CN115308184A - A method and system for super-resolution microscopy imaging with active structured light illumination - Google Patents

Abstract

The invention discloses a super-resolution microscopic imaging method and system for active structured light illumination. A spatiotemporal joint intensity modulation method is proposed, which can control the intensity of the excitation light of the illuminated sample at any spatial level, and realize the super-resolution imaging of active structured light illumination with a high modulation degree without changing the coherence of the incident light. Based on the above modulation method, the input non-uniform Gaussian distribution illumination can be modulated into uniform flat-top illumination, which meets the application requirements based on quantitative analysis of fluorescence intensity; the illumination intensity is actively adjusted according to the spatial intensity distribution characteristics of the sample to meet the requirements of high dynamic and low illumination. Dose imaging requirements; user-defined lighting area to meet the application requirements of photostimulation and fluorescence bleaching recovery. The present invention can effectively improve the imaging dynamic range of SIM and reduce the light dose without losing the temporal and spatial resolution of SIM, so as to meet the requirements of cell substructure and its interaction for super-resolution, extremely low light dose and high dynamic range of less than 100 nanometers. imaging needs.

Description

A super-resolution microscopy imaging method and system for active structured light illumination

technical field

The invention belongs to the technical field of fluorescence microscopic imaging, in particular to a super-resolution microscopic imaging system combining active lighting and structured light lighting.

Background technique

A cell is a highly complex dynamic system, in which dozens of organelles with different functions are formed through the compartmentalization of the membrane structure. The interaction of these organelles is the key to maintaining cell function and determining cell fate. The study of these organelles and their interactions requires live cell imaging with high resolution below 100 nanometers, extremely low light dose, high dynamic range, and large field of view with uniform illumination.

In the study of obtaining uniform illumination of a large field of view, the beam shaper directly converts the Gaussian propagating beam into a flat-hat beam, using a pair of aspheric lens groups, the first lens uniformly redistributes the Gaussian beam, and the second lens redistributes the Gaussian beam. collimated, resulting in flat-field illumination. However, based on the limited working distance of the refractive beam shaper, there are strict requirements on the surface processing quality and optical arrangement. Optical waveguides can provide uniform illumination over a very large field of view, but because they operate in total internal reflection mode, the illumination is limited to the vicinity of the cover glass, making it impossible to switch illumination angles to achieve different depths of illumination. Moreover, the fixed illumination size cannot match the illumination with the imaging field of view of the camera, and the whole slide illumination will cause unnecessary light damage to the cell sample. In addition, classical methods include using a pair of microlens arrays or multimode optical fibers. In order to reduce laser speckle, speckle reducers or vibrating optical fibers are added to the system, but the spatial coherence of the beam is reduced, and it is not suitable for total internal reflection. illumination. ASTER is essentially a hybrid scanning wide-field illumination device, which scans Gaussian beams in a specific pattern and provides flat-top illumination in a time-averaged manner, so that a uniform illumination field can be scanned in a relatively wide field of view. However, the above-mentioned flat-field illumination method can only be applied to super-resolution microscopy based on single-molecule localization, such as PALM and STORM, and its transformation to structured light illumination microscopy (SIM) based on multi-beam interference to generate cosine distribution fringes is still limited. , the reason is that when modulating the incident non-uniform Gaussian spot, it is necessary to ensure the same frequency, constant phase difference, and constant polarization direction among the multiple beams, so that high-modulation illumination fringes can be formed by interference on the sample surface.

In the study of expanding the dynamic range of the microscopic imaging system and reducing the light dose, Vinergoni et al. combined the multi-exposure fusion technology widely used in photography with the two-photon imaging technology based on point scanning, and applied it to large dynamic scenes in interior scenes. range of neuronal imaging. However, continuous multiple exposure acquisition not only increases the light dose, but also reduces the imaging speed. In order to overcome the defects of multi-exposure, multi-detection simultaneous imaging technology has been developed, which collects images with different exposures at the same time through multi-level spectroscopic imaging of the same scene. However, since multiple imaging optical paths are used for simultaneous detection, different detectors need to be calibrated, and the optical system is complex and expensive. As a result, active lighting technology has been developed. Acousto-optic modulators are added to the imaging system to regulate the lighting light field in a single pixel in space, which not only improves the dynamic range, but also reduces the light dose. In order to solve the problems of limited dynamic range, photobleaching and phototoxicity of confocal microscope, Hoebe et al. developed a microscopic imaging technology with spatially adjustable illumination light dose, and applied it on Nikon C1 confocal microscope. Experiments have proved that this imaging technology can not only increase the dynamic range by 2 times, but also reduce the light dose by 5 times, effectively prolonging the survival time of living cells. However, the resolution of confocal, two-photon-based low-light-dose, high-dynamic imaging methods is limited by the optical diffraction limit. In the case of using a large numerical aperture objective lens, the lateral limiting resolution is about 200nm and the axial resolution is 500nm. In addition, based on the point-scanning imaging mode, photobleaching and phototoxicity are serious and the imaging speed is slow, which cannot meet the needs of dynamic imaging of living cells.

