CN112798564B - Stochastic Optical Reconstruction and Structured Light Illumination Composite Super-resolution Imaging System - Google Patents

Abstract

The invention discloses a random optical reconstruction and structured light illumination composite super-resolution imaging system, which comprises: the light source module is used for providing a plurality of combined illumination light rays with different wavelengths, controlling the light illumination time sequence, and also used for controlling single-wavelength light illumination, or multiple-wavelength light rays for alternate illumination, or multiple-wavelength light rays for simultaneous illumination; the composite light field regulation and control module comprises a first optical regulation and control device and a second optical regulation and control device, wherein the first optical regulation and control device is used for regulating and controlling an incident light field into a cosine structure illumination light field, and the second optical regulation and control device is used for regulating and controlling the incident light field into a uniform illumination light field; the two optical regulation and control devices can work independently, alternately or simultaneously; and the fluorescence imaging module is used for acquiring a plurality of original fluorescence images of the sample and reconstructing a super-resolution image by a computer. The invention realizes the combination of two super-resolution imaging technologies of random optical reconstruction and structured light illumination on a set of optical imaging platform, and can realize multi-mode and cross-resolution scale simultaneous imaging of a complex biological system.

Description

Stochastic Optical Reconstruction and Structured Light Illumination Composite Super-resolution Imaging System

technical field

The invention belongs to the technical field of microscopic imaging, in particular to a composite super-resolution imaging system of random optical reconstruction and structured light illumination.

Background technique

Fluorescence super-resolution imaging has a wide range of application prospects. At present, there are many super-resolution imaging technologies, including stochastic optical reconstruction super-resolution imaging technology, structured light illumination super-resolution imaging technology, and stimulated radiation depletion super-resolution imaging technology. While improving the resolution, these technologies are accompanied by unfavorable factors such as the reduction of imaging speed, the increase of illumination light power, the increase of phototoxicity, and the reduction of imaging field of view.

In the prior art, the technical principle of stochastic optical reconstruction super-resolution imaging method (STORM) is: using optical switch fluorescent proteins, single molecules within the diffraction limit range are randomly activated at different times, and each fluorescent molecule can be precisely positioned and then regenerated. Recombination, superposition to obtain super-resolution images, the resolution can reach 10nm or even higher. Stochastic optical reconstruction microscopy is mainly used for intracellular single-molecule imaging and observation of fine subcellular structures, such as observing the precise localization of individual proteins in cells, observing protein-protein interactions, and intracellular microfilaments, microtubules, adhesions Spots, inclusions and other fine structures. However, the random optical reconstruction microscope needs to collect a large number of original images (typically 20,000) to reconstruct a super-resolution image. The imaging time ranges from several seconds to tens of minutes, and the time resolution is low, which is difficult to use for live Cell imaging.

The technical principle of the structured light illumination super-resolution imaging method (SIM) is to irradiate multiple interfering beams onto the sample, then extract high-resolution information from the collected fluorescence images, and reconstruct the super-resolution image. Structured light illumination super-resolution technology is mainly used for in vivo observation at the subcellular level because of its fast imaging speed, low excitation light energy, and little damage to cells, including dynamic changes in mitochondria, dynamic changes in cytoskeleton, dynamic changes in chromosomes, Inner vesicle movement, virus movement within cells, etc. However, the imaging resolution of structured light illumination can be up to 2 times higher than that of traditional fluorescence microscopy, reaching the level of 100 nm. It is difficult to perform fine biochemical reactions such as motor protein movement along the cytoskeleton and the process of pathogen particles invading cells, which limits its application in ultramicrobiology, applications in medicine and other fields.

The demand for high-end imaging technology in the fields of life science and basic medicine covers ultra-high spatial resolution, fast imaging speed, low illumination power, low phototoxicity, and large imaging field of view. These requirements are difficult to reconcile from a technical perspective. Paradoxically, there is no single technical means that can meet these needs at the same time, and the combination of different types of technologies is an important breakthrough direction to break through the relevant difficulties.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a composite super-resolution imaging system of stochastic optical reconstruction and structured light illumination, which meets the urgent needs of cross-resolution scale imaging in life science and basic medical research.

The technical solution for realizing the purpose of the present invention is: a composite super-resolution imaging system of random optical reconstruction and structured light illumination, the system comprising:

The light source module is used to provide a plurality of combined illumination lights of different wavelengths, and control the lighting sequence of the light, and is also used to control the illumination of a single wavelength of light, or the alternate illumination of multiple wavelengths of light, or the simultaneous illumination of multiple wavelengths of light;

The composite light field control module includes a first optical control device for adjusting the incident light field into a cosine structure illumination light field, and a second optical control device for adjusting the incident light field into a uniform illumination light field; two optical control devices The control devices can work independently, alternately or simultaneously;

The fluorescence imaging module is used to acquire multiple original fluorescence images of the sample and reconstruct the super-resolution images by the computer.

Further, the light source module includes: a plurality of light sources with different wavelengths, a reflection beam combining component, an acousto-optic tunable filter, a fiber coupler, and a polarization-maintaining fiber; the light emitted by the multiple light sources with different wavelengths is combined by reflection. After the bundle components are combined, the acousto-optic tunable filter is irradiated, and the first-order diffracted light of the acousto-optic tunable filter is collected by the fiber coupler into the polarization-maintaining fiber, and then the light is introduced into the composite optical field through the polarization-maintaining fiber. control module.

Further, the reflection beam combining assembly includes:

Multiple reflectors correspond to light sources of different wavelengths, and guide the light emitted by all light sources to the dichroic mirror to complete the beam combination;

a plurality of dichroic mirrors for combining light from the mirrors into a light beam and transmitting the light beam to the acousto-optic tunable filter;

Or, the light emitted by one or more light sources is directly matched with a certain dichroic mirror, and the corresponding reflector is omitted.

