Scanning structured light microscopic imaging system and method based on multi-focus diffraction-free light beams

Abstract

The invention relates to the technical field of optical microscopic imaging, and discloses a scanning structured light microscopic imaging system and method based on a multi-focus diffraction-free light beam. Exciting light emitted by a laser light source sequentially passes through an electro-optical modulator and a lens group I and then enters a spatial light modulator; the spatial light modulator is used for synchronously regulating and controlling the multi-focus array and the non-diffraction light beam to generate the multi-focus array with the non-diffraction light beam characteristic; afterwards, the light sequentially passes through a lens group II, a two-dimensional scanning galvanometer, a scanning lens, a tube lens, an emission optical filter, a double-color piece and an objective lens and then irradiates a sample to be imaged; fluorescent light generated by a to-be-imaged sample passes through the objective lens and the double-color sheet and then is transmitted to the area-array camera, and fluorescent light signals at a plurality of focal points are collected to form a fluorescent light image. According to the invention, a diffraction-free optical phase modulation mechanism is introduced, simultaneous imaging of different depth layers is realized, the imaging speed is improved, sample information missed due to limited focusing depth is avoided, and the optical power utilization rate and the imaging quality are greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of optical microscopic imaging, and in particular to a scanning structured light microscopic imaging system and method based on multi-focus non-diffraction light beams. Background Art

[0002] The breakthrough in super-resolution imaging technology has effectively overcome the diffraction limitation of traditional optical microscopes, allowing subcellular structures to be visualized with unprecedented precision. Among the many related super-resolution microscopy techniques, structured illumination microscopy (SIM) achieves optical super-resolution by restoring structural details of high spatial frequencies through spatial frequency mixing. Currently, SIM has developed into one of the most widely used super-resolution imaging techniques, with the advantages of significantly improving resolution and being independent of fluorescent labeling.

[0003] In particular, scanning structured light super-resolution microscopy (SSIM) has broken through the limitation of SIM technology that the resolution is only improved by two times by further expanding the lateral resolution of traditional wide-field structured illumination microscopy (SIM). This technology modulates the intensity of the excitation light and excites the fluorescent sample point by point, so that the high-frequency information of the sample is mixed into the microscopic system through the moiré effect. After precise detection, demodulation and image reconstruction, the ability to detect high-frequency information beyond the diffraction limit is successfully achieved. In theory, the imaging spatial resolution of SSIM technology is no longer limited by the diffraction limit and has the potential for unlimited improvement. However, in order to achieve the improvement in resolution, the system needs to collect images from multiple directions and phases for super-resolution reconstruction, which inevitably sacrifices the temporal resolution while improving the spatial resolution. In addition, although SSIM has excellent tomographic imaging capabilities, it can only obtain information on a single plane and cannot fully capture the multi-plane structure of the sample.

[0004] In order to solve the above problems, the applicant has proposed a multi-focus scanning structured light super-resolution microscope (MSSIM), which can quickly collect multiple focuses while achieving super-resolution imaging. MSSIM generates a multi-focus excitation pattern by constructing a focus array that is evenly distributed and of uniform size. However, the traditional method of generating a light focus array has many limitations, such as poor array uniformity, or light power dispersion when generating multiple focuses, resulting in poor imaging effects. Although MSSIM effectively improves the imaging speed, it still has limitations in fully capturing the multi-faceted structure of the sample. Therefore, how to solve the problems of low temporal resolution and inability to effectively capture depth information in the existing scanning structured light super-resolution microscopy technology has become an important research topic in this field. Summary of the invention

[0005] The purpose of the present invention is to provide a scanning structured light microscopy imaging system and method based on a multi-focus non-diffraction light beam. On the basis of MSSIM, a non-diffraction light phase modulation mechanism is introduced, and simultaneous imaging of different depth levels is achieved by using the non-diffraction light beam. While improving the imaging speed, it can also avoid the loss of sample information due to limited focusing depth, greatly improving the optical power utilization and imaging quality.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] On the one hand, the present invention provides a scanning structured light microscopic imaging system based on a multi-focus non-diffraction beam, the system comprising: a laser light source, an electro-optic modulator, a lens group 1, a spatial light modulator, a lens group 2, a two-dimensional scanning galvanometer, a scanning lens, a tube lens, an emission filter, a dichroic film, an objective lens, and an area array camera arranged in sequence along an excitation light path;

