A super-resolution microscopic imaging method and system using active structured light illumination

Abstract

The present invention discloses a super-resolution microscopic imaging method and system for active structured light illumination. A spatiotemporal combined intensity modulation method is proposed, which can regulate the spatial arbitrary intensity of the excitation light illuminating the sample, and realize active structured light illumination super-resolution imaging with high modulation index 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 to meet 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 light dose imaging; the user defines the illumination area to meet the application requirements of light stimulation and fluorescence bleaching recovery. The present invention can effectively improve the imaging dynamic range of SIM and reduce the light dose without losing the spatiotemporal resolution of SIM, so as to meet the imaging requirements of cell substructures and their interactions for super-resolution below 100 nanometers, extremely low light dose and high dynamic range.

Description

Technical Field

[0001] The present invention belongs to the technical field of fluorescence microscopy, and in particular to a super-resolution microscopy system combining active illumination and structured light illumination. Background Art

[0002] Cells are highly complex, dynamic systems, internally compartmentalized by membranes to form dozens of organelles with diverse functions. The interactions among these organelles are crucial for maintaining cellular function and determining cell fate. Studying these organelles and their interactions requires live-cell imaging with sub-hundred-nanometer high resolution, extremely low light dose, high dynamic range, and uniform illumination over a wide field of view.

[0003] In research aimed at achieving uniform illumination over a large field of view, beam shapers directly convert Gaussian beams into flat-top beams. Using a pair of aspheric lenses, the first lens uniformly redistributes the Gaussian beam, while the second lens recollimates it, resulting in flat-field illumination. However, refractive beam shapers have a limited working distance and impose stringent requirements on surface finish quality and optical alignment. Optical waveguides can provide uniform illumination over a very large field of view, but because they operate in total internal reflection mode, illumination is limited to the vicinity of the coverslip, making it impossible to switch the illumination angle to achieve illumination at varying depths. Furthermore, the fixed illumination size cannot be aligned with the camera's imaging field of view, and full-slide illumination can cause unnecessary photodamage to cell samples. Furthermore, classic approaches include using a pair of microlens arrays or multimode optical fibers. To reduce laser speckle, speckle reducers or vibrating optical fibers are incorporated into the system, but these methods reduce the spatial coherence of the beam and are unsuitable for total internal reflection illumination. ASTER is essentially a hybrid scanning widefield illumination device, which sweeps a Gaussian beam along a specific pattern to provide flat-top illumination in a temporally averaged manner, thereby achieving uniform illumination across 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. Its conversion to structured light illumination microscopy (SIM) based on multi-beam interference to produce cosine-distributed fringes is still limited. The reason is that when modulating the incident non-uniform Gaussian spot, it is necessary to ensure that the multiple light beams have the same frequency, constant phase difference, and consistent polarization direction, so that they interfere on the sample surface to form highly modulated illumination fringes.

[0004] In their research to expand the dynamic range of microscopic imaging systems and reduce light dose, Vinergoni et al. combined the widely used multi-exposure fusion technique in photography with point-scanning two-photon imaging and applied it to imaging neurons within a large dynamic range within a scene. However, continuous acquisition of multiple exposures not only increases the light dose but also reduces imaging speed. To overcome the limitations of multi-exposure, multi-detection simultaneous imaging was developed. This involves performing multi-level spectroscopic imaging of the same scene, simultaneously acquiring images with varying exposure doses. However, the simultaneous detection of multiple imaging light paths requires calibration of the different detectors, making the optical system complex and costly. This led to the development of active illumination technology, which incorporates an acousto-optic modulator (AOM) into the imaging system to spatially control the illumination field on a per-pixel basis. This not only improves the dynamic range but also reduces the light dose. To address the limited dynamic range, photobleaching, and phototoxicity issues of confocal microscopy, Hoebe et al. developed a microscopic imaging technique with spatially controllable illumination dose, which they applied to a Nikon C1 confocal microscope. Experimental verification shows that this imaging technology can not only increase the dynamic range by 2 times, but also reduce the light dose by 5 times, effectively extending the survival time of living cells. However, the resolution of the low-light-dose, high-dynamic imaging method based on confocal and two-photon is limited by the optical diffraction limit. When using a large numerical aperture objective lens, the lateral limit 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.