Further, the multiple light sources with different wavelengths include a first light source, a second light source, a third light source, and a fourth light source; the reflection beam combining component includes a first reflector, a second reflector, a third reflector, a fourth mirror, a fifth mirror, a first dichroic mirror, a second dichroic mirror, and a third dichroic mirror;

The light emitted by the first light source passes through the second reflecting mirror and the first reflecting mirror in sequence, and then passes through the first dichroic mirror, the second dichroic mirror and the third dichroic mirror in sequence;

The light emitted by the second light source passes through the third reflecting mirror, is reflected by the first dichroic mirror, and then passes through the second dichroic mirror and the third dichroic mirror in sequence;

The light emitted by the third light source passes through the fifth reflecting mirror, is reflected by the second dichroic mirror, and then passes through the third dichroic mirror;

The light emitted by the fourth light source passes through the fourth reflecting mirror, is reflected by the third dichroic mirror, and is combined with the light from the other three lasers and transmitted to the acousto-optic tunable filter.

Further, the composite light field control module includes:

The fiber collimation beam expander assembly includes a fiber collimator, a laser beam expander, and a first achromatic half-wave plate arranged in sequence along the optical axis where the light emitted from the light source module is located;

a first optical control device, including a spatial light modulator, a polarization beam splitter prism, a Fourier lens, a porous mask, a polarization rotator, a liquid crystal phase compensator, and a collimating lens;

The second optical control device includes a two-dimensional scanning galvanometer, a second achromatic half-wave plate, a polarization beam splitter prism, a Fourier lens, a porous mask, a polarization rotator, a liquid crystal phase compensator, and a collimating lens;

4f imaging system, including a Fourier lens and a collimating lens, that is, the back focal plane of the Fourier lens coincides with the front focal plane of the collimating lens;

The spatial light modulator and the two-dimensional scanning galvanometer are both located on the front focal plane of the 4f imaging system; the porous mask is located on the Fourier plane of the 4f imaging system;

The 4f imaging system is optically connected to the fluorescence imaging module;

The process of realizing the structured light illumination mode by the composite light field control module: the light output by the optical fiber collimating beam expanding assembly is transmitted and incident to the spatial light modulator through the polarization beam splitting prism, and then reflected by the spatial light modulator and reflected by the polarization beam splitting prism in turn. Through Fourier lens, porous mask, polarization rotator, liquid crystal phase compensator, collimating lens;

The process of realizing the uniform illumination mode by the composite light field control module: the light output from the optical fiber collimating beam expanding assembly is reflected by the polarizing beam splitter prism and then passes through the second achromatic half-wave plate and the two-dimensional scanning galvanometer in turn, and then passes through the second After being reflected by the dimensional scanning galvanometer, it passes through the second achromatic half-wave plate, the polarizing beam splitter prism, and then passes through the Fourier lens, porous mask, polarization rotator, liquid crystal phase compensator, collimating lens.

Further, a binary periodic fringe computational hologram is loaded on the spatial light modulator, and the light is diffracted to multiple orders by the spatial light modulator;

The porous mask is composed of 2N rotationally symmetrically distributed pinholes, each pinhole is a rectangular structure, and the center position of the long side corresponds to the critical angle of total internal reflection of the sample surface, where N is an integer; the porous mask is used to realize the space Filtering, allowing only light that meets a specific incident angle to enter the fluorescence imaging module to illuminate the sample, and the light passing through different positions of the pinhole has different incident angles;

The polarization rotator is used to make the polarization state of the light beam passing through any pinhole perpendicular to the plane determined by the center point of the pinhole and the optical axis of the 4f imaging system;

The fast axis direction of the phase compensator is parallel to the polarization direction of the transmitted light of the polarizing beam splitter prism, and the phase compensator is used to actively compensate the laser phase drift caused by the optical element, so that the polarization state of any beam that finally reaches the sample surface is maintained as Linear polarization state, and the polarization direction is perpendicular to the plane of incidence of the light.

Further, the first achromatic half-wave plate is installed on the electric rotating wave plate frame, and the angle between its fast axis direction and the polarization direction of the transmitted light of the polarization beam splitter is θ, and the liquid crystal space light is transmitted through the polarization beam splitter. The light intensity of the modulator is proportional to sin ² (2θ), and the light intensity of the two-dimensional scanning galvanometer reflected by the polarization beam splitter prism is proportional to cos ² (2θ).

Further, when sin ² (2θ)=1, cos ² (2θ)=0, the composite light field control module independently works in the structured light illumination mode; when sin ² (2θ)=0, cos ² (2θ)=1 When , the compound light field control module works independently in the uniform illumination mode; when θ is equal to other values, the two imaging modes work simultaneously; by controlling the value of the included angle θ, the compound light field control module can be operated in the structured light illumination mode and the Switching between the uniform illumination modes can also make the two imaging modes work at the same time.

Further, the fluorescence imaging module includes two cameras, one camera receives the fluorescence excited by the structured illumination light field and images, and the other camera receives the fluorescence excited by the uniform illumination light field and images, and the original image collected by the camera is reconstructed by computer for super-resolution. image, and the image acquisition timing of the two cameras, the composite light field control module and the light source module are controlled synchronously by the computer.

Further, the fluorescence imaging module includes:

A device for realizing structured light illumination super-resolution imaging, including a lens, a microscope objective lens, a sample stage, a fourth dichroic mirror, a fifth dichroic mirror, a first tube lens, and a first camera; The incident light passes through the lens and is then reflected by the fourth dichroic mirror to illuminate the surface of the sample placed on the sample stage, generating a cosine structure illumination light field, exciting the sample to emit fluorescence, and the excited fluorescence is collected by the microscope objective lens, and then sequentially passed through the The fourth dichroic mirror transmits, the fifth dichroic mirror transmits, and the first tube lens is then imaged to the detection surface of the first camera;

A device for realizing super-resolution imaging with uniform light illumination, including a lens, a microscope objective lens, a sample stage, a fourth dichroic mirror, a fifth dichroic mirror, a second tube lens, and a second camera; the output of the compound light field control module The incident light passes through the lens and is then reflected by the fourth dichroic mirror to illuminate the surface of the sample placed on the sample stage, generating a uniform illumination light field, exciting the sample to emit fluorescence, and the excited fluorescence is collected by the microscope objective lens, and then sequentially passed through the fourth dichroic mirror The dichroic mirror is transmitted, the fifth dichroic mirror is reflected, and the second tube lens is then imaged to the detection surface of the second camera.

