A full-wavelength limit resolution structured light microscopic imaging method and related products

Abstract

The application discloses a full-wavelength limit resolution structured light microscopic imaging method and related products, which can be applied to the technical field of optical microscopes. The method comprises the following steps: determining a target fringe period of a structured light illumination fringe based on an emission wavelength; adjusting the structured light illumination fringe to the target fringe period based on a spatial light modulator, so that a structured light microscopic imaging system reaches full-wavelength limit resolution; acquiring a first frequency domain image, a second frequency domain image and a third frequency domain image corresponding to a target sample based on the structured light illumination fringe; processing the first frequency domain image, the second frequency domain image and the third frequency domain image based on a frequency domain image multiplication algorithm, and determining a target wave vector; and performing image reconstruction on the target sample based on the target wave vector. In this way, the system maintains the limit resolution at any emission wavelength through the adjustment of the spatial light modulator, and then the frequency domain image multiplication algorithm accurately solves the target wave vector under the condition, thereby improving the accuracy of image reconstruction.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of optical microscopy, in particular to a full-wavelength limit resolution structured light microscopic imaging method and related products. BACKGROUND

[0002] Structured light microscopic imaging (SIM) is a fluorescence microscopic imaging technology, which breaks through the diffraction limit of traditional optical microscopes by using illumination light with a specific spatial structure, thereby obtaining images with higher resolution and optical sectioning capability.

[0003] The use of multiple dyes and staining schemes to achieve multicolor cell imaging is a very attractive feature of SIM imaging in cell biology applications. However, different wavelengths of emitted light caused by multicolor often require different widths of stripe periods to correspond to them in order to ensure that the structured light microscopic imaging system reaches the limit resolution. The existing structured light microscopic imaging method usually relies on a fixed phase difference assumption when solving the phase, which limits the selection range of the stripe period, and when the stripe period outside the range is selected, it affects the solving accuracy of the wave vector, thereby causing the problem of low accuracy of image reconstruction.

[0004] Therefore, how to improve the accuracy of image reconstruction is a problem that those skilled in the art need to solve. SUMMARY

[0005] Based on the above problems, the present application provides a full-wavelength limit resolution structured light microscopic imaging method and related products, which maintains the limit resolution of the system at any emitted light wavelength through the adjustment of the spatial light modulator, and then provides a frequency domain image multiplication algorithm to accurately solve the target wave vector under this condition, thereby improving the accuracy of image reconstruction.

[0006] In a first aspect, the embodiments of the present application provide a full-wavelength limit resolution structured light microscopic imaging method, comprising:

[0007] Determining a target stripe period of a structured light illumination stripe based on an emitted light wavelength;

[0008] Adjusting the structured light illumination stripe to the target stripe period based on a spatial light modulator, so that the structured light microscopic imaging system reaches the full-wavelength limit resolution;

[0009] Based on the structured light illumination stripe, obtaining a first frequency domain image, a second frequency domain image and a third frequency domain image corresponding to a target sample; the first frequency domain image, the second frequency domain image and the third frequency domain image correspond to the same stripe direction and different phases;

[0010] determine a target wave vector based on the first frequency domain image, the second frequency domain image and the third frequency domain image by using a frequency domain image multiplication algorithm;

[0011] reconstruct an image of the target sample based on the target wave vector.

[0012] Optionally, the target fringe period of the structured light illumination fringe is determined based on the wavelength of the emitted light, including:

[0013] the target spatial period size of the structured light illumination fringe is determined based on the wavelength of the emitted light;

[0014] the target fringe period of the structured light illumination fringe is determined based on the target spatial period size and a corresponding single-pixel size of the structured light microscopic imaging system.

[0015] Optionally, before the first frequency domain image, the second frequency domain image and the third frequency domain image corresponding to the target sample are acquired based on the structured light illumination fringe, the method further includes:

[0016] a target position of a target hole center point of a spatial filter is determined based on the target fringe period;

[0017] the target hole of the spatial filter is adjusted based on the target position.

[0018] Optionally, the target wave vector is determined based on the first frequency domain image, the second frequency domain image and the third frequency domain image by using the frequency domain image multiplication algorithm, including:

[0019] a first light intensity frequency domain distribution expression corresponding to the first frequency domain image, a second light intensity frequency domain distribution expression corresponding to the second frequency domain image, and a third light intensity frequency domain distribution expression corresponding to the third frequency domain image are respectively constructed;

[0020] the first light intensity frequency domain distribution expression, the second light intensity frequency domain distribution expression and the third light intensity frequency domain distribution expression are multiplied to obtain a pre-processing expression;

[0021] the pre-processing expression is optimized to obtain a target expression, with the goal of suppressing the contribution of interference terms and non-solution terms to the frequency domain energy spectrum;

[0022] the target wave vector is determined based on the target expression.

