CN111781174A - Three-dimensional super-resolution microscopy imaging method and device based on structured light illumination and supercritical angle imaging - Google Patents

Abstract

The invention discloses a three-dimensional super-resolution microscopic imaging method and device based on structured light illumination and supercritical angle imaging. The device is simple and convenient to operate; the light energy is not high, and the sample is not easy to bleach; the imaging speed is high, and the device can be used for observing living cells; the required fluorescent labeling density is low, a specific fluorescent dye is not required, and the application range is wide; the three-dimensional information is restored only by the algorithm processing of the two-dimensional image without slicing, and the high-resolution microscopic imaging in the three-dimensional space can be quickly and effectively realized.

Description

Three-dimensional super-resolution microscopy imaging method based on structured light illumination and supercritical angle imaging and device

technical field

The invention relates to the field of optical super-resolution microscopic imaging, in particular to a three-dimensional super-resolution microscopic imaging method and device based on structured light illumination and supercritical angle imaging.

Background technique

Optical microscopy is an important means for observing and studying microstructures in life sciences and other fields. However, due to the diffraction effect of light and the limited aperture of the optical system, the resolution of ordinary optical microscopes is limited to about half a wavelength, and it is impossible to detect samples with a scale of less than 200 nanometers.

In order to break through this limitation, scientists have proposed a variety of super-resolution imaging techniques to realize the observation and study of nano-scale tiny structures. One of them is stimulated emission depletion microscopy, which uses high-power depletion light to quench partially excited fluorescent molecules by stimulated emission, thereby reducing the width of the spontaneous emission fluorescence point spread function to achieve super-resolution rate microscopic imaging. However, this technique requires a high density of fluorescent labels and requires specific fluorescent dyes, and the high-power depletion light can easily bleach the sample. Another class of techniques, such as stochastic optical reconstruction and light-activated localization microscopy, uses single-molecule localization techniques to achieve super-resolution microscopy. Such techniques require the use of high-intensity lasers to bleach correctly positioned molecules, and hundreds of cycles are required to get the final result. Therefore, in addition to the same limitations as the previous stimulated emission depletion microscopy, this type of technology has the disadvantage of being unable to observe molecular dynamics due to the slow imaging speed.

Structured light illumination microscope is different from the two types of microscopes mentioned above. It is realized by changing the illumination system, projecting fringes on the surface of the sample, and modulating the spatial frequency of the sample to collect high-frequency information containing the details of the sample, and then restoring it through a later algorithm. Super-resolution imaging. Compared with stimulated emission depletion microscopy and single-molecule localization technology, structured light illumination microscope has the advantages of no need for high fluorescent labeling density, fast imaging speed of specific fluorescent dyes, low incident light power, not easy to bleach, fast imaging speed and real-time observation. However, the two-dimensional structured light illumination microscope can only obtain two-dimensional images to restore the lateral distribution information of the sample, but cannot obtain accurate axial structure information. The three-dimensional structured light microscope realized by slice slice cannot achieve fast imaging, which limits its Applications in life sciences.

SUMMARY OF THE INVENTION

The invention provides a three-dimensional super-resolution microscopic imaging method and device based on structured light illumination and supercritical angle imaging, which can realize three-dimensional super-resolution microscopic imaging.

In order to achieve the above object, the three-dimensional super-resolution microscopy imaging method based on structured light illumination and supercritical angle imaging provided by the present invention comprises the following steps:

1) The laser beam enters an illumination system that can generate structured light illumination, and after generating a structured light pattern, it is projected on the surface of the sample to illuminate and excite the sample;

2) Collect the fluorescence signal excited by the sample through the microscope objective lens, and divide it into two light rays with a beam splitter. The diaphragm is projected on another industrial camera;

3) Rotate the stripe direction of the illumination structured light pattern or move the stripes to obtain multiple patterns, and reconstruct the multiple images collected by the first industrial camera through structured light illumination to obtain a lateral super-resolution image, which is the two-dimensional image of the sample. super-resolution images;