[0005] In contrast, SIM offers the advantages of fast imaging, minimal photodamage, and no special requirements for fluorescent probes. Its resolution of hundreds of nanometers perfectly meets the requirements for observing important organelles within living cells, making it the preferred choice for dynamic imaging of living cells. However, from the perspective of illumination, SIM is essentially a wide-field fluorescence microscopy technique, with the overall illumination intensity exhibiting a Gaussian distribution across the sample. This non-uniform illumination method first reduces the available field of view. To obtain a relatively uniform fluorescence image, the central region of the Gaussian spot is typically captured for imaging, resulting in a loss of peripheral field of view. Second, Gaussian illumination, with a bright center and dark edges, results in inconsistent photobleaching characteristics within the imaging field of view, with the center being more susceptible to bleaching than the edges, limiting its application in fluorescence intensity-based quantitative analysis. Furthermore, for samples with a high dynamic range, this indiscriminate illumination method, which disregards sample labeling density, results in underexposure of weak signals, overexposure of strong signals, and ineffective exposure of background and out-of-focus signals, limiting the dynamic range of the SIM imaging system and causing unnecessary photodamage. Therefore, an active structured light illumination super-resolution imaging method (Active-SIM) based on the spatiotemporal joint modulation of a spatial light modulator was invented, such as modulating the Gaussian spot into flat-field illumination, adaptively adjusting the illumination intensity according to the sample labeling density, and illuminating any area of interest. This is crucial for expanding the application of SIM imaging technology in dynamic long-term imaging of living cells. Summary of the Invention

[0006] The purpose of the present invention is to meet the requirements of fluorescence imaging with high resolution below 100 nanometers, extremely low light dose, high dynamic range, and uniform illumination over a large field of view in the study of subcellular organelles and their interactions in living cells. A spatiotemporal intensity modulation method based on a spatial light modulator is proposed to establish a super-resolution microscopic imaging system based on active structured light illumination.

[0007] The technical solution to achieve the purpose of the present invention is: a super-resolution microscopic imaging method using active structured light illumination, the method comprising the following steps:

[0008] Step 1: Capture a fluorescence image of the sample excited by laser through a camera;

[0009] Step 2: construct the required active structured light illumination light field based on the image in step 1;

[0010] Step 3, converting the active structured light illumination light field of step 2 and loading it into a spatial light modulator to form an active structured light illumination light field with spatiotemporal joint intensity modulation;

[0011] Step 4: Take out the corresponding bitmap from the holographic fringe bitmap loaded into the spatial light modulator in step 3 and start displaying it. The laser will illuminate the sample synchronously, and the camera will expose and collect the original image data synchronously.

[0012] Step 5: Determine whether all holographic fringe plane images have been displayed. If so, the spatial light modulator stops plane display and the camera stops image acquisition. Otherwise, it switches to plane images with different spatial direction angles and phases in a preset order, returns to step 4, and continues to perform synchronous display and exposure.

[0013] Furthermore, the active structured light illumination light field in step 2 includes:

[0014] First illumination light field: modulates 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;

[0015] Alternatively, the second illumination light field actively adjusts the illumination intensity based on the sample intensity distribution characteristics, including reducing the illumination dose in strong signal areas, increasing the illumination dose in weak signal areas, and eliminating illumination in areas with no signal or background, to meet the imaging requirements of high dynamic range and low illumination dose.

[0016] Or, the third illumination light field: customize the illumination of the specified position and intensity to meet the imaging requirements of the user-defined illumination area of interest.

