CN116224561A - Polarization modulation super-resolution microscopic imaging system and method based on DMD - Google Patents

Abstract

The invention discloses a polarization modulation super-resolution microscopic imaging system and a method based on a DMD, which can realize sparse imaging of a target sample by combining polarization modulation of a photoelectric modulator with excitation illumination modulation of scattered pattern reflected light of a point array of the DMD, and improve the time resolution and the space resolution of imaging by a polarization modulation molecular positioning mode based on the DMD. The system and the method have very important significance for the exploration of biological cells with high spatial resolution and dynamic imaging, and meanwhile, the super-resolution imaging technology with high spatial and temporal resolution is beneficial to the exploration and discovery of a plurality of leading-edge fields such as life science, material science and the like.

Description

Polarization modulation super-resolution microscopic imaging system and method based on DMD

Technical Field

The invention belongs to the technical field of super-resolution microscopic imaging, and particularly relates to a polarization modulation super-resolution microscopic imaging system and method based on a Digital Micromirror Device (DMD).

Background

A digital micromirror device (Digtial Micromirror Devices, DMD) is an electronically-input, optically-output microelectromechanical system (MEMS) that consists of a number of small aluminum reflective mirrors, each mirror being referred to as a pixel. Each mirror is capable of deflecting about the diagonal of each small positive direction mirror (or pixel).

DMDs are based on semiconductor fabrication technology, consisting of high-speed digital light reflective switch arrays, and determine the imaging pattern and its characteristics by controlling the rotation of the micromirror about the fixed (yoke) and the time domain response (determining the angle of reflection and dead time of the light). DMD is a new, fully digital flat panel display device, employing MEMS technology to integrate a reflective micromirror array and a complementary metal oxide semiconductor static random access memory (CMOS SRAM) on the same chip.

Due to the superior performance of DMDs, DMD-based imaging system devices are widely used, and interest in industry and technology is stimulated. The DMD may perform full-area lithography according to the color range of an image, or may perform block exposure according to the pixel size of an image. The working process is a coordination and matching process of optical, mechanical and electrical integration.

Super-resolution microscopic imaging technology breaks through the imaging advantage of optical diffraction limit, and provides a very important observation method for researching nano-sized hyperfine structure and biological cell dynamic process. Super-resolution microscopic imaging technology has been rapidly developed since 2014, and a plurality of super-resolution microscopic imaging methods have emerged so far, and can be divided into two categories according to the space-time resolution of imaging: 1) A high spatial resolution super-resolution imaging method; 2) A high-time resolution super-resolution imaging method.

In the existing super-resolution imaging technology, the random optical reconstruction super-resolution imaging technology (stochastic optical reconstruction microscopy, STORM) can realize high spatial resolution (less than 50 nm), but the time resolution is lower, and tens of minutes are needed to shoot tens of thousands of frames of images for reconstructing one super-resolution image; structured light obvious micro-technology (structured illumination microscopy, SIM) can achieve high temporal resolution (less than one second), but the spatial resolution is limited, typically difficult to break through 100nm.

The existing super-resolution imaging technology can realize super-resolution imaging with high spatial resolution or high time resolution, but is difficult to achieve both spatial resolution and time resolution of imaging. The super-resolution imaging technology with high spatial resolution and high time resolution has very important scientific significance for exploring and discovering numerous front fields such as life science, material science and the like, and also becomes a development trend of a novel super-resolution imaging technology in recent years.

Disclosure of Invention

In view of the foregoing, it is an object of the present invention to provide a DMD-based polarization-modulated super-resolution microscopic imaging system and method to achieve both high spatial resolution and high temporal resolution super-resolution imaging.