Aiming at the imaging requirements in the fields of cell biology and clinical pathological diagnosis, the present invention proposes a composite super-resolution imaging method and system of random optical reconstruction and structured light illumination. The microscopic inspection system has the following advantages:

1) The combined use of STORM and SIM technology can achieve simultaneous imaging and observation across resolution scales. For example, in the study of motor protein movement along the cytoskeleton in living cells, nano-resolution STORM imaging of motor proteins can be achieved, and cytoskeleton can be imaged simultaneously. 100-nanometer-resolution SIM imaging. The capability of simultaneous imaging across resolution scales has broad needs in the fields of cellular material transport, pathogen infection processes, etc., and cannot be satisfied by the existing technologies.

2) In the research aiming at SIM imaging, in situ STROM ultra-high-resolution imaging can be carried out on key areas of interest, which overcomes the problems of fast SIM imaging, large imaging field but low resolution, and avoids the The problems of inaccurate positioning and sample state changes caused by secondary imaging are solved.

3) In studies targeting STORM imaging, fast SIM imaging to guide STORM imaging and analysis can significantly reduce the amount of STROM raw data and improve the imaging speed.

4) Simultaneous realization of STORM and SIM imaging on a set of imaging platforms can share most of the hardware equipment, significantly reduce the economic cost of obtaining the same imaging function, and reduce the space resources of the biological laboratory occupied by the instruments.

The present invention will be described in further detail below with reference to the accompanying drawings.

Description of drawings

FIG. 1 is a schematic structural diagram of a composite super-resolution imaging system of random optical reconstruction and structured light illumination in one embodiment.

Figure 2(a) is the structure diagram of the perforated mask, (b) is the incident light spot distribution measured at the rear pupil of the microscope objective in the uniform illumination mode; (c)-(e) are in the structured light illumination mode, Incident spot distribution measured at the rear pupil plane of a microscope objective.

FIG. 3 is a super-resolution image taken by the present invention in one embodiment, wherein (a) is the SIM imaging of the HER2 positive signal of breast cancer cells, and (b) is the STORM imaging of the breast cancer cytoskeleton.

The marks in the figure are as follows: 1 laser 647nm; 2 laser 561nm; 3 laser 488nm; 4 laser 405nm; mirrors 9, 5, 10, 11, 12; dichroic mirrors 6, 7, 8, 29; Tuning filter; 14 fiber coupler; 15 polarization maintaining fiber; 16 fiber collimator; 17 laser beam expander; 18 achromatic half wave plate; 19 polarization beam splitter prism; 20 spatial light modulator; 21 Fourier Leaf lens; 22 aperture mask; 23 polarization rotator; 24 liquid crystal phase compensator; 25 collimator lens; 26 lens; 27 microscope objective; 28 sample stage; 30 fluorescence filter; 31 tube lens; 32 camera; 33 2D scanning galvanometer; 34 achromatic half-wave plates; 35 tube lenses; 36 cameras; 37 fluorescence filters.

Detailed ways

In order to make the purpose, technical solutions and advantages of the present application more clearly understood, the present application will be described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application, but not to limit the present application.

In addition, if there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. are only used for the purpose of description, and should not be construed as indicating or implying Its relative importance or implicitly indicates the number of technical features indicated. Thus, a feature delimited with "first", "second" may expressly or implicitly include at least one of that feature. In addition, the technical solutions between the various embodiments can be combined with each other, but must be based on the realization by those of ordinary skill in the art. When the combination of technical solutions is contradictory or cannot be realized, it should be considered that the combination of such technical solutions does not exist. , is not within the scope of protection required by the present invention.

In one embodiment, the present invention provides a stochastic optical reconstruction and structured light illumination composite super-resolution imaging system, the system comprising:

The light source module is used to provide a plurality of combined illumination lights of different wavelengths, and control the lighting sequence of the light, and is also used to control the illumination of a single wavelength of light, or the alternate illumination of multiple wavelengths of light, or the simultaneous illumination of multiple wavelengths of light;

The composite light field control module includes a first optical control device for adjusting the incident light field into a cosine structure illumination light field, and a second optical control device for adjusting the incident light field into a uniform illumination light field; two optical control devices The control devices can work independently, alternately or simultaneously;

The fluorescence imaging module is used to acquire multiple original fluorescence images of the sample and reconstruct the super-resolution images by the computer.

The present invention realizes composite super-resolution imaging by combining stochastic optical reconstruction super-resolution imaging technology and structured light illumination super-resolution imaging technology, that is, SIM or STOM imaging can be independently performed; and SIM can also be used for rapid imaging to locate the focus of interest. area, and then perform STROM ultra-high-resolution imaging on key areas; SIM and STOM imaging can also be performed on samples at the same time. It has the advantages of high spatial resolution, high imaging speed, large imaging field of view, and low phototoxicity, and can meet the needs of multiple modes of imaging at the same time, especially the in situ and cross-resolution scale simultaneous imaging of the same biological sample. In addition, the composite imaging system can realize the combined use of SIM and STORM super-resolution imaging technologies on a set of imaging platforms at the same time. The two share most of the hardware equipment, which can reduce the economic cost of obtaining the same imaging function and reduce the equipment occupied. Laboratory space resources, simplifying the complexity of system maintenance.

