopting the polarization structure light modulation-based multi-dimensional tomography fluorescent microscopic imaging system;

ii, intercepting and separating different spectral detection channels of each original image, and solving wide-field optical chromatographic patterns corresponding to the spectral detection channels according to the steps d) and e);

and iii, positioning the position coordinates of the center of the fluorescent microsphere, and solving an affine transformation matrix corresponding to the pattern transformation of all the spectrum detection channels to the first spectrum detection channel according to the coordinates.

In step c), the light intensity correction matrix corresponding to different polarization modulation directions is obtained through calibration, and the method comprises the following steps:

i. preparing a dense fluorescent microsphere fixed sample wafer and adopting the polarization structure light modulation-based multi-dimensional chromatography fluorescence microscopic imaging system to image to obtain n (n is 3) groups of original images, wherein the fluorescent microspheres in the sample wafer should cover the whole imaging field as much as possible;

ii, obtaining images of each spectral detection channel without light intensity correction after processing according to the step b)

Averaging two sinusoidal illumination patterns in each polarization modulation direction to obtain an image corresponding to uniform illumination under the polarization modulation

Dividing the pattern of uniform illumination in each polarization modulation direction by the pattern of uniform illumination in the first polarization modulation direction to obtain the corresponding intensity correction matrix in each polarization modulation direction.

Finally, it is noted that the disclosed embodiments are intended to aid in further understanding of the invention, but those skilled in the art will appreciate that: various substitutions and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the invention should not be limited to the embodiments disclosed, but the scope of the invention is defined by the appended claims.