To achieve the above object, an embodiment provides a polarization modulation super-resolution microscopic imaging system based on a DMD, which includes a polarization modulation unit based on an electro-optical modulator, an illumination modulation unit based on a DMD, a microscopic imaging unit based on a detector, a data acquisition control unit, and an imaging calculation unit;

the polarization modulation unit modulates laser based on the electro-optical modulator and outputs polarized light;

the illumination modulation unit modulates polarized light based on the DMD and generates reflected light of a dot discrete array pattern;

the microscopic imaging unit irradiates reflected light of the dot discrete array pattern to a target object to form a dot discrete excitation area, and a target sample in the dot discrete excitation area is excited by the irradiated light to generate fluorescence, and the fluorescence is received and imaged by the detector;

the data acquisition control unit controls the DMD to perform illumination modulation of the dot discrete array pattern, controls the electro-optical modulator to perform polarization modulation of n polarization modulation periods in each illumination modulation, and controls the detector to acquire fluorescence generated by a target sample during each polarization modulation to form a fluorescence imaging array;

the imaging calculation unit locates all single-molecule positions of each point discrete excitation area based on the fluorescence imaging array corresponding to each point discrete excitation area, and obtains super-resolution imaging images by superposing the single-molecule positions of all point discrete excitation areas.

Preferably, the polarization modulation unit further comprises a laser, a linear polarizer and a quarter wave plate, wherein a light transmission axis of the linear polarizer is arranged along the horizontal direction, a crystal optical axis direction of the electro-optic modulator is arranged at an included angle of 45 degrees with the horizontal direction, a quarter wave plate fast axis is arranged along the horizontal direction, and laser beams emitted by the laser sequentially pass through the linear polarizer, the electro-optic modulator and the quarter wave plate and are modulated to output polarized light.

Preferably, the illumination modulation unit further comprises a beam expansion assembly, a third lens, a dichroic mirror and an objective lens, the polarized light emitted by the polarization modulation unit is expanded by the beam expansion assembly, the expanded beam fully irradiates the micro mirror array of the DMD, the reflected light with a point discrete array pattern is generated after the micro mirror array is modulated, the reflected light is converged by the third lens and then reflected to the back focal plane of the objective lens by the dichroic mirror, and the emergent light passing through the objective lens is irradiated to the target sample in parallel.

Preferably, the beam expanding assembly comprises a first lens and a second lens.

Preferably, the microscopic imaging unit further comprises an objective lens, an optical filter and an imaging lens, wherein the target sample emits fluorescence after being excited by emergent light of the objective lens, and the fluorescence is received by the detector after passing through the objective lens, the dichroic mirror optical filter and the imaging lens in sequence.

Preferably, the data acquisition control unit modulates the polarized light direction by controlling the voltage of the electro-optical modulator, changes the light polarization direction by 180 degrees into one polarization modulation period, and realizes high-speed polarization modulation by controlling the voltage of the electro-optical modulator.

Preferably, the DMD includes a micromirror array composed of micromirror units, each micromirror unit has an on state and an off state, when the micromirror unit is in the off state, reflection of polarized light cannot be achieved, when the micromirror unit is in the on state, reflection of polarized light is achieved, and the data acquisition control unit controls a dot discrete array pattern composed of the micromirror units and reflection of polarized light by the dot discrete array pattern by controlling the on state or the off state of each micromirror unit.

Preferably, when the data acquisition control unit is used for controlling the reflected light of the DMD emergent point discrete array patterns, the reflected light of each point discrete array pattern is ensured to irradiate the target sample, the point discrete excitation areas formed by the target sample are mutually independent, and no overlap occurs within the diffraction limit.

Preferably, the imaging calculation unit adopts a molecular positioning algorithm to position all single molecule positions of each point discrete excitation area based on a fluorescence imaging array corresponding to each point discrete excitation area, and the method comprises the following steps:

and defining an optimal molecular concentration range, and calculating the position of a single molecule in the area according to the fluorescence intensity change sequences of a plurality of polarization modulation periods of the point discrete excitation area when the molecular concentration in the point discrete excitation area is in the optimal molecular concentration range.