Further, in one of the embodiments, the light source module includes: a plurality of light sources with different wavelengths, a reflection beam combining component, an acousto-optic tunable filter 13, a fiber coupler 14, and a polarization maintaining fiber 15; the plurality of The light emitted by the light sources with different wavelengths is combined by the reflection beam combining component, and then irradiated to the acousto-optic tunable filter 13. The first-order diffracted light of the acousto-optic tunable filter 13 is collected by the fiber coupler 14 and enters the polarization maintaining fiber. In step 15, the light is then introduced into the composite light field control module through the polarization maintaining fiber 15.

The control of the light illumination sequence is also used to control the illumination of a single wavelength of light, or the alternate illumination of multiple wavelengths of light, or the simultaneous illumination of multiple wavelengths of light, which are all realized by controlling the acousto-optic tunable filter 13 .

Further, in one of the embodiments, the reflection beam combining assembly includes:

Multiple reflectors correspond to light sources of different wavelengths, and guide the light emitted by all light sources to the dichroic mirror to complete the beam combination;

a plurality of dichroic mirrors, which are used to combine the light from the mirror into a light beam, and transmit the light beam to the acousto-optic tunable filter 13;

Or, the light emitted by one or more light sources is directly matched with a certain dichroic mirror, and the corresponding reflector is omitted.

Further, in one of the embodiments, the multiple light sources with different wavelengths include a first light source 1, a second light source 2, a third light source 3, and a fourth light source 4; the reflection beam combining component includes a first reflector 5. The second mirror 9, the third mirror 10, the fourth mirror 11, the fifth mirror 12, the first dichroic mirror 6, the second dichroic mirror 7, and the third dichroic mirror 8;

The light emitted by the first light source 1 passes through the second reflecting mirror 9 and the first reflecting mirror 5 in sequence, and then passes through the first dichroic mirror 6 , the second dichroic mirror 7 and the third dichroic mirror 8 in sequence. ;

The light emitted by the second light source 2 passes through the third reflecting mirror 10, is reflected by the first dichroic mirror 6, and then passes through the second dichroic mirror 7 and the third dichroic mirror 8 in turn;

The light emitted by the third light source 3 passes through the fifth reflecting mirror 12, is reflected by the second dichroic mirror 7, and then passes through the third dichroic mirror 8;

The light emitted by the fourth light source 4 passes through the fourth reflecting mirror 11 , is reflected by the third dichroic mirror 8 , and is combined with the light from the other three lasers and transmitted to the acousto-optic tunable filter 13 .

Here exemplarily, in one of the embodiments, the first light source 1 , the second light source 2 , the third light source 3 , and the fourth light source 4 use lasers with wavelengths of 647 nm, 4561 nm, 488 nm, and 405 nm, respectively.

It should be pointed out that the light source is not limited to a laser, but can also be an LED light source. At the same time, the light wavelength is not limited to the above four wavelengths. There is no sequence or primary and secondary relationship between the light sources.

Further, in one of the embodiments, the composite light field control module includes:

The fiber collimation beam expander assembly includes a fiber collimator 16, a laser beam expander 17, and a first achromatic half-wave plate 18 arranged in sequence along the optical axis where the light emitted from the light source module is located;

The first optical control device includes a spatial light modulator 20, a polarization beam splitter prism 19, a Fourier lens 21, a porous mask 22, a polarization rotator 23, a liquid crystal phase compensator 24, and a collimating lens 25;

The second optical control device includes a two-dimensional scanning galvanometer 33, a second achromatic half-wave plate 34, a polarization beam splitter prism 19, a Fourier lens 21, a porous mask 22, a polarization rotator 23, and a liquid crystal phase compensation device 24, collimating lens 25;

4f imaging system, including Fourier lens 21 and collimating lens 25, namely the back focal plane of Fourier lens 21 coincides with the front focal plane of collimating lens 25;

The spatial light modulator 20 and the two-dimensional scanning galvanometer 33 are both located on the front focal plane of the 4f imaging system; the porous mask 22 is located on the Fourier plane of the 4f imaging system;

The 4f imaging system is optically connected to the fluorescence imaging module;

The process of realizing the structured light illumination mode by the composite light field control module: the light output by the optical fiber collimating beam expanding assembly is transmitted to the spatial light modulator 20 through the polarization beam splitting prism 19, and then reflected by the spatial light modulator 20 and the polarization beam splitting prism. 19 After being reflected, it passes through the Fourier lens 21, the porous mask 22, the polarization rotator 23, the liquid crystal phase compensator 24, and the collimating lens 25 in sequence;

The process of realizing the uniform illumination mode by the composite light field control module: the light output by the optical fiber collimating beam expanding assembly is reflected by the polarizing beam splitting prism 19 and then passes through the second achromatic half-wave plate 34 and the two-dimensional scanning galvanometer 33 in turn. , after being reflected by the two-dimensional scanning galvanometer 33, it passes through the second achromatic half-wave plate 34 and the polarizing beam splitting prism 19 in turn, and then transmits through the polarizing beam splitting prism 19 and then passes through the Fourier lens 21, the porous mask plate 22, Polarization rotator 23 , liquid crystal phase compensator 24 , collimating lens 25 .

Further, in one of the embodiments, a binary periodic fringe computational hologram is loaded on the spatial light modulator 20 , and the light is diffracted into multiple orders by the spatial light modulator 20 . Changing the phase and spatial orientation of the image loaded on the spatial light modulator 20 can change the orientation and phase of the two-dimensional cosine illumination light field on the sample surface.

The porous mask plate 22 is composed of 2N rotationally symmetrically distributed pinholes, each of which is a rectangular structure, and the center position of the long side corresponds to the critical angle of total internal reflection of the sample surface, where N is an integer. The perforated mask 22 is used to realize spatial filtering, allowing only light with a specific incident angle to enter the fluorescence imaging module to illuminate the sample, and light passing through different positions of the pinhole has different incident angles; at the same time, the rectangular structure makes the incident angle within a certain range The interior can be adjusted. The center position of the rectangle corresponds to the critical angle of total internal reflection. When the incident beam passes through the pinhole along the inner side, the sample can be illuminated at a large angle. When the incident light changes position along the inner side of the center position, the period of the structured light field can be adjusted. , to achieve optimal structured light illumination for samples with different structural characteristics; when the incident light beam passes through the pinhole along the outer side, the total internal reflection illumination of the sample can be achieved, and when the incident light changes its position along the outer side of the center position, the penetration of the evanescent wave can be adjusted. The depth of penetration enables illumination of samples at different depths.