[0017] Furthermore, the spatial distribution of the light intensity of the active structure illumination light field modulated in step 3 is expressed as I a (x,y):

[0018]

[0019]

[0020] Where, I g (x, y, t) represents the spatiotemporal volume of the illumination intensity of the excitation light on the sample surface within a camera exposure period T. During the SIM imaging process when only one order of light beam is allowed to pass through, 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 bit plane loaded in the spatial light modulator, 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 time weight function corresponding to each binary plane, and the sum of all time weights is one exposure cycle of the camera Point (x, y) represents any spatial coordinate in the spatial light modulator; m represents the modulation degree of the structured light, k x 、k y , φ represents the spatial frequency and initial phase.

[0021] An active structured light illumination super-resolution microscopy imaging system for implementing the above method, the system comprising 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;

[0022] The light source module is used to realize the control of at least four laser wavelengths, and can realize single wavelength independent illumination, multiple wavelengths and multi-color time-sharing and simultaneous illumination;

[0023] The fluorescence detection module is used to collect fluorescence images of samples excited by laser;

[0024] The active structured light illumination light field generation module is used to generate an active structured light illumination light field based on the fluorescent image;

[0025] 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.

[0026] Furthermore, the light source module includes a laser and a single-mode polarization-maintaining optical fiber;

[0027] The laser is used to provide lasers with multiple wavelengths and can realize single-wavelength and multi-wavelength synchronous light output control;

[0028] The single-mode polarization-maintaining optical fiber is used to transmit the multi-channel laser light coupled by the laser to the active structured light illumination light field loading module.

[0029] Furthermore, the active structured light illumination light field loading module includes an achromatic collimating beam expander objective lens, a polarization beam splitter prism, a spatial light modulator, a Fourier lens, a half glass slide, a spatial filter, a combined half glass slide, a collimating lens, and an illumination tube lens; the Fourier lens and the collimating lens constitute 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, which is used for spatial filtering, allowing only a light beam of a specific angle diffracted by the spatial light modulator to pass through, and blocking other diffraction orders; the half glass slide and the combined half glass slide constitute a polarization control component, which is used to control incident polarized light at different direction angles;

[0030] The laser light emitted by the light source module is transmitted to the achromatic collimating beam expanding objective lens, and then enters the polarization beam splitter prism after collimation and beam expansion. The transmitted vertically polarized light enters the spatial light modulator, and after modulation by the spatial light modulator, it mainly diffracts into three output light beams of order 0 and ±1. After being reflected by the polarization beam splitter prism, the three output light beams are sequentially focused on the spatial filter through the Fourier lens and the half glass slide. After filtering, the ±1-order output light beams are selected and sequentially pass through the combined half glass slide, the collimating lens, and the illumination tube lens to enter the fluorescence detection module.

[0031] Furthermore, the fluorescence detection module includes a three-dimensional translation stage, a microscope objective lens, a dichroic mirror, an imaging lens and a camera;

[0032] The excitation light output by the active structured light illumination light field loading module is reflected by the dichroic mirror and then illuminates the sample placed on the three-dimensional translation stage through the microscope objective lens. After stimulating the sample fluorescence signal, it is collected by the microscope objective lens, then transmitted through the dichroic mirror and focused on the detection surface of the camera through the imaging lens.

[0033] Furthermore, the active structured light illumination light field generation module includes an image acquisition unit, an image processing unit and a light field generation unit;

[0034] The image acquisition unit is used to acquire the illumination light field distribution characteristics of the sample surface detected by the camera;

[0035] 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;

[0036] 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 spatiotemporal joint intensity modulation that can be loaded by the spatial light modulator, and load them into the spatial light modulator in the active structured light illumination light field loading module.

[0037] Compared with the prior art, the present invention has the following significant advantages:

[0038] 1) Active structured light illumination super-resolution imaging is achieved 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.

[0039] 2) Without compromising the spatiotemporal resolution of traditional SIM imaging systems, the system offers the following advantages: It can modulate the input non-uniform Gaussian illumination field into a uniformly distributed flat-top illumination field, meeting the needs of fluorescence intensity quantitative analysis imaging applications; actively adjust illumination intensity based on the spatial distribution of sample intensity, meeting the needs of high dynamic range and low light dose imaging applications; and customize illumination at a specific location and intensity to meet the needs of users who need to image a defined illumination area of interest. By providing these three active structured light illumination fields, the system provides advanced new imaging technologies for high-resolution, extremely low light dose, high dynamic range, and large field of view uniform illumination imaging of living cells.