Based on the same inventive concept, the embodiment also provides a polarization modulation super-resolution microscopic imaging method based on the DMD, wherein the method is applied to the polarization modulation super-resolution microscopic imaging system, and the method comprises the following steps of:

step 1, modulating laser and outputting polarized light by controlling the voltage of a polarization modulation unit by using a data acquisition control unit;

step 2, modulating polarized light and generating reflected light of a dot discrete array pattern by utilizing a data acquisition control unit, and outputting a first feedback signal to the data acquisition control unit after the dot discrete array pattern is output by the DMD;

step 3, after receiving the first feedback signal, using a data acquisition control unit to control an electro-optical modulator to carry out polarization modulation of n polarization modulation periods, and controlling a detector to acquire fluorescence generated by a point discrete excitation area of a target sample during each polarization modulation to form a fluorescence imaging array;

step 4, positioning all single-molecule positions of each point discrete excitation area based on a fluorescence imaging array corresponding to each point discrete excitation area by using an imaging calculation unit, and obtaining a super-resolution imaging image by superposing the single-molecule positions of all point discrete excitation areas;

step 5, after the detector collects fluorescence generated by the point discrete excitation areas of the target sample during n polarization modulation, outputting a second feedback signal to the data collection control unit;

and 6, after receiving the second feedback signal, the data acquisition control unit modulates polarized light and generates reflected light of another point discrete array pattern by controlling the DMD, the DMD outputs the first feedback signal to the data acquisition control unit after outputting the other point discrete array pattern, and the step 3 is executed in a jumping manner until the polarization modulation of all the micromirror units in the DMD is completed in a circulating manner.

Compared with the prior art, the invention has the beneficial effects that at least the following steps are included:

according to the polarization modulation super-resolution microscopic imaging system and method provided by the invention, the polarization modulation of the photoelectric modulator is combined with the excitation illumination modulation of the light reflected by the point array discrete pattern of the DMD, so that the sparse imaging of the target sample can be realized, and the time resolution and the spatial resolution of the imaging can be improved by the polarization modulation molecular positioning mode based on the DMD. The system and the method have very important significance for the exploration of biological cells with high spatial resolution and dynamic imaging, and meanwhile, the super-resolution imaging technology with high spatial and temporal resolution is beneficial to the exploration and discovery of a plurality of leading-edge fields such as life science, material science and the like.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required in the embodiments or the description of the prior art will be briefly described, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

Fig. 1 is a schematic structural diagram of a polarization modulation super-resolution microscopic imaging system based on DMD according to an embodiment of the present invention;

FIG. 2 is a schematic diagram of a discrete array pattern of DMD dots provided by an embodiment of the present invention;

FIG. 3 is a flow chart of a DMD-based polarization modulation super-resolution microscopic imaging method according to an embodiment of the present invention;

FIG. 4 is a schematic diagram of fluorescence image generated by excitation of a discrete array pattern of DMD dots provided by an embodiment of the present invention;

FIG. 5 is a schematic illustration of a polarization modulated imaging image under excitation of a discrete array pattern of DMD dots provided by an embodiment of the present invention;

FIG. 6 is a development flow of a molecular localization algorithm provided by an embodiment of the present invention;

FIG. 7 is a schematic diagram of a data acquisition control flow provided by an embodiment of the present invention;

in fig. 1: 1-laser, 2-linear polaroid, 3-electrooptical modulator, 4-quarter wave plate, 5-first lens, 6-second lens, 7-DMD, 8-third lens, 9-dichroic mirror, 10-objective lens, 11-optical filter, 12-imaging lens, 13-detector, 14-data acquisition control unit;

Detailed Description

The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the detailed description is presented by way of example only and is not intended to limit the scope of the invention.

The embodiment provides a polarization modulation super-resolution microscopic imaging system based on a DMD, which combines DMD illumination modulation and polarization modulation, controls the discrete excitation of a target sample according to the DMD illumination modulation, modulates the polarization direction of incident light at the same time, and further carries out polarization modulation on a discrete excitation area of the target sample; and a polarization modulation super-resolution imaging molecular positioning algorithm based on the DMD is developed, and on the basis of guaranteeing high time resolution, the spatial resolution is improved, so that super-resolution imaging with high spatial resolution and high time resolution is realized.