[0023] Optionally, the pre-processing expression is optimized with the goal of suppressing the contribution of interference terms and non-solution terms to the frequency domain energy spectrum, including:

[0024] noise terms in the pre-processing expression are optimized based on a signal-to-noise ratio;

[0025] optimizing a target sample light intensity frequency domain distribution item in the pre-processing expression based on a clipping function.

[0026] Optionally, the method further comprises:

[0027] determining a fluorescent protein for the target sample;

[0028] determining an emission light wavelength based on the fluorescent protein.

[0029] In a second aspect, an embodiment of the present application provides a full-wavelength limit resolution structured light microscopic imaging device, comprising:

[0030] a determination module configured to determine a target fringe period of a structured light illumination fringe based on the emission light wavelength;

[0031] an adjustment module configured to adjust the structured light illumination fringe to the target fringe period based on a spatial light modulator, so that a structured light microscopic imaging system reaches a full-wavelength limit resolution;

[0032] an acquisition module configured to acquire a first frequency domain image, a second frequency domain image and a third frequency domain image corresponding to a target sample based on the structured light illumination fringe; the first frequency domain image, the second frequency domain image and the third frequency domain image correspond to the same fringe direction and different phases;

[0033] a processing module configured to process the first frequency domain image, the second frequency domain image and the third frequency domain image based on a frequency domain image multiplication algorithm, and determine a target wave vector;

[0034] a reconstruction module configured to reconstruct an image of the target sample based on the target wave vector.

[0035] Optionally, the determination module is specifically configured to:

[0036] determine a target spatial period size of the structured light illumination fringe based on the emission light wavelength;

[0037] determine the target fringe period of the structured light illumination fringe based on the target spatial period size and a corresponding single-pixel size of the structured light microscopic imaging system.

[0038] In a third aspect, an embodiment of the present application provides a full-wavelength limit resolution structured light microscopic imaging device, comprising:

[0039] a memory configured to store a computer program;

[0040] a processor configured to execute the computer program to implement the steps of the full-wavelength limit resolution structured light microscopic imaging method as described above.

[0041] In a fourth aspect, the embodiments of the present application provide a readable storage medium, and the readable storage medium stores a computer program. The computer program is executed by a processor to implement the steps of the full-wavelength limit resolution structured light microscopic imaging method.

[0042] From the above technical solutions, compared with the prior art, the present application has the following advantages:

[0043] The present application first determines the target fringe period of the structured light illumination fringe based on the emission wavelength. Then, the structured light microscopic imaging system reaches the full-wavelength limit resolution based on the spatial light modulator adjusting the structured light illumination fringe to the target fringe period. And based on the structured light illumination fringe, the first frequency domain image, the second frequency domain image and the third frequency domain image corresponding to the target sample are obtained. Wherein, the first frequency domain image, the second frequency domain image and the third frequency domain image have the same fringe direction and different phases. Finally, the first frequency domain image, the second frequency domain image and the third frequency domain image are processed based on the frequency domain image multiplication algorithm to determine the target wave vector, and the target sample is image reconstructed based on the target wave vector. In this way, the system maintains the limit resolution at any emission wavelength through the adjustment of the spatial light modulator, and then provides the frequency domain image multiplication algorithm to accurately solve the target wave vector under this condition, and improves the accuracy of image reconstruction. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 A flowchart of a full-wavelength limit resolution structured light microscopic imaging method provided by the embodiments of the present application;

[0045] Figure 2 An extended frequency domain provided by the embodiments of the present application;

[0046] Figure 3 A structural schematic diagram of a full-wavelength limit resolution structured light microscopic imaging device provided by the embodiments of the present application. DETAILED DESCRIPTION

[0047] As described above, the existing structured light microscopic imaging method has the problem of low accuracy of image reconstruction. Specifically, the existing structured light microscopic imaging method relies on the assumption of fixed phase difference when solving the phase, which limits the selection range of the fringe period. However, different wavelengths of emission light caused by multicolor often need to use different width of fringe period to correspond, so as to ensure that the structured light microscopic imaging system reaches the limit resolution. Therefore, when the emission wavelength requires to select the fringe period out of its range, such solving method will affect the solving accuracy of the wave vector, and further cause the problem of low accuracy of image reconstruction.

