CN112798564A - Stochastic optical reconstruction and structured light illumination composite super-resolution imaging system - Google Patents
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Abstract
本发明公开了一种随机光学重建与结构光照明复合超分辨成像系统，包括：光源模块，用于提供多个不同波长的合束照明光线，并控制光线照明时序，还用于控制单一波长光线照明，或者多个波长光线交替照明，或者多个波长光线同时照明；复合光场调控模块，包括用于将入射光场调控为余弦结构照明光场的第一光学调控装置，和用于将入射光场调控为均匀照明光场的第二光学调控装置；两种光学调控装置可以独立工作、交替工作或者同时工作；荧光成像模块，用于采集样本的多幅原始荧光图像并由计算机重建超分辨图像。本发明通过在一套光学成像平台实现随机光学重建与结构光照明两种超分辨成像技术联用，可实现对复杂生物体系的多模式、跨分辨率尺度同时成像。
The invention discloses a composite super-resolution imaging system of random optical reconstruction and structured light illumination. Illumination, or alternate illumination of multiple wavelengths of light, or simultaneous illumination of multiple wavelengths of light; a compound light field control module, comprising a first optical control device for adjusting the incident light field into a cosine structure illumination light field, and for adjusting the incident light field to a cosine structure illumination light field. Light field regulation is a second optical regulation device for uniform illumination light field; two optical regulation devices can work independently, alternately or simultaneously; fluorescence imaging module, used to collect multiple original fluorescence images of the sample and reconstruct super-resolution by computer image. 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.
Description
Technical Field
The invention belongs to the technical field of microscopic imaging, and particularly relates to a random optical reconstruction and structured light illumination composite super-resolution imaging system.
Background
Fluorescence super-resolution imaging has a wide application prospect, and various super-resolution imaging technologies exist at present, including a random optical reconstruction super-resolution imaging technology, a structured light illumination super-resolution imaging technology, a stimulated radiation loss super-resolution imaging technology and the like. These techniques, while improving resolution, are accompanied by disadvantages such as reduced imaging speed, increased illumination light power, increased phototoxicity, and a smaller imaging field of view.
In the prior art, the technical principle of the random optical reconstruction super-resolution imaging method (STORM) is as follows: by utilizing the photoswitch fluorescent protein, single molecules in the diffraction limit range are randomly activated at different time, and each fluorescent molecule is accurately positioned and recombined, so that a super-resolution image is obtained by superposition, and the resolution can reach 10nm or even higher. The random optical reconstruction microscope is mainly used for intracellular single molecule imaging and observation of fine subcellular structures, such as observation of accurate positioning of single proteins in cells, observation of interaction between proteins, and fine structures such as microwires, microtubules, adhesive plaques and inclusions in cells. However, the random optical reconstruction microscope needs to acquire a large number of original images (typically 2 ten thousand images) to reconstruct a super-resolution image, the imaging time varies from several seconds to tens of minutes, the time resolution is low, and the random optical reconstruction microscope is difficult to be used for live cell imaging.
The technical principle of the structured light illumination super-resolution imaging method (SIM) is as follows: multiple mutually interfering beams are irradiated onto the sample, and then high-resolution information is extracted from the collected fluorescence image, and a super-resolution image is reconstructed. The structured light illumination super-resolution technology is high in imaging speed, low in excitation light energy and small in damage to cells, and is mainly used for observing living body observation of a subcellular level, including dynamic changes of mitochondria, dynamic changes of cytoskeleton, dynamic changes of chromosomes, movement of vesicles in cells, movement of viruses in cells and the like. But the imaging resolution of structured light illumination is improved by 2 times compared with the traditional fluorescence microscope at the highest energy, reaches the level of 100nm, and is difficult to carry out fine biochemical reactions such as the movement of motor protein along cytoskeleton and the invasion process of pathogen particles into cells, and the like, thereby limiting the application of the structured light illumination in the fields of ultramicrobiology, medicine and the like.
The requirements of fields such as life science, basic medicine and the like on high-end imaging technology cover ultrahigh spatial resolution, high imaging speed, low illumination power, low phototoxicity, large imaging field of view and the like, and the requirements have contradictions which are difficult to reconcile from the technical aspect.
Disclosure of Invention
The invention aims to provide a random optical reconstruction and structured light illumination composite super-resolution imaging system, which meets the urgent need of cross-resolution scale imaging in life science and basic medical research.
The technical solution for realizing the purpose of the invention is as follows: a stochastic optical reconstruction and structured light illumination combined super-resolution imaging system, the system comprising:
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 the illumination of light rays with single wavelength, or the alternate illumination of light rays with a plurality of wavelengths, or the simultaneous illumination of light rays with a plurality of wavelengths;
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.