As shown in fig. 1, the DMD-based polarization modulation super-resolution microscopic imaging system provided in the embodiment includes a polarization modulation unit, an illumination modulation unit, a microscopic imaging unit, a data acquisition control unit, and an imaging calculation unit, where a solid line portion is an optical path portion, and a dotted line is a circuit portion.

In the embodiment, the polarization modulation unit modulates laser light and outputs polarized light based on the electro-optical modulator, specifically, the polarization modulation unit comprises a laser 1, a linear polarizer 2, an electro-optical modulator 3 and a quarter wave plate 4, a light transmission axis of the linear polarizer 2 is placed along a horizontal direction, a crystal optical axis direction of the electro-optical modulator 3 is placed at an included angle of 45 degrees with the horizontal direction, a fast axis of the quarter wave plate 4 is placed along the horizontal direction, and a laser beam emitted by the laser 1 sequentially passes through the linear polarizer 2, the electro-optical modulator 3 and the quarter wave plate 4, and the voltage of the electro-optical modulator 3 is controlled to modulate the polarized light direction, so that rapid polarization modulation of kilohertz frequency can be realized.

In an embodiment, the illumination modulation unit modulates polarized light based on the DMD and generates reflected light of a pattern of discrete arrays of dots. Specifically, the illumination modulation unit includes a first lens 5, a second lens 6, a DMD7, a third lens 8, a dichroic mirror 9, an objective lens 10, and the transmission light path of the quarter wave plate 4 passes through the first lens 5, the second lens 6, the DMD7, and the third lens 8 in this order. The first lens 5 and the second lens 6 are combined as a beam expanding assembly to realize beam expansion, and the expanded beam fully irradiates the micromirror array of the DMD 7. The reflected light of the DMD7 is converged by the third lens 8 and then reflected to the back focal plane of the objective lens 10 by the dichroic mirror 9, and the emergent light passing through the objective lens 10 is irradiated to the target sample in parallel, so that the wide-field illumination of the target sample is realized, and a point discrete excitation area is formed on the target sample.

As shown in fig. 2, the DMD7 includes a micromirror array composed of micromirror units, each of which has a high-speed reflection function and has an on state and an off state, when the micromirror unit is in the off state, incident polarized light cannot enter the micromirror unit, and thus cannot be reflected, and when the micromirror unit is in the on state, the incident polarized light enters the micromirror unit and is reflected to a subsequent optical path, and based on the characteristics, the data acquisition control unit controls the dot discrete array pattern composed of the micromirror units and the reflection of the dot discrete array pattern on the polarized light by controlling the on state or the off state of each of the micromirror units, and thus can control the reflected light of the dot discrete array pattern to the discrete illumination excitation region of the target sample.

The beam expansion ratio of the combination of the first lens 5 and the second lens 6 is determined according to the light emergent caliber of the laser 1 and the size of the DMD7, the selection of the dichroic mirror 9 is determined according to the laser wavelength, and the amplification ratio of the objective lens 10 is determined according to the size of the excitation area corresponding to each micro-mirror unit in the DMD 7.

In an embodiment, the microscopic imaging unit irradiates reflected light of the dot discrete array pattern to the target object to form a dot discrete excitation area, the target sample in the dot discrete excitation area is excited by the irradiated light to generate fluorescence, the fluorescence is received and imaged by the detector, specifically, the microscopic imaging unit comprises an objective lens 10, a light filter 11, an imaging lens 12 and a detector 13, the target sample irradiates to form the dot discrete excitation area through the emergent light of the objective lens 10, the target sample in the dot discrete excitation area is excited by the irradiated light to generate fluorescence, and the fluorescence sequentially passes through the objective lens 10, the dichroic mirror 9, the light filter 11 and the imaging lens 12 to the detector 13 and is received, so that a fluorescence imaging array is formed. Wherein the selection of the filter 11 is determined according to the fluorescence wavelength, and the magnification of the objective lens 10 and the focal length of the imaging lens 12 jointly determine the imaging size.