[0048] To solve the above problems, the embodiment of the present application provides a full-wavelength limit resolution structured light microscopic imaging method, which comprises the following steps: firstly, determining a target fringe period of a structured light illumination fringe based on an emission wavelength. Then, adjusting the structured light illumination fringe to the target fringe period based on a spatial light modulator, so that the structured light microscopic imaging system reaches the full-wavelength limit resolution, and acquiring a first frequency domain image, a second frequency domain image and a third frequency domain image corresponding to a target sample based on the structured light illumination fringe. Wherein, the first frequency domain image, the second frequency domain image and the third frequency domain image correspond to the same fringe direction and different phases. Finally, processing the first frequency domain image, the second frequency domain image and the third frequency domain image based on a frequency domain image multiplication algorithm, determining a target wave vector, and reconstructing an image of the target sample based on the target wave vector.

[0049] In this way, the system maintains the limit resolution at any emission wavelength through the adjustment of the spatial light modulator, and then the frequency domain image multiplication algorithm is used to accurately solve the target wave vector under this condition, thereby improving the accuracy of image reconstruction.

[0050] It should be noted that the full-wavelength limit resolution structured light microscopic imaging method and related products provided by the embodiment of the present application can be applied to the field of optical microscopy. The above is only an example and does not limit the application field of the full-wavelength limit resolution structured light microscopic imaging method and related products provided by the present application.

[0051] In order to make the purpose, technical scheme and advantages of the embodiments of the present application clearer, the technical scheme of the embodiments of the present application will be described clearly and completely below in combination with the drawings in the embodiments of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0052] According to Abbe imaging principle, the essence of optical imaging system is that the incident light is diffracted by the object plane, and then a series of diffraction spots are formed on the lens focal plane. The spherical secondary waves emitted by each diffraction spot are coherently superimposed on the image plane, thereby forming an image. That is to say, the object contains information from low frequency to high frequency, but the aperture of the lens limits the passage of high frequency information. The characteristic that it only allows a part of low frequency information to pass leads to the blurring of the details of the imaging image. Specifically, for a non-coherent imaging system, the expression of the limit frequency that the optical system can pass is as follows:

[0053] ;

[0054] Wherein, is the limit frequency, is the numerical aperture of the objective lens, represents the laser wavelength. Further, the expression of the minimum resolvable spatial interval is as follows:

[0055] ;

[0056] wherein, is the minimum resolvable spatial interval. In summary, it can be understood that when different excitation light and different types of fluorescent proteins are used for fluorescent staining, the change of the emission wavelength will cause the change of the limit frequency and the minimum resolution. Therefore, in order to improve the limit frequency and achieve super-resolution effect, it is necessary to use structured light illumination stripes to expand the spectral information that can be passed by the target sample.

[0057] Figure 1 is a flowchart of a full-wavelength limit resolution structured light microscopic imaging method provided by an embodiment of the present application. In combination with Figure 1 , it can be understood that the full-wavelength limit resolution structured light microscopic imaging method provided by an embodiment of the present application can include:

[0058] S101: determining a target stripe period of the structured light illumination stripe based on the emission wavelength.

[0059] In actual application, when the structured light illumination is used, there is a direct proportional relationship between the stripe period of the structured light illumination stripe and the emission wavelength. In the embodiment of the present application, in order to achieve the super-resolution effect of different wavelengths, it is necessary to adjust the stripe period of the structured light illumination stripe according to the emission wavelength to adapt to the limit resolution requirement of different wavelengths. Before that, it is necessary to determine the target stripe period to which the structured light illumination stripe needs to be adjusted according to the emission wavelength.

[0060] In addition, since the ways of determining the target stripe period are not the same, the embodiment of the present application can describe one possible determination method.

[0061] In one case, S101: determining the target stripe period of the structured light illumination stripe based on the emission wavelength, can specifically include:

[0062] determining a target spatial period size of the structured light illumination stripe based on the emission wavelength;

[0063] determining the target stripe period of the structured light illumination stripe based on the target spatial period size and a corresponding single-pixel size of the structured light microscopic imaging system.