[0040] 3) The present invention does not require the addition of additional optical hardware to the existing SIM imaging system. Instead, the required light field can be constructed based on the spatiotemporal joint intensity modulation method proposed by the present invention. This achieves rich imaging functions without increasing economic costs and simplifies the complexity of the system.

[0041] 4) The present invention can improve the imaging dynamic range of traditional SIM by 40dB and reduce the light dose by 10 times without losing the spatiotemporal resolution of SIM.

[0042] The present invention is further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is a flow chart of the light field modulation method for active structured light illumination.

[0044] Figure 2 Schematic diagram of the active structured light illumination super-resolution microscopy imaging system.

[0045] Figure 3 This is the principle diagram of the active structured light illumination light field modulation method, where Figure 3 (a) is a schematic diagram of arbitrary light intensity modulation at the pixel level based on ferroelectric liquid crystal spatial light modulator. Figure 3 (b) Schematic diagram of the spatiotemporal joint intensity modulation principle.

[0046] The reference numerals in the figure are as follows: 1 four-way beam-combining laser; 2 single-mode polarization-maintaining fiber; 3 achromatic collimating beam-expanding objective lens; 4 polarization beam splitter; 5 spatial light modulator; 6 Fourier lens; 7 half-glass slide; 8 spatial filter; 9 combined half-glass slide; 10 collimating lens; 11 illumination tube lens; 12 three-dimensional translation stage; 13 objective lens; 14 dichroic mirror; 15 imaging tube lens; 16 camera; 17 image acquisition unit; 18 image processing unit; 19 light field generation unit. DETAILED DESCRIPTION

[0047] In order to make the purpose, technical solutions and advantages of this application more clear, the following further describes this application in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0048] 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. are only for descriptive purposes and cannot be understood as indicating or implying their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of such features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on the ability of ordinary technicians in this field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0049] In one embodiment, combined Figure 1, provides a super-resolution microscopic imaging method using active structured light illumination, the method comprising the following steps:

[0050] Step 1: Capture a fluorescence image of the sample excited by laser through a camera;

[0051] Step 2: construct the required active structured light illumination light field based on the image in step 1;

[0052] Step 3, converting the active structured light illumination light field of step 2 and loading it into a spatial light modulator to form an active structured light illumination light field with spatiotemporal joint intensity modulation;

[0053] Step 4: Take out the corresponding bitmap from the holographic fringe bitmap loaded into the spatial light modulator in step 3 and start displaying it. The laser will illuminate the sample synchronously, and the camera will expose and collect the original image data synchronously.

[0054] Step 5: Determine whether all holographic fringe plane images have been displayed. If so, the spatial light modulator stops plane display and the camera stops image acquisition. Otherwise, it switches to plane images with different spatial direction angles and phases in a preset order, returns to step 4, and continues to perform synchronous display and exposure.

[0055] Furthermore, in one embodiment, the active structured light illumination light field in step 2 includes:

[0056] First illumination light field: modulates 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;

[0057] Alternatively, the second illumination light field actively adjusts the illumination intensity based on the sample intensity distribution characteristics, including reducing the illumination dose in strong signal areas, increasing the illumination dose in weak signal areas, and eliminating illumination in areas with no signal or background, to meet the imaging requirements of high dynamic range and low illumination dose.

[0058] Or, the third illumination light field: customize the illumination of the specified position and intensity to meet the imaging requirements of the user-defined illumination area of interest.