In the embodiment, the data acquisition control unit 14 is connected with the electro-optical modulator 3, the DMD7 and the detector 13 through a circuit and is used for synchronously controlling the polarization modulation unit, the illumination modulation unit and the microscopic imaging unit, namely, the synchronous regulation and control of the DMD illumination modulation and the polarization modulation are completed, and the fluorescent imaging array is synchronously acquired; the method specifically comprises the following steps: the DMD7 is controlled to perform illumination modulation of the dot discrete array pattern, and within each illumination modulation, the electro-optical modulator 3 is controlled to perform polarization modulation of n polarization modulation periods, and the detector 13 is controlled to collect fluorescence generated by the target sample at each polarization modulation, so as to form a fluorescence imaging array.

When the data acquisition control unit 14 controls illumination modulation, reflected light of the discrete point array pattern is output by controlling the on state or the off state of the micromirror units in the DMD7, when the current discrete illumination modulation is completed, all the micromirror units are turned off, the next discrete illumination modulation is performed, and the above process is repeated until all the micromirror units of the DMD7 complete the on control. When the data acquisition control unit 14 controls the DMD7, as shown in fig. 3, wherein black represents that the micromirror unit in the DMD7 is currently in an on state, white represents that the micromirror unit in the DMD7 is always in an off state, and gray represents that the micromirror unit in the DMD has completed once the on state and is currently in the off state.

When the data acquisition control unit is used for controlling the reflected light of the DMD emergent point discrete array patterns, the reflected light of each point discrete array pattern is ensured to irradiate the target sample, the point discrete excitation areas formed by the target sample are mutually independent, and no overlap occurs in the diffraction limit.

When the DMD7 outputs the reflected light of the discrete dot array pattern once, the data acquisition control unit completes the polarized light direction modulation of n polarized modulation periods by controlling the voltage of the electro-optical modulator 3, wherein one polarized modulation period refers to the change of 180 ° of the polarized light direction, and realizes the high-speed polarized modulation by controlling the voltage of the electro-optical modulator.

In an embodiment, the imaging calculation unit adopts a molecular positioning algorithm to position all single-molecule positions of each point discrete excitation area based on a fluorescence imaging array corresponding to each point discrete excitation area, and specifically includes: an optimal molecular concentration range is defined, wherein the molecular concentration is the number of molecules in unit volume, and the number of molecules corresponding to each point discrete excitation area can be estimated according to the molecular concentration. And each polarization modulation corresponds to one fluorescence imaging array, a plurality of polarization modulation periods form a group of fluorescence imaging sequences, and when the molecular concentration in the point discrete excitation area is in the optimal molecular concentration range, the position of a single molecule in the area is calculated according to the fluorescence intensity change sequences of the plurality of polarization modulation periods of the point discrete excitation area. The specific analysis can adopt a multi-peak Gaussian fitting mode, namely, the position of a single molecule in the region is calculated by adopting the multi-peak Gaussian fitting mode according to the fluorescence intensity change sequence. Each time the sample area is excited by the dot discrete array pattern, the single-molecule position of the current dot discrete excitation area can be calculated, and then super-resolution imaging images are obtained by superposing the single-molecule positions of all the dot discrete excitation areas.

The embodiment also provides an imaging method using the polarization modulation super-resolution microscopic imaging system, as shown in fig. 3, comprising the following steps:

step 1, the data acquisition control unit 14 is used for modulating laser and outputting polarized light by controlling the voltage of the polarization modulation unit.

In the polarization modulation list, the laser 1 emits laser, the laser is transmitted out through the linear polarizer 2, the electro-optical modulator 3 and the quarter wave plate 4 to serve as polarized light, and meanwhile, the data acquisition control unit 14 controls the voltage of the electro-optical modulator 3 to realize the polarization direction modulation;

step 2, the data acquisition control unit 14 is utilized to modulate polarized light and generate reflected light of the dot discrete array pattern by controlling the DMD, and the DMD outputs a first feedback signal to the data acquisition control unit 14 after outputting the dot discrete array pattern.