[0064] Figure 2 is an extended frequency domain diagram provided by an embodiment of the present application. In combination with Figure 2As shown, the middle circle (a) is the frequency domain information obtained without the structured light illumination stripe expansion, and the whole (three circles superimposed) is the frequency domain information obtained after the structured light illumination stripe expansion. It can be understood that under the same emission light and excitation light, the reconstructed picture resolution is different for different stripe periods, and the method provided in the embodiment of the application can achieve optimal results in the frequency domain expansion of each wavelength, so that the final result has more high-frequency information. The expression of the frequency after the structured light illumination stripe expansion and the minimum resolvable pitch is as follows:

[0065] ;

[0066] ;

[0067] wherein, is the limit frequency after the structured light illumination stripe expansion, is the limit frequency of the structured light illumination stripe, is the emission light wavelength, is the spatial period size of the structured light illumination stripe, is the minimum resolvable pitch after the structured light illumination stripe expansion, is the excitation light wavelength. At this time, the expression of the super-resolution magnification is as follows:

[0068] ;

[0069] wherein, is the super-resolution magnification, is the limit frequency without the structured light illumination stripe expansion, is the minimum resolvable pitch without the structured light illumination stripe expansion. It can be understood that in order to improve the super-resolution effect, the value of needs to be as small as possible, and needs to be met. Therefore, in combination with the above formula, when the emission light wavelength is known, the structured light microscopic imaging system reaches the full-wavelength limit resolution when , and the at this time is the target spatial period size corresponding to the emission light wavelength. Further, because the spatial period size divided by the single-pixel size is equal to the stripe period. Therefore, after the structured light microscopic imaging system is given and the target spatial period size corresponding to the emission light wavelength is obtained, the target stripe period of the structured light illumination stripe can be determined by the target spatial period size and the single-pixel size corresponding to the structured light microscopic imaging system.

[0070] In addition, because the determination method of the emission light wavelength is not the same, the embodiment of the application can be described in terms of one possible determination method.

[0071] In one case, the method further comprises:

[0072] determining a fluorescent protein for the target sample;

[0073] determining an emission light wavelength based on the fluorescent protein.

[0074] In practical applications, the relationship between the excitation light wavelength and the emission light wavelength follows the Stokes shift, and the excitation light wavelength is always smaller than the emission light wavelength. After the excitation light and the fluorescent protein used are determined, the emission light wavelength can be determined by querying the standard spectral data of the fluorescent protein.

[0075] S102: adjusting the structured light illumination fringe to the target fringe period based on the spatial light modulator, so that the structured light microscopic imaging system reaches the full-wavelength limit resolution.

[0076] In practical applications, because the spatial light modulator (SLM) can adapt to the polychromatic condition, the adjustment of the fringe period is performed by directly generating a related pattern and quickly projecting it onto the target sample, which has more advantages than the adjustment mode of replacing the grating or the digital micromirror device (DMD). Therefore, in combination with the target fringe period calculated according to the emission light wavelength, the SLM is used to quickly generate and adjust the structured light fringe pattern in the embodiments of the present application, and then the structured light illumination fringe with the target fringe period can be obtained after magnification by the optical system, so that the structured light microscopic imaging system reaches the full-wavelength limit resolution.

[0077] In addition, because the adjustment modes of the spatial filter are not the same, the embodiments of the present application can be described in terms of one possible adjustment mode.

[0078] In one case, before the first frequency domain image, the second frequency domain image and the third frequency domain image corresponding to the target sample are acquired based on the structured light illumination fringe, the method further comprises:

[0079] determining a target position of a target hole of the spatial filter from a center point based on the target fringe period;

[0080] adjusting the target hole of the spatial filter based on the target position.

[0081] In practical applications, in addition to adjusting the fringe period, the position of the hole center point of the spatial filter also needs to be appropriately modified, so as to ensure the maximum collection of image information when implementing super-resolution imaging of different wavelengths. Specifically, the expression of the hole center point position of the spatial filter is as follows:

[0082] ;

[0083] wherein, is the center point position of the pitch of the spatial filter, is the magnification of the optical system, is the focal length of the lens, , , and are the same as above, respectively the wavelength of the laser, the spatial period size of the structured light illumination fringe, the numerical aperture of the objective lens and the super-resolution magnification. It can be understood that when and are fixed, the modification of the fringe period will affect the center point position of the pitch of the spatial filter. Therefore, the position of the center point of the pitch of the spatial filter corresponding to the target fringe period can be determined based on the target fringe period obtained above. The position obtained at this time is recorded as a target position, and the hole corresponding to the target position is recorded as a target hole. Further, the target hole corresponding to the spatial filter is adjusted to the target position according to the target position, so as to complete the adjustment of the spatial filter.