In contrast, SIM has the characteristics of fast imaging speed, low light damage, no special requirements for fluorescent probes, etc., and the resolution of hundreds of nanometers just meets the observation requirements of important organelles in living cells, and has become the first choice for dynamic imaging of living cells. choose. However, from the perspective of illumination method, SIM is essentially a wide-field fluorescence microscopy imaging technique, and its overall illumination intensity presents a Gaussian shape distribution on the sample. This non-uniform illumination method first reduces the available field of view size. In order to obtain a relatively uniform fluorescence image, the central area of the Gaussian spot is usually intercepted for imaging, thus losing the peripheral field of view. Secondly, Gaussian illumination with bright center and dark edge causes inconsistent photobleaching characteristics in the imaging field of view, and the center is more likely to be bleached than the edge, which limits its application in quantitative analysis based on fluorescence intensity. In addition, for high dynamic range samples, this indiscriminate illumination method regardless of the sample label density will cause underexposure of weak signals, overexposure of strong signals, and invalid exposure of background and out-of-focus signals, which limits the dynamic range and cause unnecessary photodamage. Therefore, an active structured light illumination super-resolution imaging method (Active-SIM) based on the spatial-temporal joint modulation of the spatial light modulator is invented, such as modulating the Gaussian spot into flat-field illumination, adaptively adjusting the illumination intensity according to the sample mark density, and for any Illumination of the region of interest, etc., is crucial for expanding the application of SIM imaging technology in dynamic long-term imaging of living cells.

Contents of the invention

The purpose of the present invention is to meet the requirements of fluorescent imaging with high resolution below 100 nanometers, extremely low light dose, high dynamic range, and uniform illumination of large field of view in the study of subcellular organelles and their interactions in living cells. The spatial-temporal joint intensity modulation method of the spatial light modulator is used to establish a super-resolution microscopy imaging system based on active structured light illumination.

The technical solution for realizing the object of the present invention is: a super-resolution microscopic imaging method of active structured light illumination, said method comprising the following steps:

Step 1, collect the fluorescence image of the sample excited by the laser through the camera;

Step 2, constructing the required active structured light illumination light field based on the image in step 1;

Step 3, converting the active structured light illumination light field in step 2 and loading it into the spatial light modulator to form an active structured light illumination light field modulated by combined spatiotemporal intensity;

Step 4, take out the corresponding bit-plane map from the holographic fringe bit-plane map loaded in the spatial light modulator in step 3, and start to display, the laser emits light to illuminate the sample synchronously, and the camera synchronously exposes to collect the original image data;

Step 5, judge whether the display of all holographic fringe bit-plane images is completed, if so, the spatial light modulator stops the bit-plane display, and the camera stops image acquisition, otherwise, switch to the bit-plane images with different spatial orientation angles and phases according to the preset order, Go back to step 4 and continue to perform synchronous display and exposure.

Further, the active structured light illumination light field described in step 2 includes:

The first illumination light field: modulate the input non-uniform Gaussian distribution light field into a uniform flat-top illumination light field to meet the needs of quantitative analysis based on fluorescence intensity and large field of view stitching imaging;

Or, the second lighting light field: actively adjust the lighting intensity according to the sample intensity distribution characteristics, including reducing the light dose in the strong signal area, increasing the light dose in the weak signal area, no signal or no light in the background area, to meet the required high dynamic range, low Imaging requirements of light dose;

Or, the third illumination light field: customize the illumination at the specified position and intensity to meet the imaging needs of the user-defined illumination area of interest.