In the illumination modulation unit, polarized light is amplified to a suitable aperture by selecting focal lengths of the first lens 5 and the second lens 6 to fully illuminate the micromirror array of the DMD7, and at the same time, reflected light of a dot discrete array pattern is output from the DMD7 by the data acquisition control unit 14 and a first feedback signal is output to the data acquisition control unit 14.

And 3, after receiving the first feedback signal, using the data acquisition control unit 14 to control the electro-optical modulator to perform polarization modulation of n polarization modulation periods, and controlling the detector to acquire fluorescence generated by the point discrete excitation area of the target sample during each polarization modulation, so as to form a fluorescence imaging array.

In the microscopic imaging unit, the reflected light is converged to the back focal plane of the objective lens 10 after passing through the third lens 8 and the dichroic mirror 9, and is irradiated to the target sample in parallel, the target sample emits fluorescence after being excited by the reflected light of the parallel wide-field point discrete array pattern, and the fluorescence is received by the detector 13 after passing through the objective lens 10, the dichroic mirror 9, the optical filter 11 and the imaging lens 12 in sequence, and forms a fluorescence imaging array.

The image collected by the detector 13 is a sample fluorescent image generated by illumination excitation of discrete points, referring to fig. 4, the discrete points are mutually independent and do not overlap in the diffraction limit, so that sparse imaging of a sample area can be primarily realized by illumination modulation of a point array discrete pattern emitted by the DMD 7.

On the basis of sparse imaging generated by point discrete array pattern excitation, polarization modulation is performed, in order to realize rapid light polarization direction modulation, an electro-optical modulator 3 modulation voltage method is adopted, so that polarization direction modulation of kilohertz frequency can be realized, n-period polarization direction modulation is performed under the condition of each point discrete array pattern excitation, and single-molecule intensity modulation can be further realized for a sparse region, referring to fig. 5.

And 4, positioning all single-molecule positions of each point discrete excitation area based on the fluorescent imaging array corresponding to each point discrete excitation area by using an imaging calculation unit, and obtaining a super-resolution imaging image by superposing the single-molecule positions of all point discrete excitation areas.

For fluorescence imaging sequences corresponding to a plurality of polarization modulation periods under the excitation of each point discrete array pattern, a molecular positioning algorithm is developed, an optimal concentration range of molecules is defined through simulation analysis, and the feasibility of the algorithm is verified through experiments when the molecular positioning position is judged to be consistent with the theoretical position, wherein the algorithm flow is shown in fig. 6.

After the molecular positioning algorithm is developed, positioning all single molecule positions of each point discrete excitation area based on the fluorescence imaging array corresponding to each point discrete excitation area by using the molecular positioning algorithm, wherein the method comprises the following steps: when the concentration of the molecules in the point discrete excitation area is in the optimal molecular concentration range, the position of a single molecule in the area is calculated according to the fluorescence intensity change sequences of a plurality of polarization modulation periods of the point discrete excitation area.

And step 5, outputting a second feedback signal to the data acquisition control unit after the detector acquires fluorescence generated by the point discrete excitation area of the target sample during n polarization modulation.

And 6, after receiving the second feedback signal, the data acquisition control unit modulates polarized light and generates reflected light of another point discrete array pattern by controlling the DMD, the DMD outputs the first feedback signal to the data acquisition control unit after outputting the other point discrete array pattern, and the step 3 is executed in a jumping manner until the polarization modulation of all the micromirror units in the DMD is completed in a circulating manner.

The data acquisition control unit 14 realizes the illumination modulation of the DMD7, the polarization modulation of the electro-optical modulator 3 and the synchronous triggering control of the detector 13, the data acquisition control unit 14 acquires the input and feedback signals of the electro-optical modulator 3 and the detector 13, so that the detector 13 acquires once every time the electro-optical modulator 3 is modulated, the polarization modulation is finished after the modulation reaches a set target period value, a second feedback signal is sent to the data acquisition control unit 14, the DMD7 carries out the polarization modulation of the next dot discrete array pattern, the modulation process is as described above until the polarization modulation of all the micromirror units in the DMD7 is circularly finished, and the control process of the data acquisition unit 14 is shown in fig. 7.