[0084] S103: Based on the structured light illumination fringe, a first frequency domain image, a second frequency domain image and a third frequency domain image corresponding to the target sample are obtained; the fringe directions corresponding to the first frequency domain image, the second frequency domain image and the third frequency domain image are the same, and the phases are different.

[0085] In actual application, after adjusting the structured light illumination fringe to the target fringe period, the imaging system can obtain nine frequency domain images containing three fringe directions. Among them, each fringe direction corresponds to three frequency domain images with different phases. Therefore, in an embodiment of the present application, all the frequency domain images in each fringe direction are taken as a group, and the first frequency domain image, the second frequency domain image and the third frequency domain image corresponding to the target sample are obtained from the nine frequency domain images for subsequent calculation of the target wave vector. It can be understood that the first frequency domain image, the second frequency domain image and the third frequency domain image are the same group, that is, the fringe directions corresponding to the first frequency domain image, the second frequency domain image and the third frequency domain image are the same, and the phases are different.

[0086] S104: The first frequency domain image, the second frequency domain image and the third frequency domain image are processed based on the frequency domain image multiplication algorithm to determine the target wave vector.

[0087] In actual application, when a sinusoidal structured light fringe pattern is used as the illumination light intensity distribution, the frequency domain composition of the image of the same fringe direction can be represented by the following formula:

[0088] ;

[0089] Further, the energy spectrum expression of the frequency domain is as follows:

[0090]

[0091] In the formula, D(k) represents the light intensity frequency domain distribution collected by the detector, m represents the structure light fringe modulation, k represents the wave vector, S(k) represents the light intensity frequency domain distribution of the target sample, H(k) represents the optical transfer function, N(k) represents the noise frequency domain distribution, θ represents different wave vectors, represent different phases, represent the imaginary unit, represent the natural exponential function. In the embodiment of the present application, the main contribution term of the energy spectrum is as follows:

[0092]

[0093] The first interference term is as follows:

[0094]

[0095] The second interference term is as follows:

[0096]

[0097] And in the main contribution term of the energy spectrum, there is a term that is not helpful for solving the wave vector, that is, the non-solution term is as follows:

[0098]

[0099]

[0100] Thus, finally, the solution term is as follows:

[0101]

[0102] That is, by analyzing the energy spectrum in the frequency domain, the interference term, the non-solution term and the solution term can be determined, and the embodiment of the present application is to process the first frequency domain image, the second frequency domain image and the third frequency domain image by the frequency domain image multiplication algorithm, suppress the contribution of the interference term and the non-solution term to the frequency domain energy spectrum, and then accurately solve the wave vector, that is, the target wave vector.

[0103] In addition, since the way to determine the target wave vector is not the same, the embodiment of the present application can be described in terms of one possible determination method.

[0104] In one case, S104: processing the first frequency domain image, the second frequency domain image and the third frequency domain image based on the frequency domain image multiplication algorithm to determine the target wave vector, which can specifically include:

[0105] ​​​​​​a first light intensity frequency domain distribution expression corresponding to the first frequency domain image, a second light intensity frequency domain distribution expression corresponding to the second frequency domain image, and a third light intensity frequency domain distribution expression corresponding to the third frequency domain image are respectively constructed;

[0106] The first light intensity frequency domain distribution expression, the second light intensity frequency domain distribution expression, and the third light intensity frequency domain distribution expression are subjected to multiplication processing to obtain a pretreatment expression;

[0107] The pretreatment expression is optimized to obtain a target expression, with the goal of suppressing the contribution of interference terms and non-solution terms to the frequency domain energy spectrum;

[0108] A target wave vector is determined based on the target expression.

[0109] In actual applications, the embodiment of the present application proposes a method based on frequency domain image multiplication, which suppresses the numerical value of interference terms and improves the contribution of solution terms to the energy spectrum. Specifically, the first light intensity frequency domain distribution expression corresponding to the first frequency domain image, the second light intensity frequency domain distribution expression corresponding to the second frequency domain image, and the third light intensity frequency domain distribution expression corresponding to the third frequency domain image are as follows:

[0110]

[0111]

[0112]