Further, the spatial distribution of light intensity of the modulated active structured illumination light field in step 3 is expressed as I _a (x, y):

点(x,y)代表空间光调制器中任意空间坐标；m表示结构光的调制度，k_x、k_y、φ表示空间频率和初始相位。In the formula, I _g (x, y, t) represents the space-time volume of the illumination intensity of the excitation light on the sample surface within a camera exposure period T. In the SIM imaging process when only one order beam is allowed to pass through, its The illumination intensity presents a non-uniform Gaussian distribution in space; M _i (x, y, t) represents the i-th intensity modulation function or binary plane loaded in the spatial light modulator, and N represents the loading of the binary plane number, the light intensity modulation accuracy of each pixel on the spatial light modulator can be expressed as 1/2 ^N ; t _i represents the time weight function corresponding to each binary bit plane, and the sum of all time weights is an exposure of the camera cycle

The point (x, y) represents any spatial coordinate in the spatial light modulator; m represents the modulation degree of the structured light, and k _x , _ky , φ represent the spatial frequency and initial phase.

A super-resolution microscopic imaging system for active structured light illumination that implements the above method, the system includes a light source module, an active structured light illumination light field loading module, an active structured light illumination light field generation module, and a fluorescence detection module;

The light source module is used to realize the control of at least four laser wavelengths, and can realize single-wavelength independent illumination, multi-wavelength multi-color time-sharing and simultaneous illumination;

The fluorescence detection module is used to collect fluorescence images of samples excited by laser;

The active structured light illumination light field generating module is configured to generate an active structured light illumination light field based on the fluorescence image;

The active structured light illumination light field loading module is used to load the generated active structured light illumination light field into the SIM imaging system to complete active structured light illumination super-resolution imaging.

Further, the light source module includes a laser and a single-mode polarization-maintaining fiber;

The laser is used to provide multiple wavelengths of laser light, and can realize single-wavelength and multi-wavelength synchronous light output control;

The single-mode polarization-maintaining fiber is used to transmit the multi-channel laser coupled by the laser to the active structured light illumination light field loading module.

Further, the active structured light illumination light field loading module includes an achromatic collimating beam expander objective lens, a polarizing beam splitter, a spatial light modulator, a Fourier lens, a half slide, a spatial filter, a combined half A glass slide, collimating lens, and illuminating tube mirror; Fourier lens and collimating lens form a 4f system, the spatial light modulator is located at the front focal plane of the 4f system, and the spatial filter is located at the back focal plane of the 4f system for Spatial filtering, which only allows the light beam of a specific angle diffracted by the spatial light modulator to pass through, and blocks other diffraction orders; half of the glass and combined half of the glass constitute a polarization control component, which is used to control different direction angles The incident polarized light is adjusted;

The laser light emitted by the light source module is transmitted to the achromatic collimating beam expander objective lens, and then enters the polarization beam splitter prism after being collimated and expanded, and the transmitted vertically polarized light enters the spatial light modulator, and is mainly diffracted after being modulated by the spatial light modulator There are three output beams of 0th order and ±1st order. The three outgoing beams are reflected by the polarization beam splitter and then focused on the spatial filter through the Fourier lens and half of the glass. After filtering, the ±1st order is selected. The light beam enters the fluorescence detection module after being combined with one-half of the glass slide, collimating lens, and illuminating tube lens in sequence.

Further, the fluorescence detection module includes a three-dimensional translation stage, a microscope objective lens, a dichroic mirror, an imaging lens and a camera;

The excitation light output by the active structured light illumination light field loading module is reflected by the dichroic mirror and irradiates the sample placed on the three-dimensional displacement stage through the microscopic objective lens, and the fluorescent signal of the sample is collected by the microscopic objective lens after being excited, and then passed through the dichroic After the color mirror is transmitted, it is focused on the detection surface of the camera through the imaging lens.

Further, the active structured light illumination light field generation module includes an image acquisition unit, an image processing unit and a light field generation unit;

The image acquisition unit is used to acquire the illumination light field distribution characteristics of the sample surface detected by the camera;

The image processing unit is used to convert the illumination light field obtained by the image acquisition unit into the active structured light illumination light field required by the user;

The light field generation unit is used to convert the active structured light illumination light field into a set of binary bitmaps with different time weights based on the spatial-temporal joint intensity modulation that can be loaded by the spatial light modulator, and load it into the active structured light illumination light in the spatial light modulator in the field loading module.

Compared with the prior art, the present invention has the remarkable advantages of:

1) Realize active structured light illumination super-resolution imaging without destroying the wavefront, polarization state and coherence of the interference beam in the SIM imaging system, and ensuring high-modulation interference fringes on the sample surface.