Exemplarily here, Figure 2(a) is a 6-hole mask, which is composed of 6 pinholes with rotationally symmetrical distribution; Figures 2(c)-(e) are the positive and negative first-order beams in the SIM imaging mode in the microscope objective. The intensity distribution of the rear pupil surface; Figure 2(b) shows the intensity distribution of the incident beam on the rear pupil surface of the microscope objective in the STORM imaging mode. Faster than the camera acquisition speed (less than 100Hz), it can be equivalently considered that there are 6 light spots illuminated at the same time.

The polarization rotator 23 is used to make the polarization state of the light beam passing through any pinhole be perpendicular to the plane determined by the center point of the pinhole and the optical axis of the 4f imaging system (it can make the polarization directions of the positive and negative first-order light beams always perpendicular to the plane in which the positive and negative first-order beams are co-located);

The fast axis direction of the phase compensator 24 is parallel to the polarization direction of the transmitted light of the polarizing beam splitter prism 19, and the phase compensator 24 is used to actively compensate the collimating lens 25, the lens 26, the fourth dichroic mirror 29, and the microscope objective lens 27. Laser phase drift caused by other optical components, the amount of phase compensation, and the phase difference between the fast axis and the slow axis can be controlled by computer programming. When changing the incident azimuth angle of the incident light or changing the wavelength of the incident light, the phase can be changed synchronously. The amount of compensation, so that the polarization state of any beam of light that finally reaches the sample surface is maintained as linear polarization state, and the polarization direction is perpendicular to the incident plane of the light (the polarization direction is perpendicular to the plane where the positive and negative first-order beams are located), so as to obtain The maximum modulation degree of structured light, and since the phase shift is related to the laser wavelength, when the laser wavelength changes, the phase compensation amount of the liquid crystal phase compensator needs to be changed, which is very important for the structured light field of any wavelength to have the maximum modulation degree. It is also the key design that enables the present invention to obtain high-quality SIM super-resolution images.

Further, in one of the embodiments, the first achromatic half-wave plate 18 is installed on the electric rotating wave plate frame, and the angle between its fast axis direction and the polarization direction of the transmitted light of the polarizing beam splitter prism 19 is θ, The light intensity of the liquid crystal spatial light modulator 20 transmitted through the polarizing beam splitting prism 19 is proportional to sin ² (2θ), and the light intensity of the two-dimensional scanning galvanometer 33 reflected by the polarizing beam splitting prism 19 is proportional to cos ² (2θ).

Further, in one of the embodiments, when sin ² (2θ)=1, cos ² (2θ)=0, the composite light field control module independently works in the structured light illumination mode; when sin ² (2θ)=0, When cos ² (2θ)=1, the composite light field control module works independently in the uniform illumination mode; when θ is equal to other values, the two imaging modes work simultaneously, and the relative illumination intensity of the two modes is controllable. By controlling the value of the included angle θ, the composite light field control module can be switched between the structured light illumination mode and the uniform illumination mode, and the two imaging modes can also work at the same time.

Further, in one of the embodiments, the fluorescence imaging module includes two cameras, one camera receives the fluorescence excited by the structured illumination light field and images, and the other camera receives the fluorescence excited by the uniform illumination light field and images, and the camera collects the fluorescence. The original image is reconstructed with a super-resolution image by the computer, and the image acquisition sequence of the two cameras is controlled synchronously with the compound light field control module and the light source module by the computer.

Further, in one of the embodiments, the fluorescence imaging module includes:

A device for realizing structured light illumination super-resolution imaging, including a lens 26, a microscope objective lens 27, a sample stage 28, a fourth dichroic mirror 29, a fifth dichroic mirror 37, a first tube lens 31, and a first camera 32; The outgoing light of the composite light field control module passes through the lens 26 and is then reflected by the fourth dichroic mirror 29 to illuminate the surface of the sample placed on the sample stage 28 to generate a cosine structure illumination light field, which excites the sample to emit fluorescence, and the excited fluorescence Collected by the microscope objective lens 27, then sequentially transmitted through the fourth dichroic mirror 29, transmitted through the fifth dichroic mirror 37, and then imaged to the detection surface of the first camera 32 by the first tube lens 31;

A device for realizing uniform light illumination super-resolution imaging, including a lens 26, a microscope objective lens 27, a sample stage 28, a fourth dichroic mirror 29, a fifth dichroic mirror 37, a second tube lens 35, and a second camera 36; The outgoing light from the composite light field control module passes through the lens 26 and is then reflected by the fourth dichroic mirror 29 to illuminate the surface of the sample placed on the sample stage 28 to generate a uniform illumination light field, which excites the sample to emit fluorescence, and the excited fluorescence passes through the surface of the sample. The microscope objective lens 27 collects the images, and then is sequentially transmitted through the fourth dichroic mirror 29 , reflected by the fifth dichroic mirror 37 , and then imaged to the detection surface of the second camera 36 by the second tube lens 35 .

Here, the camera can be a two-dimensional image detector such as EMCCD, SCMOS, etc.

Here, the fluorescence imaging module can either be built independently, or a commercial microscope can be used.