[0113] wherein D(k) represents the light intensity frequency domain distribution collected by the detector, m represents the fringe modulation of the structured light, k represents the wave vector, S(k) represents the light intensity frequency domain distribution of the target sample, H(k) represents the optical transfer function, θ represents different wave vectors, represents the imaginary unit, represents the natural exponential function, and , and are the phases corresponding to the first frequency domain image, the second frequency domain image, and the third frequency domain image, respectively, and N1(k), N2(k), and N3(k) are the noise frequency domain distributions corresponding to the first frequency domain image, the second frequency domain image, and the third frequency domain image, respectively. Further, the first light intensity frequency domain distribution expression, the second light intensity frequency domain distribution expression, and the third light intensity frequency domain distribution expression are subjected to multiplication processing to obtain the following expression:

[0114]

[0115] wherein D multi (k) is a multiple multiplication light intensity frequency domain distribution, i, p, and q are index variables, Indicates containing The sum of terms. Furthermore, since the phase sum of the corresponding terms for the first, second, and third frequency domain images is 2... Therefore, the sum of the three middle terms in the above expression is zero, and we have the simplified expression, i.e., the preprocessed expression, as follows:

[0116]

[0117] Furthermore, with the goal of suppressing the contribution of interference terms and non-solvable terms to the frequency domain energy spectrum, the above preprocessing expression is optimized to obtain the target expression, and the accurate target wave vector can be obtained by solving the target expression.

[0118] Furthermore, since there are different ways to optimize preprocessed expressions, this application embodiment can describe one possible optimization method.

[0119] In one case, optimizing the preprocessing expression with the goal of suppressing the contributions of interference terms and non-solvable terms to the frequency domain energy spectrum includes:

[0120] The noise term in the preprocessing expression is optimized based on the signal-to-noise ratio;

[0121] The frequency domain distribution term of the target sample light intensity in the preprocessing expression is optimized based on the apodization function.

[0122] In practical applications, due to Indicates containing The sum of the terms represents the noise term. Therefore, increasing the signal-to-noise ratio can reduce this contribution, thereby optimizing the noise term in the preprocessing expression. Furthermore, in the preprocessing expression... Item and All items contain Item, and This causes the spectral signal to decrease from low frequency to high frequency. Therefore, this application introduces an apodization function to eliminate its extreme values ​​near zero, thereby optimizing the S(k) term in the preprocessing expression, i.e., optimizing the frequency domain distribution term of the target sample light intensity. The expression of the apodization function is as follows:

[0123] ;

[0124] In the formula, Let k denote the apodization function, and k denote the wave vector. Represents the natural constant. Let θ represent the independent variable and θ represent different wave vectors. The preprocessed expression combined with the apodization function is called the objective expression. In the objective expression, based on the first solution term: and the second solving item: will cause at and The specific value of the target wave vector can be obtained according to the characteristic that the second derivative has a maximum point at the target wave vector.

[0125] S105: image reconstruction of the target sample based on the target wave vector.

[0126] In actual application, the image reconstruction of the target sample can be realized by combining the target wave vector obtained above and the phases corresponding to the frequency domain images. The phases corresponding to the frequency domain images can be solved by any method, which is not limited in the present application.

[0127] In summary, the present application first determines the target fringe period of the structured light illumination fringe based on the wavelength of the emitted light. Then the structured light illumination fringe is adjusted to the target fringe period based on the spatial light modulator, so that the structured light microscopic imaging system reaches the full-wavelength limit resolution. Based on the structured light illumination fringe, the first frequency domain image, the second frequency domain image and the third frequency domain image corresponding to the target sample are obtained. The fringe directions of the first frequency domain image, the second frequency domain image and the third frequency domain image are the same, and the phases are different. Finally, the first frequency domain image, the second frequency domain image and the third frequency domain image are processed based on the frequency domain image multiplication algorithm to determine the target wave vector, and the target sample is image reconstructed based on the target wave vector. In this way, the system maintains the limit resolution at any wavelength of the emitted light through the adjustment of the spatial light modulator, and then provides the frequency domain image multiplication algorithm to accurately solve the target wave vector under this condition, thereby improving the accuracy of image reconstruction.

[0128] Figure 3 A structural schematic diagram of a full-wavelength limit resolution structured light microscopic imaging device provided by an embodiment of the present application is shown. As shown in Figure 3 The full-wavelength limit resolution structured light microscopic imaging device 300 can include:

[0129] A determination module 301 is configured to determine the target fringe period of the structured light illumination fringe based on the wavelength of the emitted light.

[0130] An adjustment module 302 is configured to adjust the structured light illumination fringe to the target fringe period based on the spatial light modulator, so that the structured light microscopic imaging system reaches the full-wavelength limit resolution.