4) The multiple images collected by the two industrial cameras are respectively superimposed to obtain two sample images under uniform wide-field illumination, in which the supercritical angle fluorescence component and sub-surface fluorescence components of the sample fluorescence are included in the supercritical image superposition result of the first industrial camera image. The critical angle fluorescence component, while the second-channel industrial camera image stacking result only includes the subcritical angle fluorescence component in the sample fluorescence, and the normalized supercritical angle fluorescence intensity of the sample dye molecule is obtained according to the two super-resolution images obtained by stacking. distribution, and restore the axial position information of each pixel through the exponential decay relationship between the fluorescence intensity of the supercritical angle and the distance between the dye molecule and the interface;

5) Combine the restored axial position information with the two-dimensional super-resolution image obtained in step 3) to obtain three-dimensional distribution information of the sample.

Further, the size of the diaphragm in step 2) satisfies ρ=n _o fsinθ _c , where n _o is the refractive index of the immersion medium of the microscope objective, f is the focal length of the objective lens, θ _c =arcsin(nm _{/ g} ), where n _m is the refractive index of the sample medium and n _g is the refractive index of the glass interface.

Further, for the detection light of fluorescent molecules far from the plane, which only contains subcritical angle fluorescence components but not supercritical angle fluorescence components, the subcritical angle fluorescence is acquired by a CCD placed on the equivalent back focal plane of the objective lens. According to the angular distribution diameter, the diaphragm diameter is designed according to the angular distribution diameter, and then the CCD is replaced with the diaphragm, the diaphragm is placed in the optical path, and the position of the diaphragm is adjusted so that the diaphragm and the optical path are coaxial.

Further, in step 4), the normalized supercritical angle fluorescence ratio distribution of the sample dye molecule is obtained according to the two super-resolution images obtained by superposition, which is specifically as follows: the superposition result of the first path of light and the second path of light are obtained. The supercritical angle fluorescence component of the sample fluorescence is obtained by differencing the superposition results; the supercritical angle fluorescence component of the second path light is the subcritical angle fluorescence component of the sample fluorescence; for each pixel point of the supercritical angle fluorescence component and the subcritical angle fluorescence component , select the average intensity of the pixel points within an Elephant range with this point as the center as the average intensity of the point, and generate the average supercritical angle fluorescence component map and the subcritical angle fluorescence component average map; the supercritical angle fluorescence component average map; The component average map is divided by the subcritical angle fluorescence component average map to obtain the supercritical angle fluorescence ratio distribution of the sample image, which decreases exponentially with the axial distance of the sample molecules from the interface.

In order to realize the above method, the imaging device provided by the present invention includes:

Transverse super-resolution module: including a laser for generating excitation light; a single-mode fiber for transmitting laser light; a lens group for reflection and collimation; a structured light illumination module for generating fringes, which is the core part.

Axial super-resolution module: including a microscope objective lens for collecting the fluorescent signal emitted by the sample; a dichroic mirror for transmitting the illumination light and reflecting the fluorescent signal; a supercritical angle fluorescence detection module for obtaining axial position information, This module is the core part, including a 4f lens group for obtaining the equivalent back focal plane of the objective lens, a half mirror for beam splitting, a diaphragm for limiting the beam (only used in the second optical path), and a Filters for filtering out stray light, lenses for imaging sample fluorescence signals, and industrial cameras for receiving sample fluorescence signals.

Further, the structured light illumination module in the lateral super-resolution module is mainly to generate a light source with a certain regular pattern, and any device that can achieve this function can be used. Commonly used methods include polarizing and splitting the light source, so that the two beams of polarized light interfere with each other to produce fringes when projected on the surface of the sample; or add a beam splitting device to the optical path, such as spatial light modulator, grating or digital micromirror array, etc. to produce streaks. Saturated structured light illumination and nonlinear structured light illumination are also applicable in this system. The resulting structured light illumination fringe group requires superposition just for uniform widefield illumination.