Based on the above-mentioned embodiments, the steps of realizing structured light illumination super-resolution imaging in the system of the present invention include: making the laser emit light and performing wavelength gating and illumination timing control through the acousto-optic tunable filter 13; loading binary light on the spatial light modulator 20 Periodic fringe calculation hologram, the light is diffracted to multiple orders by the liquid crystal spatial light modulator 20, reflected by the polarization beam splitter prism 19, and then passes through the Fourier lens 21, the porous mask 22, the polarization rotator 23, the liquid crystal phase in turn The compensator 24, the collimating lens 25 and the fluorescence imaging module are irradiated to the sample surface, so that the positive and negative first-order diffracted light interferes on the sample surface (the light diffracted by the spatial light modulator 20 is filtered by the porous mask 22, so that only the positive and negative first-order diffracted light interferes on the sample surface. The first-order diffracted light can be irradiated to the sample surface to cause interference) to generate a cosine structure illumination light field to excite the sample to emit fluorescence; the fluorescence emitted by the sample is collected by the fluorescence imaging module, transmitted through the fourth dichroic mirror 29, and the fifth dichroic The mirror 37 transmits, and the first tube lens 31 forms an image on the detection surface of the camera 32 . Changing the phase and spatial orientation of the image loaded on the spatial light modulator can change the orientation and phase of the structured illumination light field on the sample surface, and excite fluorescence to acquire images. The above process is repeated to collect fluorescence images of 3 directions and 3 phases, and finally the 9 original images collected by the camera are reconstructed into super-resolution images by image processing algorithms.

The system of the present invention realizes uniform light illumination super-resolution imaging, that is, the steps of random optical reconstruction illumination super-resolution imaging include: controlling the propagation direction of light through the two-dimensional scanning galvanometer 33, and the light reflected by the two-dimensional scanning galvanometer 33 passes through the second Achromatic half-wave plate 34, polarizing beam splitter prism 19, Fourier lens 21, porous mask 22, polarization rotator 23, liquid crystal phase compensator 24, collimating lens 25 and imaging module are irradiated onto the sample surface. The incident angle is α, when α is greater than the critical angle of total internal reflection, the incident light undergoes total internal reflection at the sample surface, forming a beam of evanescent wave illumination light field on the sample surface, and the fluorescence emitted by the sample is collected by the objective lens, passing through the fourth dichroic The chromatic mirror 29 transmits, the fifth dichroic mirror 37 reflects, and the second tube lens 35 forms an image on the detection surface of the second camera 36 .

The step of random optical reconstruction illumination super-resolution imaging further includes: rapidly switching the deflection direction of the two-dimensional scanning galvanometer 33 to change the angular distribution of the incident light. The optical axis of the micro-objective is rotated by an integer multiple of 360 degrees, and the light trajectories are distributed on a cone with an apex angle of 2α, which can make the distribution of the evanescent wave light field uniform and eliminate the negative impact of laser speckle on the imaging quality. At the same time, by controlling the size of the α angle, the penetration depth of the evanescent wave can be precisely controlled.

Exemplarily, FIG. 3 shows the super-resolution image taken by the present invention, wherein (a) is the SIM imaging of the HER2 positive signal of breast cancer cells, and (b) is the STORM imaging of the breast cancer cytoskeleton.

Further, in one of the embodiments, the system of the present invention can also perform two-color labeling on the sample, excite one of the proteins with a light source of one wavelength for fast super-resolution imaging under structured light illumination, and excite the light source with another wavelength. The second protein performed single-molecule stochastic optical reconstruction ultra-high-resolution imaging.

Combining the above embodiments, the composite super-resolution imaging system of random optical reconstruction and structured light illumination proposed by the present invention includes a light source module that provides multi-wavelength combined illumination light, modulates the light field into a two-dimensional cosine structure illumination light field and uniform illumination light. Field compound light field control device, imaging module for collecting fluorescence signal imaging and reconstructing super-resolution images. The acousto-optic tunable filter performs wavelength gating and timing control of the multi-wavelength combined light beams, so as to realize single-wavelength light illumination, multiple wavelength light alternate illumination, and multiple wavelength light simultaneous illumination. The incident light is diffracted into multiple orders by the spatial light modulator, filtered by a porous mask, only the positive and negative first-order diffracted light is allowed to reach the surface of the sample and interfere to generate a cosine structured light field; the polarization direction of the positive and negative first-order diffracted light is determined by Controlled by the polarization rotator, it is always perpendicular to its incident surface; a liquid crystal phase compensator is set behind the polarization rotator to actively compensate for the random phase shift caused by the optical element, so that the light finally reaching the sample surface remains linearly polarized and the polarization direction is perpendicular to the incident surface noodle. The azimuth angle of the illumination light is quickly scanned by the two-dimensional scanning galvanometer, and the multi-angle incident average is realized within one exposure time of the camera, resulting in a uniform illumination light field and eliminating the adverse effect of laser speckle on imaging. By controlling the rotation angle of the achromatic half-wave plate, structured light field illumination, or uniform light field illumination, or simultaneous illumination of structured light field and uniform light field but with different wavelengths can be achieved. The imaging module is provided with two cameras, the fluorescence excited by structured light field illumination is detected and imaged by one camera, and the fluorescence excited by uniform light field illumination is detected and imaged by another camera.

To sum up, the system of the present invention has the following characteristics:

1. Quickly switch the incident azimuth angle of the illumination light through the two-dimensional scanning galvanometer, and make the light trajectory quickly sweep through the cone with the optical axis of the microscope objective lens as the axis within one exposure integration time of the camera, and the total illumination light intensity received by the sample is equal to different angles. The sum of the incident light intensity, so the random laser speckle can be effectively suppressed during the summation process, thereby forming a uniform illumination light field on the surface of the sample. At the same time, the incident angle of the incident light can also be controlled by the two-dimensional scanning galvanometer, thereby controlling the penetration depth of the evanescent wave illumination light field.

2. By controlling the computational hologram loaded on the spatial light modulator, a structured illumination light intensity with any light intensity distribution can be formed on the sample surface. The structured illumination light field is generated based on double-beam interference, and the system includes an active phase compensator, which can keep the incident beam as linearly polarized light, and the polarization direction is perpendicular to the incident surface, so that the structured light field can obtain the maximum modulation degree. At the same time, since the laser phase shift introduced by optical elements such as lenses, dichroic mirrors, and microscope objective lenses is related to the laser wavelength, the liquid crystal phase compensator can actively adjust the phase compensation amount in real time when changing the incident wavelength, so that the amount of phase compensation can always be adjusted for different wavelengths. achieve optimal modulation.