Further, the light source module includes: the device comprises a plurality of light sources with different wavelengths, a reflection beam combination component, an acousto-optic tunable filter, an optical fiber coupler and a polarization maintaining optical fiber; light rays emitted by the light sources with different wavelengths are combined through the reflection beam combination assembly and then irradiate the acousto-optic tunable filter, first-order diffraction light of the acousto-optic tunable filter is collected through the optical fiber coupler and enters the polarization maintaining optical fiber, and then the light rays are introduced into the composite light field regulation and control module through the polarization maintaining optical fiber.
Further, the reflective beam combining assembly comprises:
the plurality of reflectors respectively correspond to the light sources with different wavelengths and guide the light rays emitted by all the light sources to the dichroic mirror to complete beam combination;
the plurality of dichroic mirrors are used for combining the light rays from the reflecting mirrors into one 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 reflecting mirror is omitted.
Further, the plurality of light sources with different wavelengths include a first light source, a second light source, a third light source and a fourth light source; the reflecting beam combining assembly comprises a first reflecting mirror, a second reflecting mirror, a third reflecting mirror, a fourth reflecting mirror, a fifth reflecting mirror, a first dichroic mirror, a second dichroic mirror and a third dichroic mirror;
the light rays emitted by the first light source sequentially pass through the second reflecting mirror and the first reflecting mirror and then sequentially pass through the first dichroic mirror, the second dichroic mirror and the third dichroic mirror;
after passing through the third reflector, the light emitted by the second light source is reflected by the first dichroic mirror and then sequentially passes through the second dichroic mirror and the third dichroic mirror;
after passing through a fifth reflector, light rays emitted by the third light source are reflected by the second dichroic mirror and then pass through the third dichroic mirror;
and light rays emitted by the fourth light source are reflected by the third dichroic mirror after passing through the fourth reflector, and are combined with light rays from other three lasers and then transmitted to the acousto-optic tunable filter.
Further, the composite light field regulation module comprises:
the optical fiber collimation beam expanding assembly comprises an optical fiber collimator, a laser beam expander and a first achromatic half wave plate which are sequentially arranged along an optical axis where the emergent light of the light source module is located;
the first optical regulation and control device comprises a spatial light modulator, a polarization beam splitter prism, a Fourier lens, a porous mask plate, a polarization rotator, a liquid crystal phase compensator and a collimating lens;
the second optical regulation and control device comprises a two-dimensional scanning galvanometer, a second achromatic half wave plate, a polarization splitting prism, a Fourier lens, a porous mask plate, a polarization rotator, a liquid crystal phase compensator and a collimating lens;
the 4f imaging system comprises a Fourier lens and a collimating lens, wherein the rear focal plane of the Fourier lens is superposed with the front focal plane of the collimating lens;
the spatial light modulator and the two-dimensional scanning galvanometer are both positioned on the front focal plane of the 4f imaging system; the porous mask plate is arranged on a Fourier surface of a 4f imaging system;
the 4f imaging system is optically connected with the fluorescence imaging module;
the process of the composite light field regulation and control module for realizing the structured light illumination mode is as follows: light output by the optical fiber collimation beam expanding assembly is transmitted by the polarization beam splitting prism to be incident to the spatial light modulator, then is reflected by the spatial light modulator and the polarization beam splitting prism and then sequentially passes through the Fourier lens, the porous mask plate, the polarization rotator, the liquid crystal phase compensator and the collimating lens;
the process of the composite light field regulation and control module for realizing the uniform illumination mode is as follows: the light output by the fiber collimation beam expanding assembly passes through the second achromatic half wave plate and the two-dimensional scanning galvanometer after being reflected by the polarization splitting prism, passes through the second achromatic half wave plate and the polarization splitting prism after being reflected by the two-dimensional scanning galvanometer, is transmitted by the polarization splitting prism and passes through the Fourier lens, the porous mask plate, the polarization rotator, the liquid crystal phase compensator and the collimating lens.
Furthermore, a binary periodic fringe calculation hologram is loaded on the spatial light modulator, and light is diffracted to a plurality of orders by the spatial light modulator;
the porous mask plate is composed of 2N pinholes which are rotationally and symmetrically distributed, each pinhole is of a rectangular structure, the central position of a long edge corresponds to the critical angle of total internal reflection of a sample surface, and N is an integer; the porous mask plate is used for realizing spatial filtering, only light rays meeting a specific incident angle are allowed to enter the fluorescence imaging module to illuminate a sample, and light rays passing through different positions of the pinhole have different incident angles;
the polarization rotator is used for enabling the polarization state of the light beam passing through any pinhole to be vertical to a plane determined by the central 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 polarization beam splitter prism, and the phase compensator is used for actively compensating laser phase drift caused by the optical element, so that the polarization state of any beam of light finally reaching the sample surface is kept in a linear polarization state, and the polarization direction is vertical to the incident surface of the light.