Further, in order to collect all the fluorescence signals emitted by the sample to the maximum extent, a larger numerical aperture should be used for the microscope objective, and the numerical aperture NA should be greater than or equal to 1.49.

The beneficial effects of the invention are as follows: the device of the invention is simple and the operation is convenient; the illumination light energy is not high, and the sample is not easy to bleach; the imaging speed is fast, and it can be used to observe living cells; Wide; without slice, only through the algorithm processing of the two-dimensional image to restore the three-dimensional information, can quickly and effectively achieve high-resolution microscopic imaging in the three-dimensional space.

Description of drawings

1 is a schematic diagram of a three-dimensional super-resolution microscopy imaging device according to an embodiment of the present invention;

2 is a schematic diagram of supercritical angle fluorescence detection according to an embodiment of the present invention;

FIG. 3 is a schematic diagram of a three-dimensional super-resolution microscopic image calculation and processing flow diagram according to an embodiment of the present invention;

FIG. 4 is a schematic diagram of fluorescent light emission of dye molecules;

FIG. 5 is a schematic diagram showing the change of the supercritical angle fluorescence intensity of dye molecules with the axial position.

Detailed ways

In order to make the above objects, features and advantages of the present invention more clearly understood, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

Many specific details are set forth in the following description to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can do so without departing from the connotation of the present invention. Similar promotion, therefore, the present invention is not limited by the specific embodiments disclosed below.

The three-dimensional super-resolution microscopic imaging method based on structured light illumination and supercritical angle imaging provided by the present invention comprises the following steps:

1) The laser beam enters an illumination system that can generate structured light illumination, and after generating a structured light pattern, it is projected on the surface of the sample to illuminate and excite the sample;

2) Collect the fluorescence signal excited by the sample through the microscope objective lens, and divide it into two light rays with a beam splitter. The diaphragm is projected on another industrial camera;

3) Rotate the stripe direction of the illumination structured light pattern or move the stripes to obtain multiple patterns, and reconstruct the multiple images collected by the first industrial camera through structured light illumination to obtain a lateral super-resolution image, which is the two-dimensional image of the sample. super-resolution images;

4) The multiple images collected by the two industrial cameras are respectively superimposed to obtain two sample images under uniform wide-field illumination, in which the supercritical angle fluorescence component and sub-surface fluorescence components of the sample fluorescence are included in the supercritical image superposition result of the first industrial camera image. The critical angle fluorescence component, while the second-channel industrial camera image stacking result only includes the subcritical angle fluorescence component in the sample fluorescence, and the normalized supercritical angle fluorescence intensity of the sample dye molecule is obtained according to the two super-resolution images obtained by stacking. distribution, and restore the axial position information of each pixel point through the exponential decay relationship between the supercritical angle fluorescence intensity and the distance between the dye molecule and the interface shown in Figure 5;

5) Combine the restored axial position information with the two-dimensional super-resolution image obtained in step 3) to obtain three-dimensional distribution information of the sample.

Further, the size of the diaphragm in step 2) satisfies ρ=n _o fsinθ _c , where n _o is the refractive index of the immersion medium of the microscope objective, _f is the focal length of the objective lens, and θ _c =arcsin(n _m /ng )( where n _m is the refractive index of the sample medium and n _g is the refractive index of the glass interface).

Further, for the detection light of fluorescent molecules far from the plane, which only contains subcritical angle fluorescence components but not supercritical angle fluorescence components, the subcritical angle fluorescence is acquired by a CCD placed on the equivalent back focal plane of the objective lens. According to the angular distribution diameter, the diaphragm diameter is designed according to the angular distribution diameter, and then the CCD is replaced with the diaphragm, the diaphragm is placed in the optical path, and the position of the diaphragm is adjusted so that the diaphragm and the optical path are coaxial.