[0059] Furthermore, in one embodiment, in order to realize the active illumination structured light field with spatiotemporal combined intensity modulation, the present invention utilizes the fast switching characteristics of each independent pixel in the spatial light modulation based on ferroelectric liquid crystal between "on" and "off", so as to realize the single-pixel level regulation of the illumination intensity within a camera integration period, such as Figure 3 In the SIM imaging system, the illumination intensity on the sample surface presents a Gaussian distribution as a whole, which can be expressed as:

[0060]

[0061] Since the polarization state of the incident light on each pixel in the ferroelectric liquid crystal spatial light modulator can be individually controlled, the light intensity can be "switched" in combination with a polarization beam splitter. In this way, by adjusting the lighting switch at each time point during the camera's exposure cycle, arbitrary light intensity control can be achieved. Therefore, the active illumination light field can be expressed as:

[0062]

[0063] In order to convert to digital modulation, digital pulse width modulation method is introduced. Assume that N binary bit planes M are loaded in the spatial light modulator. i (x, y, t), each plane is assigned a different lighting time weight t i =(1 / 2 i )·T, such as Figure 3 By continuously loading N binary bitmaps with different time weights within a camera exposure period T, the arbitrary light intensity of a single pixel in the incident light field can be controlled, as shown in the following expression:

[0064]

[0065] Therefore, the illumination intensity distribution of the active structured light field after modulation in step 3 is expressed as I a (x,y):

[0066]

[0067]

[0068] Where I0 represents the peak intensity, σ is the waist radius of the Gaussian beam, and I g (x, y, t) represents the spatiotemporal volume of the illumination intensity of the excitation light on the sample surface within a camera exposure period T. During the SIM imaging process when only one order of light beam is allowed to pass through, 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 bit plane loaded in the spatial light modulator, 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 time weight function corresponding to each binary plane, and the sum of all time weights is one exposure cycle of the camera Point (x, y) represents any spatial coordinate in the spatial light modulator; m represents the modulation degree of the structured light, k x 、k y , φ represents the spatial frequency and initial phase.

[0069] In one embodiment, the super-resolution microscopic imaging system using active structured light illumination to implement the above method is as follows: Figure 2 As shown, the system includes a light source module, an active structured light illumination light field loading module, an active structured light illumination light field generating module and a fluorescence detection module;

[0070] The light source module is used to realize the control of at least four laser wavelengths, and can realize single wavelength independent illumination, multiple wavelengths and multi-color time-sharing and simultaneous illumination;

[0071] The fluorescence detection module is used to collect fluorescence images of samples excited by laser;

[0072] The active structured light illumination light field generation module is used to generate an active structured light illumination light field based on the fluorescent image;

[0073] 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.

[0074] Furthermore, in one embodiment, the light source module includes a laser 1 and a single-mode polarization-maintaining optical fiber 2;

[0075] The laser 1 is used to provide lasers of multiple wavelengths and can realize single-wavelength and multi-wavelength synchronous light output control;

[0076] The single-mode polarization-maintaining optical fiber 2 is used to transmit the multi-channel laser light coupled by the laser 1 to the active structured light illumination light field loading module.

[0077] Here preferably, the laser 1 provides laser light with four wavelengths, which are 405 nm, 488 nm, 561 nm and 637 nm respectively.

[0078] Furthermore, in one embodiment, 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 glass slide 7, a spatial filter 8, a combined half glass slide 9, a collimating lens 10, and an illumination tube lens 11; the Fourier lens 6 and the collimating lens 10 constitute a 4f system, the spatial light modulator 5 is located at the front focal plane of the 4f system, and the spatial filter 8 is located at the back focal plane of the 4f system, which is used to perform spatial filtering, allowing only a light beam of a specific angle diffracted by the spatial light modulator 5 to pass through, and blocking other diffraction orders; the half glass slide 7 and the combined half glass slide 9 constitute a polarization control component, which is used to control incident polarized light of different direction angles;

[0079] The laser emitted by the light source module is transmitted to the achromatic collimating beam expanding objective lens 3, and after collimation and beam expansion, it is incident on the polarization beam splitter prism 4. The transmitted vertically polarized light is incident on the spatial light modulator 5, and after modulation by the spatial light modulator, it diffracts into three output light beams of order 0, ±1, and the three output light beams are reflected by the polarization beam splitter prism 4 and focused on the spatial filter 8 in sequence through the Fourier lens 6 and the half glass slide 7. After filtering, the ±1-order output light beams are selected and sequentially pass through the combined half glass slide 9, the collimating lens 10, and the illumination tube lens 11 to enter the fluorescence detection module.