Furthermore, the first achromatic half-wave plate is arranged on the electric rotating wave plate frame, the included angle between the fast axis direction of the first achromatic half-wave plate and the polarization direction of the transmission light of the polarization beam splitter prism is theta, and the light intensity and sin of the liquid crystal spatial light modulator are transmitted through the polarization beam splitter prism to illuminate2(2 theta) is in direct proportion, and the intensity of the light reflected by the polarization beam splitter prism and illuminating the two-dimensional scanning galvanometer is related to cos2(2 θ) is proportional.
Further, when sin2(2θ)＝1，cos2When the (2 theta) is 0, the composite light field regulation and control module independently works in a structured light illumination mode; when sin2(2θ)＝0，cos2When the (2 theta) is 1, the composite light field regulation and control module independently works in a uniform illumination mode; when theta is equal to other values, the two imaging modes work simultaneously; by controlling the value of the included angle theta, the composite light field regulation and control module can be switched between the structured light illumination mode and the uniform illumination mode, and can also be switchedThe two imaging modes operate simultaneously.
Furthermore, the fluorescence imaging module comprises two cameras, one camera receives and images fluorescence excited by the structure illumination light field, the other camera receives and images fluorescence excited by the uniform illumination light field, the original images acquired by the cameras are reconstructed into super-resolution images through a computer, and the image acquisition time sequences of the two cameras, the composite light field regulation and control module and the light source module are synchronously controlled through the computer.
Further, the fluorescence imaging module includes:
the device for realizing the structured light illumination super-resolution imaging comprises a lens, a microscope objective, a sample stage, a fourth dichroic mirror, a fifth dichroic mirror, a first tube lens and a first camera; emergent light of the composite light field regulation and control module penetrates through the lens, is reflected by the fourth dichroic mirror and then irradiates the surface of a sample placed on the sample stage, a cosine structure illumination light field is generated, the sample is excited to emit fluorescence, the excited fluorescence is collected by the microscope objective, and then is sequentially transmitted by the fourth dichroic mirror, transmitted by the fifth dichroic mirror and imaged on a detection surface of the first camera through the first tube lens;
the device for realizing uniform light illumination super-resolution imaging comprises a lens, a microscope objective, a sample stage, a fourth dichroic mirror, a fifth dichroic mirror, a second tube lens and a second camera; emergent light of the composite light field regulation and control module penetrates through the lens, is reflected by the fourth dichroic mirror and then irradiates the surface of a sample placed on the sample stage, an even illumination light field is generated, the sample is excited to emit fluorescence, the excited fluorescence is collected by the microscope objective, and then is sequentially transmitted by the fourth dichroic mirror, reflected by the fifth dichroic mirror and imaged on a detection surface of the second camera through the second tube lens.
Aiming at the imaging requirements in the fields of cell biology, clinical pathological diagnosis and the like, the invention provides a random optical reconstruction and structured light illumination composite super-resolution imaging method and system, which essentially integrates the advantages of two technologies and has the following advantages compared with the traditional microscopic detection system:
1) the simultaneous imaging observation of cross-resolution scale can be realized by combining the STORM technology and the SIM technology, for example, in the research of the movement of living cell motor protein along cytoskeleton, the nano-resolution STORM imaging of the motor protein can be realized, and meanwhile, the hundred-nano-resolution SIM imaging of the cytoskeleton can be realized. The cross-resolution scale simultaneous imaging capability has wide requirements in the fields of cell mass transport, pathogen infection process and the like, and the prior art cannot meet the requirements.
2) In the research aiming at SIM imaging, in-situ STROM ultrahigh resolution imaging can be carried out on an interested key area, the problems of high SIM imaging speed, large imaging field of view but low resolution are solved, and the problems of inaccurate positioning, sample state change and the like caused by secondary imaging are avoided.
3) In the research aiming at STORM imaging, the STORM imaging and analysis are guided by quick SIM imaging, so that the amount of STROM original data can be obviously reduced, and the imaging speed is improved.
4) STORM and SIM imaging are realized on one set of imaging platform, most hardware equipment can be shared, the economic cost for obtaining the same imaging function is obviously reduced, and the biological laboratory space resource occupied by the instrument is reduced.
The present invention is described in further detail below with reference to the attached drawing figures.