Further, in step 4), the normalized supercritical angle fluorescence ratio distribution of the sample dye molecule is obtained according to the two super-resolution images obtained by superposition, which is specifically as follows: the superposition result of the first path of light and the second path of light are obtained. The supercritical angle fluorescence component of the sample fluorescence is obtained by differencing the superposition results; the supercritical angle fluorescence component of the second path light is the subcritical angle fluorescence component of the sample fluorescence; for each pixel point of the supercritical angle fluorescence component and the subcritical angle fluorescence component , select the average intensity of the pixel points within an Elephant range with this point as the center as the average intensity of the point, and generate the average supercritical angle fluorescence component map and the subcritical angle fluorescence component average map; the supercritical angle fluorescence component average map; The component average map is divided by the subcritical angle fluorescence component average map to obtain the supercritical angle fluorescence ratio distribution of the sample image, which decreases exponentially with the axial distance of the sample molecules from the interface.

In order to realize the above method, the imaging device provided by the present invention includes:

Transverse super-resolution module: including a laser for generating excitation light; a single-mode fiber for transmitting laser light; a lens group for reflection and collimation; a structured light illumination module for generating fringes, which is the core part.

Axial super-resolution module: including a microscope objective lens for collecting the fluorescent signal emitted by the sample; a dichroic mirror for transmitting the illumination light and reflecting the fluorescent signal; a supercritical angle fluorescence detection module for obtaining axial position information, This module is the core part, including a 4f lens group for obtaining the equivalent back focal plane of the objective lens, a half mirror for beam splitting, a diaphragm for limiting the beam (only used in the second optical path), and a Filters for filtering out stray light, lenses for imaging sample fluorescence signals, and industrial cameras for receiving sample fluorescence signals.

Further, the structured light illumination module in the lateral super-resolution module is mainly to generate a light source with a certain regular pattern, and any device that can achieve this function can be used. Commonly used methods include polarizing and splitting the light source, so that the two beams of polarized light interfere with each other to produce fringes when projected on the surface of the sample; or add a beam splitting device to the optical path, such as spatial light modulator, grating or digital micromirror array, etc. to produce streaks. Saturated structured light illumination and nonlinear structured light illumination are also applicable in this system. The resulting structured light illumination fringe group requires superposition just for uniform widefield illumination.

In order to maximize the collection of all the fluorescent signals emitted by the sample, the microscope objective should adopt a larger numerical aperture, and the numerical aperture NA should be greater than or equal to 1.49.

Further, regarding the method of calculating the axial position, the spatial position of the fluorescence on each pixel point is restored mainly through the relationship between the supercritical angle fluorescence (SAF) intensity of a single dye molecule along the axis. The fluorescence emitted by a single dye molecule when excited contains a subcritical angle fluorescence (UAF) part (the gray area in Figure 4, that is, the part where the angle between the emission angle and the optical axis is less than the critical angle of total reflection) and a supercritical angle fluorescence (SAF) part. (As shown in the dashed area in Figure 4, that is, the part where the angle between the light-emitting angle and the optical axis is greater than the critical angle of total reflection). Subcritical angle fluorescence (UAF) intensity emitted by a single dye molecule does not vary with axial distance, whereas supercritical angle fluorescence (SAF) is an evanescent wave whose intensity decreases exponentially with axial distance from the interface.