3. Stochastic optical reconstruction/structured light illumination composite super-resolution imaging method:

a) Independent working mode: the present invention can independently carry out STORM imaging and SIM imaging, or switch between them quickly. Quickly image the sample with a large field of view through structured light illumination imaging, find the key area of interest, switch to the random optical reconstruction mode, and perform ultra-high-resolution imaging of this area. This is very important. In many cases, only a small part of the sample is of interest, and super-resolution imaging of the entire sample area is not required. How to quickly locate the key area and super-resolve the area Imaging is a technical difficulty. The invention adopts wide-field structured light illumination to rapidly and high-resolution imaging of the entire sample, and then uses a two-dimensional scanning galvanometer to rapidly scan the key areas with random optical reconstruction of ultra-high-resolution imaging, thereby solving this problem.

b) Simultaneous working mode: the present invention can simultaneously perform structured light illumination super-resolution imaging and random optical reconstruction super-resolution imaging on the sample, can collect more information, and complete the imaging process that cannot be carried out by a single imaging method, such as double-color labeling of the sample , excite one of the proteins with a laser of one wavelength for fast super-resolution imaging with structured light illumination, and excite the second protein with a laser of another wavelength for single-molecule stochastic optical reconstruction super-resolution imaging. The kinetic processes at these precise sites are important.

The present invention can realize multi-mode and cross-resolution scale simultaneous imaging of complex biological systems by realizing the combination of two super-resolution imaging technologies of random optical reconstruction and structured light illumination on a set of optical imaging platforms.

The description and application of the present invention herein is illustrative, and is not intended to limit the scope of the present invention to the above-described embodiments. Variations and variations of the embodiments disclosed herein are possible, and alternative and equivalent various components of the embodiments are known to those of ordinary skill in the art. It should be apparent to those skilled in the art that the present invention may be implemented in other forms, structures, arrangements, proportions, and with other components, materials and components without departing from the spirit or essential characteristics of the invention. Other modifications and changes of the embodiments disclosed herein may be made without departing from the scope and spirit of the invention. The various embodiments in this specification are described in a progressive manner, and each embodiment focuses on the points that are different from other embodiments, and the similar parts between the various embodiments can be referred to each other. The above description of the disclosed embodiments enables any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims (3)