Drawings
FIG. 1 is a schematic structural diagram of a random optical reconstruction and structured light illumination combined super-resolution imaging system in an embodiment.
FIG. 2(a) is a structure diagram of a porous mask plate, and (b) is an incident light spot distribution measured at the rear pupil plane of a microscope in a uniform illumination mode; (c) and (e) the distribution of the incident light spots measured on the back pupil surface of the microscope objective in the structured light illumination mode.
Fig. 3 is a super-resolution image taken by the present invention in one embodiment, in which (a) breast cancer cell HER2 positive signal SIM is imaged and (b) breast cancer cytoskeleton STORM is imaged.
The respective symbols in the figure are as follows: 1 laser 647 nm; 2, 561nm of laser; 3, 488nm of laser; 4, 405nm of laser; mirrors 9, 5, 10, 11, 12; dichroic mirrors 6, 7, 8, 29; 13 acousto-optic tunable filter; 14 a fiber coupler; 15 polarization maintaining fiber; 16 fiber collimator; 17 a laser beam expander; 18 an achromatic half wave plate; 19 polarization beam splitting prism; 20 a spatial light modulator; 21 a Fourier lens; 22 a porous mask plate; 23 a polarization rotator; 24 liquid crystal phase compensator; 25 a collimating lens; 26 lenses; 27 microscope objective lens; 28 sample stage; 30 a fluorescent color filter; a 31-tube lens; 32 cameras; 33 two-dimensional scanning galvanometer; 34 an achromatic half wave plate; a 35 tube lens; 36 cameras; 37 fluorescent color filter.
Detailed Description
In order to make the objects, technical solutions and advantages of the present application more apparent, the present application is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the present application.
In addition, if there is a description of "first", "second", etc. in an embodiment of the present invention, the description of "first", "second", etc. is for descriptive purposes only and is not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In addition, technical solutions between various embodiments may be combined with each other, but must be realized by a person skilled in the art, and when the technical solutions are contradictory or cannot be realized, such a combination should not be considered to exist, and is not within the protection scope of the present invention.
In one embodiment, the present invention provides a stochastic optical reconstruction and structured light illumination combined super-resolution imaging system, the system comprising:
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 the illumination of light rays with single wavelength, or the alternate illumination of light rays with a plurality of wavelengths, or the simultaneous illumination of light rays with a plurality of wavelengths;
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 composite super-resolution imaging by combining the random optical reconstruction super-resolution imaging technology and the structured light illumination super-resolution imaging technology, namely SIM or STOM imaging can be independently carried out; or firstly, quickly imaging through the SIM, positioning an interested key area, and then carrying out STROM ultrahigh-resolution imaging on the key area; SIM and STOM imaging can also be performed simultaneously on the sample. The system has the advantages of high spatial resolution, high imaging speed, large imaging field of view, low phototoxicity and the like, can meet the imaging requirements of multiple modes at the same time, and particularly can realize in-situ cross-resolution scale simultaneous imaging of the same biological sample. In addition, the composite imaging system can simultaneously realize the combination of two super-resolution imaging technologies of SIM and STORM on one imaging platform, the SIM and the STORM share most hardware equipment, the economic cost for obtaining the same imaging function can be reduced, the laboratory space resource occupied by the equipment is reduced, and the complexity of system maintenance is simplified.
Further, in one embodiment, the light source module includes: a plurality of light sources with different wavelengths, a reflection beam combination component, an acousto-optic tunable filter 13, an optical fiber coupler 14 and a polarization maintaining optical fiber 15; light rays emitted by the light sources with different wavelengths are combined by the reflection beam combination component and then irradiate the acousto-optic tunable filter 13, first-order diffraction light of the acousto-optic tunable filter 13 is collected by the optical fiber coupler 14 and enters the polarization maintaining optical fiber 15, and then the light rays are introduced into the composite light field regulation and control module through the polarization maintaining optical fiber 15.
The control of the light illumination time sequence is also used for controlling the illumination of light with single wavelength, or the alternate illumination of light with multiple wavelengths, or the simultaneous illumination of light with multiple wavelengths, which are all realized by controlling the acousto-optic tunable filter 13.
Further, in one embodiment, the reflective beam combiner assembly includes:
the plurality of reflectors respectively correspond to the light sources with different wavelengths and guide the light rays emitted by all the light sources to the dichroic mirror to complete beam combination;
a plurality of dichroic mirrors for combining the light from the reflecting mirrors into one 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 certain dichroic mirror, and the corresponding reflecting mirror is omitted.