Subcritical angle fluorescence (UAF) and supercritical angle fluorescence (SAF) emitted by a single dye molecule can be distinguished at the back focal plane of a microscope objective. Since the objective lens satisfies the Abbe sine relationship: the light emission angle θ appears as a circle with a radius of ρ=n _o fsinθ on the back focal plane, where n _o is the refractive index of the immersed medium of the microscope objective, and f is the focal length of the objective lens. Therefore, the UAF light is located in a circle with a radius of n _o fsinθ _c , θ _c =arcsin(n _m /ng ) (where m is the medium and _g is the glass interface), and the SAF light part is surrounded by the UAF light with a radius of Ring of f·NA. Therefore, a diaphragm with a radius of n _o fsinθ _c can block the supercritical angle fluorescence component in the fluorescence at the back focal plane. In the experimental device, we design a diaphragm that meets the above requirements. For the detection light of fluorescent molecules far from the plane, it only contains subcritical angle fluorescent components but not supercritical angle fluorescent components, so it can be placed on the objective lens, etc. The CCD on the effective back focal plane obtains the angular distribution diameter of the subcritical angle fluorescence, and then design the aperture diameter according to the angular distribution diameter, then replace the CCD with an aperture, put the aperture into the optical path, and adjust the position of the aperture Make the diaphragm coaxial with the optical path. The first light in the detection path of the device does not have a diaphragm, and the signal collected by the industrial camera includes the subcritical angle fluorescence (UAF) intensity signal and the supercritical angle fluorescence (SAF) intensity signal; the second light in the device detection path is set. With the diaphragm, the signal collected by the industrial camera only contains the subcritical angle fluorescence (UAF) intensity signal. Therefore, the supercritical angle fluorescence (SAF) component can be obtained by differentiating the two signals.

Since the excitation intensity of each dye molecule is different, the fluorescence intensity is also different, so the image must be normalized. Since the subcritical angle fluorescence (UAF) intensity emitted by a single dye molecule does not vary with axial distance, the supercritical angle fluorescence (SAF) intensity decreases exponentially with the axial distance from the interface. Because of the image noise and the mutual influence of adjacent points, for each pixel, we select a range of an Elliott centered on this pixel, and divide the average value of the supercritical angle fluorescence (SAF) intensity within the range by By averaging the subcritical angle fluorescence (UAF) intensities in the range, the supercritical angle fluorescence ratio distribution can be obtained, which decreases exponentially with the axial distance of the dye molecules from the interface (Figure 5). Then, through this exponential decreasing relationship, one-to-one correspondence can be obtained, and the axial position of each pixel point can be obtained according to the ratio of the supercritical angle fluorescence (SAF) component of each pixel point. In the actual experiment, considering that the imaging has a certain signal-to-noise ratio, a corresponding axial position interval is obtained, and the size of the interval decreases as the signal-to-noise ratio increases.

A specific implementation example of the present invention is given below, but is not limited thereto. The 3D super-resolution microscopy imaging device of this example is shown in Figure 1, including a laser 1, a structured light illumination module 2, a first mirror 3, a dichroic mirror 4, a microscope objective 5, a sample 6 and a supercritical angle fluorescence detection module 7. The supercritical angle fluorescence detection module 7 is shown in FIG. 2. After passing through the first lens 8, the beam splitter 9 divides the detection light into two paths, and the first path passes through the second lens 10, the first filter 11 and the The first imaging lens 12 is imaged at the first industrial camera 13, wherein the first lens 8 and the second lens 10 form a 4f system; the second path passes through the second mirror 14, the third lens 15, the diaphragm 16, The second filter 17 and the second imaging lens 18 are imaged at the second industrial camera 19, wherein the first lens 8 and the third lens 15 form a 4f system, and the diaphragm 16 is placed behind the equivalent of the microscope objective 5 on the focal plane. Filters, imaging lenses and industrial cameras are the same in both optical paths.