[0080] Furthermore, in one embodiment, the fluorescence detection module includes a three-dimensional translation stage 12, a microscope objective 13, a dichroic mirror 14, an imaging lens 15 and a camera 16;

[0081] The active structured light field output by the active structured light illumination light field loading module is reflected by the dichroic mirror 14 and then illuminates the sample placed on the three-dimensional translation stage 12 through the microscope objective 13. The sample illumination tube lens 11 and the microscope objective 13 form another pair of 4f systems, which are used to conjugate the structured light illumination light field to the sample surface, excite the sample fluorescence signal, and then be collected by the microscope objective 13. After that, it is transmitted through the dichroic mirror 14 and then focused onto the detection surface of the camera 16 through the imaging lens 15.

[0082] Furthermore, in one embodiment, 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;

[0083] The image acquisition unit 17 is used to acquire the illumination light field distribution characteristics of the sample surface detected by the camera 16;

[0084] The image processing unit 18 is used 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;

[0085] The light field generating 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 spatiotemporal joint intensity modulation that can be loaded by the spatial light modulator, and load them into the spatial light modulator in the active structured light illumination light field loading module.

[0086] Before operation, the above system requires spatial coordinate calibration of the spatial light modulator and camera. This is used to match the input and output light fields with the modulated light field, generating an active illumination light field with pixel-level matching accuracy. The specific process is as follows: 1) A "cross" of evenly spaced intersection points is loaded into the spatial light modulator; 2) The fluorescence detection module captures an image of the uniformly distributed fluorescent dye signal; 3) Based on the image loaded by the spatial light modulator and the image captured by the camera, a calibration matrix (including scaling, rotation, and translation) is calculated.

[0087] The system's workflow is as follows:

[0088] Laser 1 selects one of the four lasers of 405 nm, 488 nm, 561 nm or 673 nm to enter the single-mode polarization-maintaining optical fiber 2 .

[0089] Furthermore, the excitation light is transmitted to the achromatic collimating objective lens 3 of the light field loading module through a single-mode beam. The light beam after collimation and expansion illuminates the polarization beam splitter prism 4, and the transmitted vertically polarized light (s polarization) illuminates the spatial light modulator 5. After the light beam is modulated by the spatial light modulator, it mainly diffracts into three output beams of order 0, ±1, and the reflected output beam passes through the polarization beam splitter prism 4 again. Only the modulated horizontally polarized light (p polarization) can enter the imaging system; as shown in the principle of light intensity modulation Figure 3 As shown in Figure a, the time-weighted bit plane is loaded in the spatial light modulator. By modulating the "pixel on" and "pixel off" of the incident polarized light, the light intensity control of a single pixel can be achieved. For example, the non-uniform Gaussian distribution of excitation light can be modulated into a uniformly distributed flat-top beam ( Figure 3 b) The three diffracted beams (0th order, ±1st order) undergoing single-pixel intensity modulation are incident on Fourier lens 6. They pass through a half-glass slide 7 for polarization compensation. The modulated polarized beams are finally focused on spatial filter 8. After spatial filtering, the 0th order beam is blocked, and only the ±1st order beams pass through. The ±1st order beams pass through a combined half-glass slide 9, which is used to rotate the polarization direction of the incident light at different angles, thereby obtaining interference fringes with maximum modulation on the sample surface. The ±1st order beams pass through collimating lens 10, interfering at its rear focal plane. Furthermore, the beams pass through the illumination tube lens and enter the fluorescence detection module.

[0090] The illumination beam is then reflected by a dichroic mirror 14 and enters the microscope objective 13, ultimately exciting the fluorescent sample held by the translation stage 12. The fluorescent sample, illuminated by the active structured light, emits fluorescence with a wavelength longer than the excitation light. This fluorescence is collected by the high-numerical-aperture objective 13 and enters the fluorescence detection module. The collected fluorescence signal is then transmitted through the dichroic mirror 14 and enters the imaging lens 15 before being detected by the camera 16.