According to the polarization modulation super-resolution microscopic imaging system and the method, the DMD discrete illumination modulation is combined with the incident light polarization modulation, the point discrete array pattern emitted by the DMD is actively regulated and controlled to realize the discrete excitation of a target sample, the sample imaging areas excited by the light source are controlled to be mutually independent, the polarization light direction is modulated in each discrete excitation process, the polarization modulation is further realized on the discrete excitation areas, the polarization modulation molecular positioning algorithm based on the DMD is developed, the spatial resolution is obviously improved on the basis of guaranteeing the high time resolution, and the super-resolution imaging requirement of the high time-space resolution is met. The system introduces a polarization modulation optical element into an incident light path to realize the polarization direction modulation of incident light; meanwhile, a DMD device for illumination modulation is arranged in an incident light path to realize discrete excitation of a sample area; the polarization imaging of the discrete excitation area of the sample is controlled by combining the polarization modulation optical element and the DMD device, so that dynamic and high-spatial resolution super-resolution imaging can be realized, and the method has very important scientific significance and wide application prospect for exploring and discovering numerous front-edge fields such as life science, material science and the like.

The foregoing detailed description of the preferred embodiments and advantages of the invention will be appreciated that the foregoing description is merely illustrative of the presently preferred embodiments of the invention, and that no changes, additions, substitutions and equivalents of those embodiments are intended to be included within the scope of the invention.

Claims (10)

1. The polarization modulation super-resolution microscopic imaging system based on the DMD is characterized by comprising a polarization modulation unit based on an electro-optic modulator, an illumination modulation unit based on the DMD, a microscopic imaging unit based on a detector, a data acquisition control unit and an imaging calculation unit;

the polarization modulation unit modulates laser based on the electro-optical modulator and outputs polarized light;

the illumination modulation unit modulates polarized light based on the DMD and generates reflected light of a dot discrete array pattern;

the microscopic imaging unit irradiates reflected light of the dot discrete array pattern to a target object to form a dot discrete excitation area, and a target sample in the dot discrete excitation area is excited by the irradiated light to generate fluorescence, and the fluorescence is received and imaged by the detector;

the data acquisition control unit controls the DMD to perform illumination modulation of the dot discrete array pattern, controls the electro-optical modulator to perform polarization modulation of n polarization modulation periods in each illumination modulation, and controls the detector to acquire fluorescence generated by a target sample during each polarization modulation to form a fluorescence imaging array;

the imaging calculation unit locates all single-molecule positions of each point discrete excitation area based on the fluorescence imaging array corresponding to each point discrete excitation area, and obtains super-resolution imaging images by superposing the single-molecule positions of all point discrete excitation areas.

2. The DMD-based polarization-modulating super-resolution microscopic imaging system according to claim 1, wherein the polarization-modulating unit further comprises a laser, a linear polarizer and a quarter-wave plate, wherein the light transmission axis of the linear polarizer is arranged along the horizontal direction, the crystal optical axis of the electro-optic modulator is arranged at an included angle of 45 degrees with the horizontal direction, the fast axis of the quarter-wave plate is arranged along the horizontal direction, and the laser beam emitted by the laser sequentially passes through the linear polarizer, the electro-optic modulator and the quarter-wave plate and is modulated to output polarized light.

3. The DMD-based polarization modulation super-resolution microscopic imaging system of claim 1, wherein the illumination modulation unit further comprises a beam expanding assembly, a third lens, a dichroic mirror and an objective lens, the polarized light emitted by the polarization modulation unit is expanded by the beam expanding assembly, the expanded beam is fully irradiated to the micromirror array of the DMD, the reflected light with a dot discrete array pattern is generated after being modulated by the micromirror array, the reflected light is converged by the third lens and then reflected to the back focal plane of the objective lens by the dichroic mirror, and the emergent light passing through the objective lens is irradiated to the target sample in parallel.