Further, in one embodiment, the plurality of light sources with different wavelengths includes a first light source 1, a second light source 2, a third light source 3, and a fourth light source 4; the reflecting beam combining component comprises a first reflecting mirror 5, a second reflecting mirror 9, a third reflecting mirror 10, a fourth reflecting mirror 11, a fifth reflecting mirror 12, a first dichroic mirror 6, a second dichroic mirror 7 and a third dichroic mirror 8;
the light emitted by the first light source 1 sequentially passes through the second reflecting mirror 9 and the first reflecting mirror 5, and then sequentially passes through the first dichroic mirror 6, the second dichroic mirror 7 and the third dichroic mirror 8;
after passing through the third reflector 10, the light emitted by the second light source 2 is reflected by the first dichroic mirror 6, and then sequentially passes through the second dichroic mirror 7 and the third dichroic mirror 8;
after passing through the fifth reflector 12, the light emitted by the third light source 3 is reflected by the second dichroic mirror 7 and then passes through the third dichroic mirror 8;
light emitted by the fourth light source 4 passes through the fourth reflector 11, is reflected by the third dichroic mirror 8, and is combined with light from other three lasers and then transmitted to the acousto-optic tunable filter 13.
Here, exemplarily, in one embodiment, the first light source 1, the second light source 2, the third light source 3, and the fourth light source 4 employ lasers, and the wavelengths are 647nm, 4561nm, 488nm, and 405nm, respectively.
It should be noted that the light source is not limited to a laser, but may also be an LED light source, and the light wavelength is not limited to the above four wavelengths, and the light sources do not have a primary-secondary relationship, and parameter selection, number increase and decrease, or replacement may be performed according to requirements.
Further, in one embodiment, the composite light field modulation module comprises:
the optical fiber collimation and beam expansion assembly comprises an optical fiber collimator 16, a laser beam expander 17 and a first achromatic half wave plate 18 which are sequentially arranged along an optical axis where the emergent light of the light source module is located;
the first optical regulation and control device comprises a spatial light modulator 20, a polarization beam splitter prism 19, a Fourier lens 21, a porous mask plate 22, a polarization rotator 23, a liquid crystal phase compensator 24 and a collimating lens 25;
the second optical regulation and control device comprises 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 plate 22, a polarization rotator 23, a liquid crystal phase compensator 24 and a collimating lens 25;
the 4f imaging system comprises a Fourier lens 21 and a collimating lens 25, namely, the back focal plane of the Fourier lens 21 is coincided with the front focal plane of the collimating lens 25;
the spatial light modulator 20 and the two-dimensional scanning galvanometer 33 are both positioned on the front focal plane of the 4f imaging system; the reticle 22 is in the fourier plane of the 4f imaging system;
the 4f imaging system is optically connected with the fluorescence imaging module;
the process of the composite light field regulation and control module for realizing the structured light illumination mode is as follows: light output by the fiber collimation and beam expansion assembly is transmitted by the polarization beam splitter prism 19 and enters the spatial light modulator 20, then is reflected by the spatial light modulator 20 and the polarization beam splitter prism 19, and then 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 in sequence;
the process of the composite light field regulation and control module for realizing the uniform illumination mode is as follows: the light output by the fiber collimation beam expanding assembly is reflected by the polarization splitting prism 19 and then sequentially passes through the second achromatic half wave plate 34 and the two-dimensional scanning galvanometer 33, is reflected by the two-dimensional scanning galvanometer 33 and then sequentially passes through the second achromatic half wave plate 34 and the polarization splitting prism 19, is transmitted by the polarization splitting prism 19 and then sequentially 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.
Further, in one embodiment, the spatial light modulator 20 is loaded with a binary periodic fringe computer hologram, and light is diffracted into a plurality of orders by the spatial light modulator 20. Changing the phase and spatial orientation of the loaded image on spatial light modulator 20 changes the orientation and phase of the two-dimensional cosine illumination field on the sample plane.
The porous mask plate 22 is composed of 2N rotationally symmetrically distributed pinholes, each pinhole is of a rectangular structure, the central position of a long edge corresponds to the critical angle of total internal reflection of the sample surface, and N is an integer. The porous mask plate 22 is used for realizing spatial filtering, only light rays meeting a specific incident angle are allowed to enter the fluorescence imaging module to illuminate a sample, and light rays passing through different positions of the pinhole have different incident angles; meanwhile, the incident angle of the rectangular structure can be adjusted within a certain range, the central position of the rectangle corresponds to the critical angle of total internal reflection, when an incident beam passes through a pinhole along the inner side, large-angle incident illumination on a sample can be realized, when the position of the incident beam is changed along the inner side of the central position, the period size of a structured light field can be adjusted, and the illumination on the samples with different structural characteristics by optimal structured light is realized; when the incident light beam passes through the pinhole along the outer side, the total internal reflection illumination of the sample can be realized, and when the position of the incident light beam is changed along the outer side of the central position, the penetration depth of evanescent waves can be adjusted, so that the illumination of samples with different depths is realized.