2) On the premise of not reducing the temporal and spatial resolution of the traditional SIM imaging system, it has the following advantages: including the modulation of the input non-uniform Gaussian distribution illumination light field into a uniformly distributed flat-top illumination light field, which meets the requirements of quantitative analysis imaging based on fluorescence intensity According to the application requirements of the sample intensity spatial distribution characteristics, the illumination light intensity is actively adjusted to meet the application requirements of high dynamic range and low light dose imaging; the illumination at the specified position and intensity can be customized to meet the imaging requirements of the user-defined illumination area of interest. By providing these three types of active structured light illumination light fields, it provides advanced new imaging technology means for imaging of living cells with high resolution, extremely low light dose, high dynamic range, and large field of view uniform illumination.

3) In the original SIM imaging system, the present invention does not need to add additional optical hardware, but only needs to construct the required light field based on the space-time joint intensity modulation method proposed by the present invention, and obtains rich imaging functions without increasing economic costs. Simplify the complexity of the system.

4) The present invention can increase the imaging dynamic range of the traditional SIM by 40dB and reduce the light dose by 10 times without losing the temporal and spatial resolution of the SIM.

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

Description of drawings

Fig. 1 is a flowchart of a light field modulation method for active structured light illumination.

Fig. 2 is a schematic diagram of an active structured light illumination super-resolution microscopy imaging system.

Figure 3 is a schematic diagram of the light field modulation method for active structured light illumination, in which Figure 3(a) is a schematic diagram of pixel-level arbitrary light intensity modulation based on a ferroelectric liquid crystal spatial light modulator, and Figure 3(b) is a schematic diagram of the principle of spatiotemporal joint intensity modulation.

The reference signs in the figure are as follows: 1 four-way beam combining laser; 2 single-mode polarization-maintaining optical fiber; 3 achromatic collimating beam expander objective lens; 4 polarization beam splitting prism; 5 spatial light modulator; 6 Fourier lens; 1 slide; 8 spatial filter; 9 combined half slide; 10 collimator lens; 11 illumination tube mirror; 12 three-dimensional translation stage; 13 objective lens; 14 dichroic mirror; ; 17 image acquisition unit; 18 image processing unit; 19 light field generation unit.

Detailed ways

In order to make the purpose, technical solution and advantages of the present application clearer, the present application will be further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described here are only used to explain the present application, and are not intended to limit the present application.

It should be noted that if there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. Implying their relative importance or implying the number of technical features indicated. Thus, the features defined as "first" and "second" may explicitly or implicitly include at least one of these features. In addition, the technical solutions of the various embodiments can be combined with each other, but it must be based on the realization of those skilled in the art. When the combination of technical solutions is contradictory or cannot be realized, it should be considered that the combination of technical solutions does not exist , nor within the scope of protection required by the present invention.

In one embodiment, with reference to FIG. 1 , a super-resolution microscopic imaging method of active structured light illumination is provided, the method comprising the following steps:

Step 1, collect the fluorescence image of the sample excited by the laser through the camera;

Step 2, constructing the required active structured light illumination light field based on the image in step 1;

Step 3, converting the active structured light illumination light field in step 2 and loading it into the spatial light modulator to form an active structured light illumination light field modulated by combined spatiotemporal intensity;

Step 4, take out the corresponding bit-plane map from the holographic fringe bit-plane map loaded in the spatial light modulator in step 3, and start to display, the laser emits light to illuminate the sample synchronously, and the camera synchronously exposes to collect the original image data;

Step 5, judge whether the display of all holographic fringe bit-plane images is completed, if so, the spatial light modulator stops the bit-plane display, and the camera stops image acquisition, otherwise, switch to the bit-plane images with different spatial orientation angles and phases according to the preset order, Go back to step 4 and continue to perform synchronous display and exposure.

Further, in one of the embodiments, the active structured light illumination light field in step 2 includes:

The first illumination light field: modulate the input non-uniform Gaussian distribution light field into a uniform flat-top illumination light field to meet the needs of quantitative analysis based on fluorescence intensity and large field of view stitching imaging;

Or, the second lighting light field: actively adjust the lighting intensity according to the sample intensity distribution characteristics, including reducing the light dose in the strong signal area, increasing the light dose in the weak signal area, no signal or no light in the background area, to meet the required high dynamic range, low Imaging requirements of light dose;

Or, the third illumination light field: customize the illumination at the specified position and intensity to meet the imaging needs of the user-defined illumination area of interest.