1. a random optical reconstruction and structured light illumination composite super-resolution imaging system, is characterized in that, described system comprises: The light source module is used to provide a plurality of combined illumination lights of different wavelengths, and control the lighting sequence of the light, and is also used to control the illumination of a single wavelength of light, or the alternate illumination of multiple wavelengths of light, or the simultaneous illumination of multiple wavelengths of light; The composite light field control module includes a first optical control device for adjusting the incident light field into a cosine structure illumination light field, and a second optical control device for adjusting the incident light field into a uniform illumination light field; two optical control devices The control devices can work independently, alternately or simultaneously; The fluorescence imaging module is used to collect multiple original fluorescence images of the sample and reconstruct the super-resolution images by the computer; The light source module includes: a plurality of light sources with different wavelengths, a reflection beam combining component, an acousto-optic tunable filter (13), a fiber coupler (14), and a polarization-maintaining fiber (15); the plurality of light sources with different wavelengths After the emitted light is combined by the reflection beam combining component, it is irradiated to the acousto-optic tunable filter (13), and the first-order diffracted light of the acousto-optic tunable filter (13) is collected by the optical fiber coupler (14) and enters the protection device. In the polarization fiber (15), the light is then introduced into the composite light field control module through the polarization maintaining fiber (15); The reflection beam combining assembly includes: Multiple reflectors correspond to light sources of different wavelengths, and guide the light emitted by all light sources to the dichroic mirror to complete the beam combination; a plurality of dichroic mirrors for combining the light from the mirror into a light beam, and transmitting the light beam to the acousto-optic tunable filter (13); Or, the light emitted by one or more light sources is directly matched with a dichroic mirror, and the corresponding mirror is omitted; The multiple light sources with different wavelengths include a first light source (1), a second light source (2), a third light source (3), and a fourth light source (4); the reflection beam combining assembly includes a first reflection mirror (5) ), the second mirror (9), the third mirror (10), the fourth mirror (11), the fifth mirror (12), the first dichroic mirror (6), the second dichroic mirror (7), the third dichroic mirror (8); The light emitted by the first light source (1) passes through the second reflecting mirror (9) and the first reflecting mirror (5) in sequence, and then passes through the first dichroic mirror (6) and the second dichroic mirror ( 7), the third dichroic mirror (8); The light emitted by the second light source (2) passes through the third reflecting mirror (10), is reflected by the first dichroic mirror (6), and then passes through the second dichroic mirror (7), the third Chromatic mirror (8); The light emitted by the third light source (3) passes through the fifth reflecting mirror (12), is reflected by the second dichroic mirror (7), and then passes through the third dichroic mirror (8); The light emitted by the fourth light source (4) passes through the fourth reflecting mirror (11), is reflected by the third dichroic mirror (8), and is combined with the light from the other three lasers and then transmitted to the acousto-optic sensor. Tuning filter (13); The composite light field control module includes: an optical fiber collimating beam expanding assembly, comprising an optical fiber collimator (16), a laser beam expander (17), and a first achromatic half-wave plate (18) arranged in sequence along the optical axis where the light emitted from the light source module is located; A first optical control device, comprising a spatial light modulator (20), a polarization beam splitter prism (19), a Fourier lens (21), a porous mask (22), a polarization rotator (23), and a liquid crystal phase compensator (24) ), a collimating lens (25); The second optical control device includes a two-dimensional scanning galvanometer (33), a second achromatic half-wave plate (34), a polarizing beam splitter prism (19), a Fourier lens (21), and a porous mask (22) ), a polarization rotator (23), a liquid crystal phase compensator (24), a collimating lens (25); 4f imaging system, comprising a Fourier lens (21) and a collimating lens (25), that is, the back focal plane of the Fourier lens (21) coincides with the front focal plane of the collimating lens (25); The spatial light modulator (20) and the two-dimensional scanning galvanometer (33) are both located on the front focal plane of the 4f imaging system; the porous mask plate (22) is located on the Fourier plane of the 4f imaging system; The 4f imaging system is optically connected to the fluorescence imaging module; The process in which the composite light field control module realizes the structured light illumination mode: the light output by the optical fiber collimating beam expanding assembly is transmitted and incident on the spatial light modulator (20) through the polarization beam splitting prism (19), and then passes through the spatial light modulator (20). ) reflection, the polarization beam splitting prism (19) is reflected and successively passes through the Fourier lens (21), the porous mask plate (22), the polarization rotator (23), the liquid crystal phase compensator (24), and the collimating lens (25); The process of realizing the uniform illumination mode by the composite light field control module: the light output by the optical fiber collimating beam expanding assembly is reflected by the polarization beam splitting prism (19) and then passes through the second achromatic half-wave plate (34), the two-dimensional The scanning galvanometer (33) is reflected by the two-dimensional scanning galvanometer (33) and then passes through the second achromatic half-wave plate (34) and the polarizing beam splitting prism (19) in turn, and is then transmitted through the polarizing beam splitting prism (19) and then pass through a Fourier lens (21), a porous mask plate (22), a polarization rotator (23), a liquid crystal phase compensator (24), and a collimating lens (25) in sequence; The spatial light modulator (20) is loaded with a binary periodic fringe computational hologram, and the light is diffracted to multiple orders by the spatial light modulator (20); The porous mask plate (22) is composed of 2N rotationally symmetrically distributed pinholes, each pinhole has a rectangular structure, and the center position of the long side corresponds to the critical angle of total internal reflection of the sample surface, wherein N is an integer; the porous mask plate ( 22) It is used to realize spatial filtering, allowing only light that meets a specific incident angle to enter the fluorescence imaging module to illuminate the sample, and the light passing through different positions of the pinhole has different incident angles; The polarization rotator (23) is used to make the polarization state of the light beam passing through any pinhole perpendicular to the plane defined by the pinhole center point and the optical axis of the 4f imaging system; The fast axis direction of the liquid crystal phase compensator (24) is parallel to the polarization direction of the transmitted light of the polarizing beam splitter prism (19), and the liquid crystal phase compensator (24) is used for actively compensating the laser phase drift caused by the optical element, so that it finally reaches the sample The polarization state of any beam of light on the surface is maintained as linear polarization state, and the polarization direction is perpendicular to the incident surface of the light; The fluorescence imaging module includes two cameras, one camera receives the fluorescence excited by the structured illumination light field and images, and the other camera receives the fluorescence excited by the uniform illumination light field and images, the original image collected by the camera is reconstructed by a computer super-resolution image, and The image acquisition sequence of the two cameras, the composite light field control module and the light source module are controlled synchronously by the computer; The fluorescence imaging module includes: A device for realizing structured light illumination super-resolution imaging, comprising a lens (26), a microscope objective lens (27), a sample stage (28), a fourth dichroic mirror (29), a fifth dichroic mirror (37), a third A tube lens (31) and a first camera (32); the light emitted from the composite light field control module passes through the lens (26) and is then reflected by a fourth dichroic mirror (29) before being irradiated and placed on the sample stage (28) On the sample surface, a cosine structure illumination light field is generated, and the sample is excited to emit fluorescence. The excited fluorescence is collected by the microscope objective lens (27), and then transmitted through the fourth dichroic mirror (29) and the fifth dichroic mirror ( 37) transmitting and imaging the first tube lens (31) to the detection surface of the first camera (32); A device for realizing super-resolution imaging under uniform light illumination, comprising a lens (26), a microscope objective lens (27), a sample stage (28), a fourth dichroic mirror (29), a fifth dichroic mirror (37), a third A two-tube lens (35) and a second camera (36); the light emitted from the compound light field control module passes through the lens (26) and is then reflected by the fourth dichroic mirror (29) before being irradiated and placed on the sample stage (28) A uniform illumination light field is generated on the surface of the sample on the surface, and the sample is excited to emit fluorescence. The excited fluorescence is collected by the microscope objective lens (27), and then transmitted through the fourth dichroic mirror (29) and the fifth dichroic mirror (37) in turn. ) is reflected and then imaged by the second tube lens (35) to the detection surface of the second camera (36). 2. The stochastic optical reconstruction and structured light illumination composite super-resolution imaging system according to claim 1, wherein the first achromatic half-wave plate (18) is mounted on an electric rotating wave plate holder, The angle between the fast axis direction and the polarization direction of the transmitted light of the polarizing beam splitting prism (19) is θ, and the light intensity of the liquid crystal spatial light modulator (20) transmitted through the polarizing beam splitting prism (19) is proportional to sin ² (2θ), The light intensity of the polarization beam splitting prism (19) reflecting and illuminating the two-dimensional scanning galvanometer (33) is proportional to cos ² (2θ). 3. The composite super-resolution imaging system of random optical reconstruction and structured light illumination according to claim 2, characterized in that, when sin ² (2θ)=1, cos ² (2θ)=0, the composite light field control module is independent Works in structured light illumination mode; when sin ² (2θ)=0, cos ² (2θ)=1, the composite light field control module independently works in uniform illumination mode; when θ is equal to other values, the two imaging modes work simultaneously ; By controlling the value of the included angle θ, the composite light field control module can be switched between the structured light illumination mode and the uniform illumination mode, and the two imaging modes can also work at the same time.

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