When the device is in operation, the laser light generated by the laser 1 passes through the structured light illumination module 2 to generate stripes, and is transmitted by the dichroic mirror 4 through the objective lens 5 to hit the surface of the sample 6 to excite the dye molecules in the sample 6 to generate fluorescence. All the fluorescence generated by the dye molecules is reflected by the dichroic mirror 4 through the objective lens 5 and enters the supercritical angle fluorescence detection module 7, and the illumination excitation light will not be reflected. The detection light entering the supercritical angle fluorescence detection module 7 passes through the first lens 8 and is divided into two upper and lower paths by the beam splitter 9. The first transmission path passes through the second lens 10, and the stray light is filtered out by the first filter 11. , is imaged on the first industrial camera 13 by the first imaging lens 12, and the image captured by the first industrial camera 13 contains all the UAF and SAF information; The diaphragm 16 filters out the SAF light in it, and after filtering out the stray light by the second filter 17, the second imaging lens 18 forms an image on the second industrial camera 19, and the image captured by the second industrial camera 19 only contains UAF information.

Adjust the components that control the stripes in the structured light illumination module 2 to change the phase of the illumination stripes or rotate the stripe direction. The first industrial camera 13 and the second industrial camera 19 respectively capture an image of each illuminated structured light stripe in the selected area of the sample. After transforming multiple illumination stripes, two sets of images are obtained.

As shown in FIG. 3 , the first group of images is restored by the structured light illumination reconstruction algorithm, and the restoration result of the image group of the first industrial camera 13 is the horizontal super-resolution image. Because the structured light illumination used is uniform wide-field illumination after superimposition, the two sets of images obtained by the two industrial cameras are superimposed separately to obtain two fluorescence images of the sample under wide-field illumination. The superposition result of the image group of the first industrial camera 13 includes the UAF and SAF parts of the fluorescence emitted by the sample dye molecules, while the superposition result of the image group of the second industrial camera 19 only includes the UAF part of the fluorescence emitted by the sample dye molecules. Therefore, the SAF part of the fluorescence emitted by the sample dye molecules can be obtained by making a difference between the superposition result of the image group of the first industrial camera 13 and the superposition result of the image group of the second industrial camera 19 . For each pixel point, the ratio of the SAF signal is obtained by dividing the average value of the SAF image in the range by the average value of the UAF image in the range, and then the axial position information of each pixel point is correspondingly obtained from Figure 5. The three-dimensional distribution information of the sample can be obtained by loading the axial position information of each pixel point to the restoration result of the image group of the first industrial camera 13 .

The above descriptions are only preferred embodiments of the present invention. Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art, without departing from the scope of the technical solution of the present invention, can make many possible changes and modifications to the technical solution of the present invention by using the methods and technical contents disclosed above, or modify them into equivalents of equivalent changes. Example. Therefore, any simple modifications, equivalent changes and modifications made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solutions of the present invention still fall within the protection scope of the technical solutions of the present invention.

Claims (7)