[0131] An acquisition module 303 is configured to acquire the first frequency domain image, the second frequency domain image and the third frequency domain image corresponding to the target sample based on the structured light illumination fringe. The fringe directions of the first frequency domain image, the second frequency domain image and the third frequency domain image are the same, and the phases are different.

[0132] The processing module 304 is configured to process the first frequency domain image, the second frequency domain image and the third frequency domain image based on a frequency domain image multiplication algorithm, and determine a target wave vector.

[0133] The reconstruction module 305 is configured to perform image reconstruction on the target sample based on the target wave vector.

[0134] As an implementation form, for how to determine the target fringe period, the determination module 301 is specifically configured to:

[0135] determine a target spatial period size of the structured light illumination fringe based on the wavelength of the emitted light;

[0136] determine the target fringe period of the structured light illumination fringe based on the target spatial period size and a corresponding single-pixel size of the structured light microscopic imaging system.

[0137] As an implementation form, for how to adjust the optical system, the full-wavelength-limited resolution structured light microscopic imaging apparatus 300 further includes an adjustment module.

[0138] The adjustment module is configured to determine a target position of a target hole distance center point of a spatial filter based on the target fringe period.

[0139] adjust the target hole corresponding to the spatial filter based on the target position.

[0140] As an implementation form, for how to determine the target wave vector, the processing module 304 includes a construction module, a multiplication module, an optimization module and a calculation module.

[0141] The construction module is configured to construct a first light intensity frequency domain distribution expression corresponding to the first frequency domain image, a second light intensity frequency domain distribution expression corresponding to the second frequency domain image, and a third light intensity frequency domain distribution expression corresponding to the third frequency domain image, respectively.

[0142] The multiplication module is configured to perform multiplication processing on the first light intensity frequency domain distribution expression, the second light intensity frequency domain distribution expression and the third light intensity frequency domain distribution expression to obtain a pre-processing expression.

[0143] The optimization module is configured to optimize the pre-processing expression to obtain a target expression, with the goal of suppressing the contribution of interference terms and non-solution terms to frequency domain energy spectrum.

[0144] The calculation module is configured to determine a target wave vector based on the target expression.

[0145] As an implementation form, for how to optimize the pre-processing expression, the optimization module is specifically configured to:

[0146] Optimizing a noise term in the pre-processing expression based on a signal-to-noise ratio;

[0147] Optimizing a target sample light intensity frequency domain distribution term in the pre-processing expression based on an apodization function.

[0148] As an implementation, the full-wavelength-limited-resolution structured light microscopic imaging apparatus 300 further comprises a determination sub-module for determining how to determine the emission light wavelength.

[0149] The determination sub-module is configured to determine a fluorescent protein for the target sample.

[0150] The emission light wavelength is determined based on the fluorescent protein.

[0151] In summary, the present application first determines the target fringe period of the structured light illumination fringe based on the emission light wavelength. Then, the structured light illumination fringe is adjusted to the target fringe period based on the spatial light modulator, so that the structured light microscopic imaging system reaches the full-wavelength-limited-resolution, and the first frequency domain image, the second frequency domain image and the third frequency domain image corresponding to the target sample are obtained based on the structured light illumination fringe. The first frequency domain image, the second frequency domain image and the third frequency domain image correspond to the same fringe direction and different phases. Finally, the first frequency domain image, the second frequency domain image and the third frequency domain image are processed based on the frequency domain image multiplication algorithm to determine the target wave vector, and the target sample is image reconstructed based on the target wave vector. In this way, the system maintains the limited resolution at any emission light wavelength through the adjustment of the spatial light modulator, and then provides the frequency domain image multiplication algorithm to accurately solve the target wave vector under this condition, thereby improving the accuracy of image reconstruction.

[0152] In addition, the present application further provides a full-wavelength-limited-resolution structured light microscopic imaging device, comprising a memory for storing a computer program, and a processor for executing the computer program to realize the steps of the full-wavelength-limited-resolution structured light microscopic imaging method as described above.

[0153] In addition, the present application further provides a readable storage medium having a computer program stored thereon, and the computer program is executed by a processor to realize the steps of the full-wavelength-limited-resolution structured light microscopic imaging method as described above.