Further, in one of the embodiments, in order to realize the active illumination structured light field with joint intensity modulation of time and space, the present invention utilizes ferroelectric liquid crystal-based spatial light modulation in which each independent pixel can be “on” and “off”. The fast switching feature realizes the adjustment of the illumination light intensity at the single pixel level within one camera integration cycle, as shown in Figure 3a. In the SIM imaging system, the illumination intensity on the sample surface presents a Gaussian distribution as a whole, which can be expressed as:

Since the polarization state of the incident light on each pixel in the ferroelectric liquid crystal spatial light modulator can be adjusted independently, combined with the polarization beam splitter prism, the "switching" operation of the light intensity can be realized, so that each time point can be adjusted during the exposure period of the camera. The lighting switch can realize the regulation of arbitrary light intensity. Therefore, the active illumination light field can be expressed as:

In order to convert to digital modulation, a digital pulse width modulation method is introduced. Assuming that N binary bit planes M _i (x, y, t) are loaded in the spatial light modulator, each bit plane is assigned a different lighting time weight t _i =(1/2 ⁱ )·T, as shown in Figure 3b . By continuously loading N binary bitmaps with different time weights within the exposure period T of a camera, the control of any light intensity of a single pixel in the incident light field can be realized, expressed as follows:

Therefore, the illumination intensity distribution of the modulated active structured light field in step 3 is expressed as I _a (x, y):

点(x,y)代表空间光调制器中任意空间坐标；m表示结构光的调制度，k_x、k_y、φ表示空间频率和初始相位。In the formula, I ₀ represents the peak intensity, σ is the beam waist radius of the Gaussian beam, I _g (x, y, t) represents the space-time volume of the illumination intensity of the excitation light on the sample surface within a camera exposure period T, in During the SIM imaging process when only one secondary beam is allowed to pass through, its illumination intensity presents a non-uniform Gaussian distribution in space; M _i (x, y, t) represents the ith intensity modulation loaded in the spatial light modulator function or binary bit plane, N represents the number of loaded binary bit planes, and the light intensity modulation accuracy of each pixel on the spatial light modulator can be expressed as 1/2 ^N ; t _i represents the corresponding The time weight function, all time weights sum to one exposure cycle of the camera

The point (x, y) represents any spatial coordinate in the spatial light modulator; m represents the modulation degree of the structured light, and k _x , _ky , φ represent the spatial frequency and initial phase.

In one embodiment, a super-resolution microscopic imaging system for active structured light illumination that implements the above method is shown in Figure 2, the system includes a light source module, an active structured light illumination light field loading module, an active structured light illumination light field generation module, and Fluorescence detection module;

The light source module is used to realize the control of at least four laser wavelengths, and can realize single-wavelength independent illumination, multi-wavelength multi-color time-sharing and simultaneous illumination;

The fluorescence detection module is used to collect fluorescence images of samples excited by laser;

The active structured light illumination light field generating module is configured to generate an active structured light illumination light field based on the fluorescence image;

The active structured light illumination light field loading module is used to load the generated active structured light illumination light field into the SIM imaging system to complete active structured light illumination super-resolution imaging.

Further, in one of the embodiments, the light source module includes a laser 1 and a single-mode polarization-maintaining fiber 2;

The laser 1 is used to provide multiple wavelengths of laser light, and can realize single-wavelength and multi-wavelength synchronous light output control;

The single-mode polarization-maintaining fiber 2 is used to transmit the multi-channel laser coupled by the laser 1 to the active structured light illumination light field loading module.

Here, preferably, the laser 1 provides laser light with four wavelengths, the four wavelengths are 405 nm, 488 nm, 561 nm, and 637 nm respectively.

Further, in one of the embodiments, the active structured light illumination light field loading module includes an achromatic collimating beam expander objective lens 3, a polarization beam splitter prism 4, a spatial light modulator 5, a Fourier lens 6, a half Slide 7, spatial filter 8, combined half slide 9, collimator lens 10, illumination tube lens 11; Fourier lens 6 and collimator lens 10 form a 4f system, and the spatial light modulator 5 is located in the 4f system the front focal plane of the 4f system, the spatial filter 8 is located at the back focal plane of the 4f system, and is used for spatial filtering, allowing only the light beam of a specific angle diffracted by the spatial light modulator 5 to pass through, blocking other diffraction orders; A glass slide 7 and a combined half of the glass slide 9 constitute a polarization control component, which is used to control incident polarized light at different orientation angles;

The laser light emitted by the light source module is transmitted to the achromatic collimating beam expander objective lens 3, and then enters the polarization beam splitter 4 after being collimated and expanded, and the transmitted vertically polarized light enters the spatial light modulator 5, and is modulated by the spatial light modulator After diffracting three outgoing beams of 0th order and ±1st order, the three outgoing beams are reflected by the polarizing beam splitter 4 and then focused on the spatial filter 8 by the Fourier lens 6 and one-half glass slide 7. After filtering The outgoing light beams of ±1st order are gated and enter the fluorescence detection module after being combined with one-half glass slide 9, collimating lens 10, and illuminating tube lens 11 in sequence.