Here, fig. 2(a) is an exemplary 6-hole mask plate, which is composed of 6 rotationally symmetrically distributed pinholes; FIGS. 2(c) - (e) are the intensity distributions of the positive and negative primary beams at the back pupil plane of the microscope objective in the SIM imaging mode; fig. 2(b) shows the intensity distribution of the incident beam at the back pupil plane of the microscope objective in the STORM imaging mode, and these 6 light spots are formed by fast scanning 1 light spot, the scanning speed is 8kHz, which is much faster than the camera acquisition speed (less than 100Hz), and it can be equivalently considered that there are 6 light spots illuminating at the same time.
The polarization rotator 23 is used for enabling the polarization state of the light beam passing through any pinhole to be perpendicular to the plane determined by the central point of the pinhole and the optical axis of the 4f imaging system (the polarization directions of the positive and negative primary light beams can be always perpendicular to the plane where the positive and negative primary light beams are located together);
the fast axis direction of the phase compensator 24 is parallel to the polarization direction of the transmitted light of the polarization beam splitter 19, the phase compensator 24 is used for actively compensating the laser phase drift caused by optical elements such as the collimating lens 25, the lens 26, the fourth dichroic mirror 29, the microscope objective 27, etc., the phase compensation amount, the phase difference between the fast axis and the slow axis can be controlled by computer programming, when the incident azimuth angle of the incident light is changed, or the wavelength of the incident light is changed, the phase compensation amount is synchronously changed, so that the polarization state of any beam of light finally reaching the sample surface is kept in a linear polarization state, and the polarization direction is perpendicular to the incident surface of the beam (the polarization direction is perpendicular to the plane where the positive and negative primary beams are jointly located), thereby obtaining the maximum structural light modulation degree, and because the phase drift is related to the laser wavelength, when the laser wavelength is changed, the phase compensation amount of the liquid crystal phase compensator needs to be changed, this is very important to make the structured light field with any wavelength have the maximum modulation degree, and is also the key design to make the present invention obtain high quality SIM super-resolution image.
Further, in one embodiment, the first achromatic half waveplate 18 is mounted on the electric rotating waveplate frame, the fast axis direction of the first achromatic half waveplate and the polarization direction of the transmitted light of the polarization beam splitter 19 form an included angle θ, and the light intensity and sin of the liquid crystal spatial light modulator 20 transmitted through the polarization beam splitter 19 are transmitted and illuminated2(2 theta) is proportional to cos, and the intensity of light reflected by the polarizing beam splitter 19 and illuminating the two-dimensional scanning galvanometer 332(2 θ) is proportional.
Further, in one embodiment, sin is used2(2θ)＝1，cos2When the (2 theta) is 0, the composite light field regulation and control module independently works in a structured light illumination mode; when sin2(2θ)＝0，cos2When the (2 theta) is 1, the composite light field regulation and control module independently works in a uniform illumination mode; when theta 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 theta, the composite light field regulation and control module can be positionedThe structured light illumination mode and the uniform illumination mode are switched, and the two imaging modes can work simultaneously.
Further, in one embodiment, the fluorescence imaging module includes two cameras, one camera receives and images fluorescence excited by the structure illumination light field, the other camera receives and images fluorescence excited by the uniform illumination light field, the original images acquired by the cameras are reconstructed into super-resolution images through a computer, and the image acquisition time sequence of the two cameras, the composite light field regulation and control module and the light source module are synchronously controlled through the computer.
Further, in one embodiment, the fluorescence imaging module comprises:
the device for realizing the structured light illumination super-resolution imaging comprises a lens 26, a microscope objective 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; emergent light of the composite light field regulation module penetrates through the lens 26, is reflected by the fourth dichroic mirror 29 and then irradiates the surface of a sample placed on the sample stage 28, a cosine structure illumination light field is generated, the sample is excited to emit fluorescence, the excited fluorescence is collected by the microscope objective 27, and then is sequentially transmitted by the fourth dichroic mirror 29, transmitted by the fifth dichroic mirror 37 and imaged on a detection surface of the first camera 32 through the first tube lens 31;
the device for realizing uniform light illumination super-resolution imaging comprises a lens 26, a microscope objective 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; emergent light of the composite light field regulation module penetrates through the lens 26, is reflected by the fourth dichroic mirror 29 and then irradiates the surface of a sample placed on the sample stage 28, a uniform illumination light field is generated, the sample is excited to emit fluorescence, the excited fluorescence is collected by the microscope objective 27, and then is transmitted by the fourth dichroic mirror 29, reflected by the fifth dichroic mirror 37 and imaged on a detection surface of the second camera 36 after passing through the second tube lens 35 in sequence.