[0154] The above description of disclosed embodiments enables one of ordinary skill in the art to make or use the application. Various modifications to these embodiments will be readily apparent to those of ordinary skill in the art, and the generic principles defined herein can be applied to other embodiments without departing from the spirit or scope of the application. Thus, the present application is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A full-wavelength-limited resolution structured light microscopy imaging method, characterized in that, The method includes: Determine the target fringe period of structured light illumination fringes based on the emitted light wavelength; The structured light illumination fringes are adjusted to the target fringe period using a spatial light modulator, enabling the structured light microscopy imaging system to achieve full-wavelength limit resolution. Based on the structured light illumination stripes, a first frequency domain image, a second frequency domain image, and a third frequency domain image corresponding to the target sample are obtained; the stripes in the first frequency domain image, the second frequency domain image, and the third frequency domain image have the same direction but different phases; The first frequency domain image, the second frequency domain image, and the third frequency domain image are processed based on the frequency domain image accumulation multiplication algorithm to determine the target wave vector; The target sample is reconstructed based on the target wave vector.

2. The method according to claim 1, characterized in that, The determination of the target fringe period of structured light illumination fringes based on the emitted light wavelength includes: The target spatial period size of the structured light illumination stripes is determined based on the emitted light wavelength. The target fringe period of the structured light illumination stripes is determined based on the target spatial period size and the corresponding single pixel size of the structured light microscopy imaging system.

3. The method according to claim 1, characterized in that, Before acquiring the first frequency domain image, second frequency domain image, and third frequency domain image corresponding to the target sample based on the structured light illumination stripes, the method further includes: The target position of the spatial filter corresponding to the target aperture distance from the center point is determined based on the target fringe period. Adjust the target aperture corresponding to the spatial filter based on the target position.

4. The method according to claim 1, characterized in that, The frequency domain image accumulation algorithm processes the first frequency domain image, the second frequency domain image, and the third frequency domain image to determine the target wave vector, including: Construct the first light intensity frequency domain distribution expression corresponding to the first frequency domain image, the second light intensity frequency domain distribution expression corresponding to the second frequency domain image, and the third light intensity frequency domain distribution expression corresponding to the third frequency domain image, respectively. The first light intensity frequency domain distribution expression, the second light intensity frequency domain distribution expression, and the third light intensity frequency domain distribution expression are multiplied together to obtain a preprocessed expression. With the goal of suppressing the contribution of interference terms and non-solvable terms to the frequency domain energy spectrum, the preprocessed expression is optimized to obtain the target expression; The target wave vector is determined based on the target expression.

5. The method according to claim 4, characterized in that, The optimization of the preprocessing expression, aimed at suppressing the contributions of interference terms and non-solvable terms to the frequency domain energy spectrum, includes: The noise term in the preprocessing expression is optimized based on the signal-to-noise ratio; The frequency domain distribution term of the target sample light intensity in the preprocessing expression is optimized based on the apodization function.

6. The method according to claim 1, characterized in that, The method further includes: Identify the fluorescent protein for the target sample; The wavelength of emitted light is determined based on the fluorescent protein.

7. A full-wavelength-limiting resolution structured light microscopy imaging device, characterized in that, include: A determination module is used to determine the target fringe period of structured light illumination fringes based on the emitted light wavelength; An adjustment module is used to adjust the structured light illumination stripes to the target stripe period based on the spatial light modulator, so that the structured light microscopy imaging system can achieve the full wavelength limit resolution. The acquisition module is used to acquire a first frequency domain image, a second frequency domain image, and a third frequency domain image corresponding to the target sample based on the structured light illumination stripes; the stripes in the first frequency domain image, the second frequency domain image, and the third frequency domain image have the same direction but different phases. The processing module is used to process the first frequency domain image, the second frequency domain image, and the third frequency domain image based on the frequency domain image accumulation algorithm to determine the target wave vector; The reconstruction module is used to reconstruct the image of the target sample based on the target wave vector.

8. The full-wavelength-limited resolution structured light microscopy imaging device according to claim 7, characterized in that, The determining module is specifically used for: The target spatial period size of the structured light illumination stripes is determined based on the emitted light wavelength. The target fringe period of the structured light illumination stripes is determined based on the target spatial period size and the corresponding single pixel size of the structured light microscopy imaging system.

9. A full-wavelength-limiting resolution structured light microscopy imaging device, characterized in that, include: Memory, used to store computer programs; A processor, configured to execute the computer program to implement the steps of the full-wavelength limit resolution structured light microscopy imaging method as described in any one of claims 1 to 6.

10. A readable storage medium, characterized in that, The readable storage medium stores a computer program that, when executed by a processor, implements the steps of the full-wavelength limit resolution structured light microscopy imaging method as described in any one of claims 1 to 6.

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