Further, in one of the embodiments, the fluorescence detection module includes a three-dimensional translation stage 12, a microscope objective lens 13, a dichroic mirror 14, an imaging lens 15 and a camera 16;

The active illumination structured light field output by the active structured light illumination light field loading module is reflected by the dichroic mirror 14 and then irradiated by the microscopic objective lens 13 to the sample illumination tube lens 11 placed on the three-dimensional translation stage 12 and the microscopic objective lens 13. Another pair of 4f systems are used to conjugate the structured light illumination light field to the sample surface, collect the fluorescence signal of the sample after being collected by the microscope objective lens 13, transmit it through the dichroic mirror 14, and then focus it to the camera 16 through the imaging lens 15 on the detection surface.

Further, in one of the embodiments, the active structured light illumination light field generation module includes an image acquisition unit 17, an image processing unit 18 and a light field generation unit 19;

The image acquisition unit 17 is configured to acquire the illumination light field distribution characteristics of the sample surface detected by the camera 16;

The image processing unit 18 is configured to convert the illumination light field obtained by the image acquisition unit 17 into the active structured light illumination light field required by the user;

The light field generation unit 19 is used to convert the active structured light illumination light field into a set of binary bitmaps with different time weights based on the spatial-temporal joint intensity modulation that can be loaded by the spatial light modulator, and load it into the active structured illumination The bright light field is loaded into the spatial light modulator in the module.

Before the above system works, it is necessary to calibrate the spatial coordinates of the spatial light modulator and the camera to match the spatial coordinates of the input and output light fields and the modulated light field to generate an active illumination light field with pixel-level matching accuracy. The specific process is as follows: 1) load the "cross" intersection points composed of equidistant distribution in the spatial light modulator; 2) the fluorescent detection module collects the uniformly distributed fluorescent dye signal image; 3) based on the image loaded by the spatial light modulator and The image collected by the camera is used to calculate the calibration matrix (including zoom ratio, rotation, and translation).

The workflow of the system is as follows:

One of the four lasers of 405nm or 488nm or 561nm or 673nm is gated through the laser module 1 and enters into the polarization-maintaining single-mode fiber 2 .

Further, the excitation light transmits the light beam to the achromatic collimating objective lens 3 of the light field loading module through the single-mode light beam. The beam collimated and expanded beam irradiates the polarization beam splitter 4, and the transmitted vertically polarized light (s polarization) is irradiated on the spatial light modulator 5. After the beam is modulated by the spatial light modulator, it mainly diffracts 0-order, ±1-order The reflected outgoing beam passes through the polarization beam splitter 4 again, and only the modulated horizontally polarized light (p polarization) can enter the imaging system; The time-weighted bit plane can realize the light intensity control of a single pixel by modulating the "pixel on" and "pixel off" of the incident polarized light, such as modulating the non-uniform Gaussian distributed excitation light into a uniformly distributed flat-top beam (Fig. 3b). Three diffracted beams of 0th order and ±1 after single-pixel intensity modulation are incident on the Fourier lens 6, pass through the half glass 7 for polarization compensation, and the modulated polarized beams are finally focused on the spatial filter 8 position, after spatial filtering, the 0-order beam is blocked, and only the ±1-order beam passes through; the ±1-order beam passes through the combination of two divided glass slides 9, which are used to rotate the polarization directions of incident light with different orientation angles, so that in The interference fringes with the maximum modulation degree are obtained on the sample surface; after the ±1st-order light beam passes through the collimating lens 10, it interferes at the rear focal plane; further, the light beam enters the fluorescence detection module after passing through the illuminating tube lens.

Further, the illuminating light beam enters the microscope objective lens 13 after being reflected by the dichroic mirror 14 , and finally excites the fluorescent sample held at the translation stage 12 . Fluorescence samples illuminated by structured light under active illumination emit fluorescence with a wavelength longer than that of the excitation light, which is collected by the high numerical aperture objective lens 13 and enters the fluorescence detection module. The collected fluorescence signals are transmitted through the dichroic mirror 14 and then enter imaging The lens 15 is finally detected by a camera 16 .

Further, the image acquisition unit of the active structured light illumination light field generation module collects the images collected by the camera 16 and sends them to the image processing unit 18. The image processing unit calculates the collected fluorescence images and the user-preset light field, and then generates the user-preset The modulated light field required by the light field is further sent to the light field generation unit 19 to generate a binary bit-plane holographic fringe pattern with different time weights that can be loaded by the spatial light modulator 5 .