Here, the camera may be an EMCCD, SCMOS, or other two-dimensional image detector.
Here, the fluorescence imaging module can be built by itself, and a commercial microscope can also be used.
Based on the embodiment, the step of the system for realizing the structured light illumination super-resolution imaging comprises the following steps: the laser emits light and wavelength gating and illumination time sequence control are carried out through the acousto-optic tunable filter 13; loading a binary periodic fringe calculation hologram on a spatial light modulator 20, diffracting light rays into multiple orders through a liquid crystal spatial light modulator 20, reflecting the light rays through a polarization beam splitter prism 19, sequentially passing through a Fourier lens 21, a porous mask plate 22, a polarization rotator 23, a liquid crystal phase compensator 24, a collimating lens 25 and a fluorescence imaging module, irradiating the light rays onto the surface of a sample, and enabling positive and negative first-order diffracted light to interfere on the surface of the sample (the light diffracted by the spatial light modulator 20 passes through the porous mask plate filter 22, so that only the positive and negative first-order diffracted light can be irradiated onto the surface of the sample to interfere), generating a cosine structure illumination light field and exciting the sample to emit fluorescence; the fluorescence emitted by the sample is collected by the fluorescence imaging module, transmitted by fourth dichroic mirror 29, transmitted by fifth dichroic mirror 37, and imaged on the detection surface of camera 32 by first tube lens 31. The phase and the spatial orientation of the loaded image on the spatial light modulator are changed, the orientation and the phase of the sample surface structure illumination light field can be changed, and fluorescence is excited to obtain the image. Repeating the above process, collecting the fluorescence images of 3 direction angles and 3 phases, and finally reconstructing the super-resolution image from the 9 original images collected by the camera by using an image processing algorithm.
The system of the invention realizes uniform light illumination super-resolution imaging, namely random optical reconstruction illumination super-resolution imaging, and comprises the following steps: the propagation direction of the light is controlled by 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, the polarization splitting prism 19, the fourier lens 21, the porous mask plate 22, the polarization rotator 23, the liquid crystal phase compensator 24, the collimating lens 25 and the imaging module in sequence and irradiates the surface of the sample. The incident angle is alpha, the alpha is made to be larger than the critical angle of total internal reflection, the incident light is subjected to total internal reflection at the surface of the sample, a beam evanescent wave illumination light field is formed on the surface of the sample, the fluorescence emitted by the sample is collected by the objective lens, and is transmitted by the fourth dichroic mirror 29, reflected by the fifth dichroic mirror 37 and imaged on the detection surface of the second camera 36 by the second tube lens 35.
The step of random optical reconstruction illumination super-resolution imaging further comprises the steps of rapidly switching the deflection direction of the two-dimensional scanning galvanometer 33, changing the angular distribution of incident light, rotating the incident surface of the incident light by integral multiples of 360 degrees along the optical axis of the microscope objective within the one-time imaging integration time of the camera, and distributing light ray tracks on a cone with the vertex angle of 2 alpha, so that the evanescent wave light field is uniformly distributed, and the negative effect of laser speckle on the imaging quality is eliminated. Meanwhile, the penetration depth of evanescent waves can be accurately controlled by controlling the size of the alpha angle.
Exemplarily, fig. 3 shows a super-resolution image taken by the present invention, wherein (a) is a SIM image of a positive signal of HER2 for breast cancer cells, and (b) is a STORM image of breast cancer cytoskeleton.
Further, in one embodiment, the system of the present invention can also perform two-color labeling on the sample, and a light source with one wavelength is used to excite one of the proteins to perform structured light illumination for fast super-resolution imaging, while a light source with another wavelength is used to excite a second protein to perform single-molecule random optical reconstruction for ultrahigh-resolution imaging.