[0091] Furthermore, the image acquisition unit of the active structured light illumination light field generation module collects the image captured by the camera 16 and sends it to the image processing unit 18. The image processing unit calculates the collected fluorescence image and the user-preset light field, and then generates the modulated light field required by the user-preset light field, and further sends it to the light field generation unit 19 to generate a binary plane holographic fringe pattern with different time weights that can be loaded by the spatial light modulator 5.

[0092] The light field generation module then reconstructs the generated binary bitmap and loads it into the spatial light modulator 5 in the light field loading module. The sample is illuminated via devices 6 to 11, 14, 13, and 12, and the emitted fluorescence is received by the fluorescence detection modules (13, 14-15, and 16). The image collected by the camera is received by the light field generation module, which generates an active structured light illumination field. This is then fed back into the spatial light modulator in the light field loading module until the user-defined illumination light field is generated.

[0093] In summary, the present invention, by inventing a spatiotemporal combined intensity modulation method, can regulate the excitation light illuminating the sample surface to any intensity in a specified space, and realize super-resolution imaging of active structured light illumination with high modulation index without changing the coherence of the incident light. Based on the spatiotemporal combined modulation method proposed by the present invention, the input non-uniform Gaussian distribution light field can be modulated into a flat-top light field with uniform illumination, meeting the application requirements based on quantitative analysis of fluorescence intensity; according to the spatial distribution characteristics of the sample intensity, the illumination light intensity is actively adjusted, that is, the illumination dose in the strong signal area is reduced, the illumination dose in the weak signal area is increased, and there is no illumination in the signal or background area, meeting the application requirements of high dynamic range and low light dose; the user customizes the illumination area to meet the application requirements of light stimulation, fluorescence bleaching recovery, etc. The present invention is expected to increase the dynamic range of traditional structured light illumination microscopes by 40dB and reduce the illumination dose by 10 times without losing the SIM spatial resolution (90nm) and temporal resolution (100fps@512*512), providing an advanced new imaging technology for long-term dynamic observation of living cells.

[0094] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only illustrative of the principles of the present invention. Without departing from the spirit and scope of the present invention, any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A super-resolution microscopic imaging method using active structured light illumination, characterized in that: The method comprises the following steps: Step 1: Capture a fluorescence image of the sample excited by laser through a 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 of step 2 and loading it into a spatial light modulator to form an active structured light illumination light field with spatiotemporal joint intensity modulation; Step 4: Take out the corresponding bitmap from the holographic fringe bitmap loaded into the spatial light modulator in step 3 and start displaying it. The laser will illuminate the sample synchronously, and the camera will expose and collect the original image data synchronously. Step 5: Determine whether all holographic fringe plane images have been displayed. If so, the spatial light modulator stops plane display and the camera stops image acquisition. Otherwise, the camera switches to plane images with different spatial orientation angles and phases in a preset sequence, and returns to step 4 to continue synchronous display and exposure. The active structured light illumination light field in step 2 includes: First illumination light field: modulates 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; Alternatively, the second illumination light field actively adjusts the illumination intensity based on the sample intensity distribution characteristics, including reducing the illumination dose in strong signal areas, increasing the illumination dose in weak signal areas, and eliminating illumination in areas with no signal or background, to meet the imaging requirements of high dynamic range and low illumination dose. Or, the third illumination light field: customize the illumination of the specified position and intensity to meet the imaging requirements of the user-defined illumination area of interest.

2. The super-resolution microscopy imaging method using active structured light illumination according to claim 1, characterized in that: The spatial distribution of the light intensity of the active structure illumination light field modulated in step 3 is expressed as I a (x,y): Where, I g (x, y, t) represents the spatiotemporal volume of the illumination intensity of the excitation light on the sample surface within a camera exposure period T. During the SIM imaging process when only one order of light beam is allowed to pass through, 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 bit plane loaded in the spatial light modulator, N represents the number of binary bit planes loaded, and the light intensity modulation accuracy of each pixel on the spatial light modulator is expressed as 1 / 2 N ; t i Represents the time weight function corresponding to each binary plane, and the sum of all time weights is one exposure cycle of the camera Point (x, y) represents any spatial coordinate in the spatial light modulator; m represents the modulation degree of the structured light, k x 、k y , φ represents the spatial frequency and initial phase.