4. The DMD-based polarization-modulated super-resolution microscopy imaging system of claim 3, wherein the beam expanding assembly comprises a first lens and a second lens.

5. The DMD-based polarization-modulating super-resolution microscopic imaging system of claim 1, wherein the microscopic imaging unit further comprises an objective lens, an optical filter, and an imaging lens, wherein the object sample emits fluorescence after being excited by the outgoing light of the objective lens, and the fluorescence is received by the detector after passing through the objective lens, the dichroic mirror optical filter, and the imaging lens in sequence.

6. The DMD-based polarization-modulating super-resolution microscopic imaging system of claim 1, wherein the data acquisition control unit modulates the polarization direction of the polarized light by controlling the voltage of the electro-optical modulator, and changes the polarization direction of the light by 180 ° to one polarization modulation period, and realizes high-speed polarization modulation by controlling the voltage of the electro-optical modulator.

7. The DMD-based polarization-modulating super-resolution microscopic imaging system according to claim 1, wherein the DMD comprises a micromirror array composed of micromirror units, each micromirror unit has an on state and an off state, reflection of polarized light cannot be achieved when the micromirror unit is in the off state, reflection of polarized light is achieved when the micromirror unit is in the on state, and the data collection control unit controls the dot discrete array pattern composed of the micromirror units and reflection of polarized light by the dot discrete array pattern by controlling the on state or the off state of each micromirror unit.

8. The DMD-based polarization-modulating super-resolution microscopy imaging system of claim 1 or 7, wherein when the data acquisition control unit controls the reflected light of the DMD exit point discrete array pattern, it is ensured that the reflected light of each point discrete array pattern irradiates the target sample and the point discrete excitation regions formed by the target sample are independent of each other, and do not overlap within the diffraction limit.

9. The DMD-based polarization-modulated super-resolution microscopic imaging system of claim 1 or 7, wherein the imaging computational unit employs a molecular positioning algorithm to position all single molecule positions of each point discrete excitation region based on a corresponding fluorescent imaging array of each point discrete excitation region, comprising:

and defining an optimal molecular concentration range, and calculating the position of a single molecule in the area according to the fluorescence intensity change sequences of a plurality of polarization modulation periods of the point discrete excitation area when the molecular concentration in the point discrete excitation area is in the optimal molecular concentration range.

10. A DMD-based polarization-modulating super-resolution microscopy imaging method, characterized in that it employs a polarization-modulating super-resolution microscopy imaging system according to any one of claims 1-9, comprising the steps of:

step 1, modulating laser and outputting polarized light by controlling the voltage of a polarization modulation unit by using a data acquisition control unit;

step 2, modulating polarized light and generating reflected light of a dot discrete array pattern by utilizing a data acquisition control unit, and outputting a first feedback signal to the data acquisition control unit after the dot discrete array pattern is output by the DMD;

step 3, after receiving the first feedback signal, using a data acquisition control unit to control an electro-optical modulator to carry out polarization modulation of n polarization modulation periods, and controlling a detector to acquire fluorescence generated by a point discrete excitation area of a target sample during each polarization modulation to form a fluorescence imaging array;

step 4, positioning all single-molecule positions of each point discrete excitation area based on a fluorescence imaging array corresponding to each point discrete excitation area by using an imaging calculation unit, and obtaining a super-resolution imaging image by superposing the single-molecule positions of all point discrete excitation areas;

step 5, after the detector collects fluorescence generated by the point discrete excitation areas of the target sample during n polarization modulation, outputting a second feedback signal to the data collection control unit;

and 6, after receiving the second feedback signal, the data acquisition control unit modulates polarized light and generates reflected light of another point discrete array pattern by controlling the DMD, the DMD outputs the first feedback signal to the data acquisition control unit after outputting the other point discrete array pattern, and the step 3 is executed in a jumping manner until the polarization modulation of all the micromirror units in the DMD is completed in a circulating manner.

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