Further, the light field generation module reconstructs and loads the generated binary bitmap into the spatial light modulation 5 in the light field loading module, irradiates the sample through devices 6 to 11, 14, 13, and 12, and the emitted fluorescence is detected by the fluorescence Modules (13, 14-15, 16) receive. The image collected by the camera is received by the light field generation module to generate an active structured light illumination light field, which is then sent to the spatial light modulator of the light field loading module until a user-preset illumination light field is generated.

In summary, by inventing a space-time joint intensity modulation method, the present invention can regulate the intensity of the excitation light illuminating the sample surface in a specified space, and realize high-modulation active structured light without changing the coherence of the incident light. Super-resolution imaging of illumination. Based on the spatio-temporal joint modulation method proposed by the present invention, the input non-uniform Gaussian distribution light field can be modulated into a uniformly illuminated flat-top light field, which meets the application requirements based on quantitative analysis of fluorescence intensity; according to the spatial distribution characteristics of sample intensity, the illumination can be adjusted actively Light intensity, that is, reduce the light dose in the strong signal area, increase the light dose in the weak signal area, no signal or no light in the background area, to meet the application requirements of high dynamic range and low light dose; user-defined lighting area, to meet the photostimulation, fluorescence bleaching Restoration and other application requirements. Without losing the spatial resolution (90nm) and temporal resolution (100fps@512*512) of the SIM, the present invention is expected to increase the dynamic range of the traditional structured light illumination microscope by 40dB, reduce the light dose by 10 times, and provide Dynamic long-term observation provides advanced new imaging technology means.

The basic principles, main features and advantages of the present invention have been shown and described above. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments. The above-mentioned embodiments and descriptions only illustrate the principles of the present invention. Within the spirit and principles, any modifications, equivalent replacements, improvements, etc., shall be included in the protection scope of the present invention.

Claims (9)

1. An active structured light illuminated super-resolution microscopic imaging method, characterized in that the method comprises the following steps:

step 1, collecting a fluorescence image of a sample excited by laser through a camera;

step 2, constructing a required active structured light illumination light field based on the image in the step 1;

step 3, converting the active structured light illumination field of the step 2 and loading the converted active structured light illumination field into a spatial light modulator to form an active structured light illumination field modulated by space-time joint intensity;

step 4, taking out the corresponding bit plane diagram from the holographic fringe bit plane diagram loaded on the spatial light modulator in the step 3, starting to display, synchronously emitting light by a laser to illuminate a sample, and synchronously exposing by a camera to acquire original image data;

and 5, judging whether all the holographic fringe phase surface images are completely displayed, if so, stopping the phase surface display by the spatial light modulator and stopping the image acquisition by the camera, otherwise, switching to the phase surface images with different spatial direction angles and phases according to a preset sequence, returning to the step 4, and continuously executing synchronous display and exposure.

2. The method of claim 1, wherein the step 2 of illuminating the light field with the active structured light comprises:

first illumination light field: the input non-uniform Gaussian distribution light field is modulated into a uniform flat-top illumination light field, and the requirements of quantitative analysis based on fluorescence intensity and large-field splicing imaging are met;

or, the second illumination field: actively adjusting the illumination intensity according to the sample intensity distribution characteristics, wherein the illumination intensity comprises that the illumination dose is reduced in a strong signal area, the illumination dose is increased in a weak signal area, and no signal or background area has no illumination, so that the imaging requirements of a required high dynamic range and low illumination dose are met;

or, the third illumination field: the lighting of the designated position and the strength is customized, and the imaging requirement of the user for customizing the interested lighting area is met.

3. The active structured light illuminated super-resolution microscopy imaging method as claimed in claim 1, wherein the spatial distribution of the light intensity of the active structured light field modulated in step 3 is represented as I _a (x,y)：

In the formula I _g (x, y, T) represents the spatiotemporal volume of the illumination intensity of the excitation light on the sample plane during one camera exposure period T, the illumination intensity of which exhibits a spatially non-uniform gaussian distribution during SIM imaging when only one secondary beam is allowed to pass; m _i (x, y, t) represents the ith intensity modulation function or binary bit plane loaded in the spatial light modulator, N represents the number of binary bit planes loaded, and the intensity modulation precision of each pixel on the spatial light modulator is represented as 1/2 ^N ；t _i Representing the time weight function corresponding to each binary bit plane, and summing all the time weights into one exposure period of the camera

Point (x, y) represents any spatial coordinate in the spatial light modulator; m represents the degree of modulation of structured light, k _x 、k _y And phi denotes the spatial frequency and the initial phase.