3. A super-resolution microscopic imaging system using active structured light illumination to implement the method according to any one of claims 1 to 2, characterized in that: The system includes a light source module, an active structured light illumination light field loading module, an active structured light illumination light field generating 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, multiple wavelengths and 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 generation module is used to generate an active structured light illumination light field based on the fluorescent 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.

4. The super-resolution microscopy imaging system with active structured light illumination according to claim 3, characterized in that: The light source module comprises a laser (1) and a single-mode polarization-maintaining optical fiber (2); The laser (1) is used to provide lasers of multiple wavelengths and can realize single-wavelength and multi-wavelength synchronous light output control; The single-mode polarization-maintaining optical fiber (2) is used to transmit the multi-channel laser light coupled by the laser (1) to the active structured light illumination light field loading module.

5. The active structured light illumination super-resolution microscopy imaging system according to claim 4, characterized in that: The laser (1) provides laser light with four wavelengths, which are 405 nm, 488 nm, 561 nm and 637 nm respectively.

6. The super-resolution microscopy imaging system with active structured light illumination according to claim 4, characterized in that: The active structured light illumination light field loading module comprises an achromatic collimating beam expanding objective lens (3), a polarization beam splitting prism (4), a spatial light modulator (5), a Fourier lens (6), a half glass slide (7), a spatial filter (8), a combined half glass slide (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 located at the front focal plane of the 4f system, and the spatial filter (8) is located at the back focal plane of the 4f system, and is used for spatial filtering, allowing only a light beam of a preset angle diffracted by the spatial light modulator (5) to pass through, and blocking other diffraction orders; the half glass slide (7) and the combined half glass slide (9) constitute a polarization control component, which is used to control incident polarized light of different direction angles; The laser light emitted by the light source module is transmitted to the achromatic collimating beam expanding objective lens (3), and is incident on the polarization beam splitting prism (4) after collimation and beam expansion. The transmitted vertically polarized light is incident on the spatial light modulator (5), and is modulated by the spatial light modulator to diffract into three output light beams of order 0, order ±1, and order ±1. The three output light beams are reflected by the polarization beam splitting prism (4) and then sequentially focus on the spatial filter (8) through the Fourier lens (6) and the half glass slide (7). After filtering, the selected ±1-order output light beams sequentially pass through the combined half glass slide (9), the collimating lens (10), and the illumination tube lens (11) and then enter the fluorescence detection module.

7. The active structured light illumination super-resolution microscopy imaging system according to claim 6, characterized in that: The fluorescence detection module includes a three-dimensional displacement stage (12), a microscope objective lens (13), a dichroic mirror (14), an imaging lens (15) and a camera (16); The excitation light output by the active structured light illumination light field loading module is reflected by a dichroic mirror (14) and then irradiates a sample placed on a three-dimensional displacement stage (12) through a microscope objective lens (13). After exciting the sample fluorescence signal, it is collected by the microscope objective lens (13), then transmitted through the dichroic mirror (14) and focused on the detection surface of the camera (16) through an imaging lens (15).

8. The super-resolution microscopy imaging system with active structured light illumination according to claim 7, characterized in that: 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 used to acquire the illumination light field distribution characteristics of the sample surface detected by the camera (16); The image processing unit (18) is used 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 generating unit (19) is used to convert the active structured light illumination light field into a set of binary bitmaps carrying different time weights based on spatiotemporal joint intensity modulation that can be loaded by the spatial light modulator, and load the binary bitmaps into the spatial light modulator in the active structured light illumination light field loading module.

9. The active structured light illumination super-resolution microscopy imaging system according to claim 8, characterized in that: Before the system works, the spatial light modulator and camera need to be calibrated in space to match the input and output light fields and the modulated light field in space, and generate an active illumination light field with pixel-level matching accuracy.

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