By integrating the above embodiments, the random optical reconstruction and structured light illumination composite super-resolution imaging system provided by the invention comprises a light source module for providing multi-wavelength combined illumination light, a composite light field regulation and control device for modulating a light field into a two-dimensional cosine structure illumination light field and a uniform illumination light field, and an imaging module for acquiring fluorescence signal imaging and reconstructing a super-resolution image. Wavelength gating and time sequence control of the multi-wavelength combined light rays are realized through the acousto-optic tunable filter, and single-wavelength light ray illumination, multiple-wavelength light ray alternate illumination and multiple-wavelength light ray simultaneous illumination are realized. Incident light is diffracted to a plurality of orders through a spatial light modulator, and only positive and negative first-order diffracted light is allowed to reach the surface of a sample and interfere to generate a cosine structure light field through filtering of a porous mask plate; the polarization direction of the positive and negative first-order diffracted light is controlled by the polarization rotator and is always vertical to the incident surface of the polarization rotator; and a liquid crystal phase compensator is arranged behind the polarization rotator and actively compensates random phase shift caused by the optical element, so that the light finally reaching the sample surface is kept in linear polarization and the polarization direction is vertical to the incident surface. The azimuth angle of the illumination light is rapidly scanned through the two-dimensional scanning galvanometer, multi-angle incidence averaging is achieved within one-time exposure time of the camera, an even illumination light field is generated, and adverse effects of laser speckles on imaging are eliminated. By controlling the rotation angle of the achromatic half wave plate, the illumination of a structural light field, or the illumination of a uniform light field, or the simultaneous illumination of the structural light field and the uniform light field but with different wavelengths can be realized. The imaging module is provided with two cameras, fluorescence excited by the structure light field illumination is detected and imaged by one camera, and fluorescence excited by the uniform light field illumination is detected and imaged by the other camera.
In summary, the system of the present invention has the following features:
1. the incident azimuth angle of illumination light is rapidly switched through the two-dimensional scanning galvanometer, a light ray track is rapidly swept in one-time exposure integration time of the camera to form a cone with the optical axis of the microscope objective as an axis, and the total illumination light intensity received by the sample is equal to the sum of the light intensity of the incident light rays at different angles, so that the random laser speckles can be effectively inhibited in the summing process, and an even illumination light field is formed on the surface of the sample. Meanwhile, the incident angle of incident light can be controlled through the two-dimensional scanning galvanometer, so that the penetration depth of an evanescent wave illumination light field is controlled.
2. The computer hologram loaded on the spatial light modulator is controlled, and the structure illumination light intensity with any light intensity distribution can be formed on the sample surface. The structure illumination light field is generated based on double-beam interference, and the system comprises an active phase compensator, so that an incident beam can be kept as linearly polarized light all the time, and the polarization direction is vertical to an incident surface, and the structure illumination light field obtains the maximum modulation degree. Meanwhile, as the phase shift of laser introduced by optical elements such as a lens, a dichroic mirror, a microscope objective and the like is related to the wavelength of the laser, the phase compensation amount can be actively adjusted in real time by the liquid crystal phase compensator when the incident wavelength is changed, so that the optimal modulation degree can be always realized for different wavelengths.
3. The random optical reconstruction/structured light illumination composite super-resolution imaging method comprises the following steps:
a) independent working mode: the invention can independently carry out STORM imaging and SIM imaging or quickly switch between the STORM imaging and the SIM imaging. The structured light illumination imaging is used for imaging the sample quickly and in a large view field, an interested key area is found, a random optical reconstruction mode is switched, and ultrahigh resolution imaging is carried out on the area. This is important, and in many cases, it is only a small portion of the sample that is of interest, and it is not necessary to perform ultra-high resolution imaging on the entire sample area, and how to quickly locate the focal region and super-resolution image the region is a technical difficulty. The invention adopts wide-field structured light to perform fast high-resolution imaging on the whole sample, and then uses a two-dimensional scanning galvanometer to enable light to fast scan a heavy spot area and randomly optically reconstruct ultrahigh-resolution imaging, thereby solving the problem.
b) And simultaneously, the working mode is as follows: the invention can simultaneously carry out 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 which can not be carried out by a single imaging method, for example, carrying out two-color marking on the sample, exciting one protein by using laser with one wavelength to carry out structured light illumination rapid super-resolution imaging, and exciting the second protein by using laser with the other wavelength to carry out single-molecule random optical reconstruction super-resolution imaging, which is very important for observing the dynamic process at certain accurate sites.
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.
The description and applications of the invention herein are illustrative and are not intended to limit the scope of the invention to the embodiments described above. Variations and modifications of the embodiments disclosed herein are possible, and alternative and equivalent various components of the embodiments will be apparent to those skilled in the art. It will be clear to those skilled in the art that the present invention may be embodied in other forms, structures, arrangements, proportions, and with other components, materials, and parts, without departing from the spirit or essential characteristics thereof. Other variations and modifications of the embodiments disclosed herein may be made without departing from the scope and spirit of the invention. The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and similar parts between the embodiments are referred to each other. The previous description of the disclosed embodiments is provided to enable 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 applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended 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.
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