4. The active structured light illuminated super-resolution microscopic imaging system for realizing the method of any one of claims 1 to 3, wherein the system comprises a light source module, an active structured light illuminated light field loading module, an active structured light illuminated light field generating module and a fluorescence detection module;

the light source module is used for realizing the control of at least four laser wavelengths, and can realize single-wavelength independent illumination, multi-wavelength multi-color time-sharing and simultaneous illumination;

the fluorescence detection module is used for collecting a fluorescence image of the sample after laser excitation;

the active structured light illumination light field generation module is used for generating an active structured light illumination light field based on the fluorescence image;

and the active structured light illumination light field loading module is used for loading the generated active structured light illumination light field into the SIM imaging system to complete active structured light illumination super-resolution imaging.

5. The actively structured light illuminated super-resolution microscopy imaging system according to claim 4, wherein the light source module comprises a laser (1), a single mode polarization maintaining fiber (2);

the laser (1) is used for providing laser with multiple wavelengths and can realize single-wavelength and multi-wavelength synchronous light-emitting control;

the single-mode polarization maintaining optical fiber (2) is used for transmitting the multi-path laser coupled by the laser (1) to the active structured light illumination light field loading module.

6. The active structured light illuminated super resolution microscopy imaging system as claimed in claim 5, characterized in that the laser (1) provides laser light at four wavelengths, respectively 405nm, 488nm, 561nm, 637nm.

7. The active structured light illuminated super-resolution microscopic imaging system according to claim 5, wherein the active structured light illuminated light field loading module comprises an achromatic collimating beam expander objective (3), a polarization beam splitter prism (4), a spatial light modulator (5), a Fourier lens (6), a half glass (7), a spatial filter (8), a combined half glass (9), a collimating lens (10) and an illumination tube lens (11); the Fourier lens (6) and the collimating lens (10) form a 4f system, the spatial light modulator (5) is positioned on the front focal plane of the 4f system, the spatial filter (8) is positioned on the rear focal plane of the 4f system and used for spatial filtering, only the light beams with specific angles diffracted by the spatial light modulator (5) are allowed to pass through, and other diffraction orders are blocked; the half glass slide (7) and the combined half glass slide (9) form a polarization regulation and control component for regulating and controlling incident polarized light with different direction angles;

laser emitted by the light source module is transmitted to an achromatic collimation and beam expansion objective lens (3), the laser is incident to a polarization beam splitter prism (4) after being collimated and expanded, transmitted vertical polarized light is incident to a spatial light modulator (5), three emergent light beams of 0 grade and +/-1 grade are diffracted after being modulated by the spatial light modulator, the three emergent light beams are reflected by the polarization beam splitter prism (4), focused on a spatial filter (8) through a Fourier lens (6) and a half glass (7) in sequence, and the +/-1 grade emergent light beams are gated after filtering and enter a fluorescence detection module after sequentially passing through a combined half glass (9), a collimating lens (10) and an illumination cone lens (11).

8. The active structured light illuminated super-resolution microscopy imaging system according to claim 7, characterized in that the fluorescence detection module comprises a three-dimensional displacement stage (12), a microscope objective (13), a dichroic mirror (14), an imaging lens (15) and a camera (16);

the active structured light illuminates exciting light output by the light field loading module, the exciting light is reflected by a dichroic mirror (14) and then irradiates a sample placed on a three-dimensional displacement table (12) through a microscope objective (13), a fluorescence signal of the sample is excited and then collected by the microscope objective (13), and the fluorescence signal is transmitted by the dichroic mirror (14) and then focused on a detection surface of a camera (16) through an imaging lens (15).

9. The active structured light illuminated super-resolution microscopy imaging system according to claim 8, wherein the active structured light illuminated light field generating module comprises an image acquisition unit (17), an image processing unit (18) and a light field generating unit (19);

the image acquisition unit (17) is used for acquiring the illumination light field distribution characteristics of the sample surface detected by the camera (16);

the image processing unit (18) is used for converting the illumination light field obtained by the image acquisition unit (17) into an active structured light illumination light field required by a user;

the light field generating unit (19) is used for converting the active structured light illuminating light field into a group of binary bit plane maps which are loaded by the spatial light modulator and carry different time weights based on space-time joint intensity modulation, and loading the binary bit plane maps into the spatial light modulator in the active structured light illuminating light field loading module.

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