1. a three-dimensional super-resolution microscopy imaging method based on structured light illumination and supercritical angle imaging, is characterized in that, comprises the following steps: 1) The laser beam enters an illumination system that can generate structured light illumination, and after generating a structured light pattern, it is projected on the surface of the sample to illuminate and excite the sample. 2) Collect the fluorescence signal excited by the sample through the microscope objective lens, and divide it into two light rays with a beam splitter. The diaphragm is projected on another industrial camera. 3) Rotate the stripe direction of the illumination structured light pattern or move the stripes to obtain multiple patterns, and reconstruct the multiple images collected by the first industrial camera through structured light illumination to obtain a lateral super-resolution image, which is the two-dimensional image of the sample. super-resolved images. 4) The multiple images collected by the two industrial cameras are respectively superimposed to obtain two sample images under uniform wide-field illumination, in which the supercritical angle fluorescence component and sub-surface fluorescence components of the sample fluorescence are included in the supercritical image superposition result of the first industrial camera image. The critical angle fluorescence component, while the second-channel industrial camera image stacking result only includes the subcritical angle fluorescence component in the sample fluorescence, and the normalized supercritical angle fluorescence intensity of the sample dye molecule is obtained according to the two super-resolution images obtained by stacking. distribution, and restore the axial position information of each pixel through the exponential decay relationship of the fluorescence intensity at the supercritical angle with the distance of the dye molecule from the interface. 5) Combine the restored axial position information with the two-dimensional super-resolution image obtained in step 3) to obtain three-dimensional distribution information of the sample. 2. a kind of three-dimensional super-resolution microscopy imaging method based on structured light illumination and supercritical angle imaging according to claim 1, is characterized in that, in step 2), the size of diaphragm satisfies ρ=n _o fsinθ _c , wherein n _o is the refractive index of the immersion medium of the microscope objective, f is the focal length of the objective lens, θ _c =arcsin(nm _{/ ng ), where nm is the refractive index of the sample medium, and n g is the} refractive index of the glass interface. 3. a kind of three-dimensional super-resolution microscopy imaging method based on structured light illumination and supercritical angle imaging according to claim 1, is characterized in that, for the probe light of fluorescent molecules farther from the plane, wherein only contains subcritical The angular fluorescence component does not include the supercritical angle fluorescence component. A CCD placed on the equivalent back focal plane of the objective lens obtains the angular distribution diameter of the subcritical angle fluorescence, and then designs the aperture diameter according to the angular distribution diameter. The CCD is replaced with a diaphragm, the diaphragm is placed in the optical path, and the position of the diaphragm is adjusted so that the diaphragm is coaxial with the optical path. 4. a kind of three-dimensional super-resolution microscopy imaging method based on structured light illumination and supercritical angle imaging according to claim 1, is characterized in that, in step 4), obtain normalization according to two super-resolution images obtained by superposition The obtained sample dye molecule supercritical angle fluorescence ratio distribution is specifically: the supercritical angle fluorescence component of the sample fluorescence is obtained by making a difference between the superposition result of the first path light and the superposition result of the second path light; the superposition of the second path light The result is the subcritical angle fluorescence component of the sample fluorescence; for each pixel point of the supercritical angle fluorescence component and the subcritical angle fluorescence component, the average value of the intensity on the pixel point within an Elephant range with the point as the center is selected. As the average intensity of this point, the average supercritical angle fluorescence component map and the subcritical angle fluorescence component average map are generated; the supercritical angle fluorescence component average map is divided by the subcritical angle fluorescence component average map to obtain the supercritical angle fluorescence of the sample image. ratio distribution, which decreases exponentially with the axial distance of the sample molecules from the interface. 5. A three-dimensional super-resolution microscopic imaging device based on structured light illumination and supercritical angle imaging, characterized in that, comprising: Transverse super-resolution module: including a laser for generating excitation light; a single-mode fiber for transmitting laser light; a lens group for reflection and collimation; a structured light illumination module for generating fringes; Axial super-resolution module: including a microscope objective lens for collecting the fluorescent signal emitted by the sample; a dichroic mirror for transmitting the illumination light and reflecting the fluorescent signal; a supercritical angle fluorescence detection module for obtaining the axial position information; The supercritical angle fluorescence detection module includes a 4f lens group for acquiring the equivalent back focal plane of the objective lens, a half mirror for beam splitting, a diaphragm for limiting the beam, and a filter for filtering out stray light Slices, lenses for imaging sample fluorescence signals, industrial cameras for receiving sample fluorescence signals. 6. The three-dimensional super-resolution microscopic imaging device based on structured light illumination and supercritical angle imaging according to claim 5, wherein the structured light illumination module in the lateral super-resolution module is used to generate a certain The light source with a regular pattern can be realized by polarizing the light source and splitting the beam, so that the two beams of polarized light can interfere with each other to produce fringes when projected on the surface of the sample; The structured light illumination stripe group requires superposition just for uniform widefield illumination. 7. A three-dimensional super-resolution microscopic imaging device based on structured light illumination and supercritical angle imaging according to claim 5, wherein the microscope objective lens is used to collect all the fluorescence signals emitted by the sample to the maximum extent , the numerical aperture NA should be greater than or equal to 1.49.

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