[0008] The laser light source is used to emit excitation light; the excitation light emitted by the laser light source passes through the electro-optic modulator and the lens group 1 in sequence and is incident on the spatial light modulator, and the spatial light modulator is used to synchronously control the multi-focal array and the non-diffraction light beam to generate a multi-focal array with non-diffraction light beam characteristics, and control the generation positions of multiple focal points and the propagation characteristics of the excitation light through phase modulation;

[0009] The excitation light modulated by the spatial light modulator passes through lens group 2, a two-dimensional scanning galvanometer, a scanning lens, a tube lens, an emission filter, a dichroic plate, and an objective lens in sequence, and then irradiates the sample to be imaged. The excitation light interacts with the fluorescent marker in the sample to be imaged, and excites the sample to be imaged to generate fluorescence.

[0010] The fluorescence generated by the sample to be imaged passes through the objective lens and the dichroic film and is then transmitted to the area array camera. The area array camera collects fluorescence signals at multiple focal points to form a fluorescence image.

[0011] Furthermore, the method for generating the multi-focal array is as follows:

[0012] First, a light focus array of a set size is generated according to the size of the spatial light modulator;

[0013] Next, a zero-frequency point in the light focus array is selected, and the light intensity at the point is weakened to a preset value;

[0014] Then, the weakened light intensity is evenly distributed to other light focal points in the light focal point array.

[0015] After the light intensity is redistributed, the WGS iterative algorithm is entered to optimize the phase distribution of the light field through multiple phase modulations and Fourier transforms, and finally the required intensity distribution of the light focus array is achieved to obtain a multi-focus array.

[0016] Furthermore, the generating of the multi-focal array having the non-diffraction beam characteristic specifically comprises the following steps:

[0017] First, a diffraction-free beam is generated based on a set phase pattern;

[0018] Secondly, through phase modulation, the diffraction-free beam is superimposed on each focal position in the multi-focal array, so that each focal position has the characteristics of a diffraction-free beam, and finally a multi-focal array with the characteristics of a diffraction-free beam is generated.

[0019] Furthermore, a first half-wave plate is arranged between the laser light source and the electro-optical modulator, and the first half-wave plate is used to adjust the polarization state of the excitation light; a first reflector is also arranged between the electro-optical modulator and lens group one, and the excitation light adjusted by the electro-optical modulator is reflected to lens group one through the first reflector.

[0020] Furthermore, the lens group 1 includes a first lens and a second lens, the radius of the first lens is smaller than the radius of the second lens, and the excitation light incident on the lens group 1 passes through the first lens and the second lens in sequence to be magnified and collimated;

[0021] Furthermore, the lens group two includes a third lens and a fourth lens, the radius of the third lens is greater than the radius of the fourth lens, and the lens group two is used to reduce the size and collimate the excitation light that is the superposition of the Airy beam and the excitation light, so that the beam size of the excitation light is the same as the size of the excitation light before passing through the lens group one.

[0022] Furthermore, a second half-wave plate is arranged between the lens group 1 and the spatial light modulator, and the second half-wave plate is used to adjust the polarization direction of the excitation light emitted through the lens group 1 so that the polarization direction of the excitation light is parallel to the liquid crystal plane of the spatial light modulator.

[0023] Furthermore, a second reflector is provided between the lens group 2 and the two-dimensional scanning galvanometer, and the second reflector is used to change the optical path direction of the excitation light emitted through the lens group 2 so that the optical path direction of the excitation light is spatially conjugate with the two-dimensional scanning galvanometer.

[0024] Furthermore, a bandpass filter and a converging lens are arranged between the dichroic film and the area array camera. The fluorescence signal emitted by the dichroic film is focused to the area array camera through the converging lens after stray light is filtered out by the bandpass filter.

[0025] On the other hand, the present invention further provides a scanning structured light microscopic imaging method based on a multi-focus non-diffraction beam, which is applied to the above-mentioned scanning structured light microscopic imaging system based on a multi-focus non-diffraction beam, and includes the following steps:

[0026] The laser light source emits excitation light;

[0027] The excitation light is modulated by an electro-optic modulator and then magnified and collimated by a lens group 1;

[0028] The excitation light passing through the first lens group is incident on a spatial light modulator loaded with an Airy beam mask, and is modulated by the spatial light modulator using zero-order light to generate a multi-focal array with non-diffraction beam characteristics. The generation positions of multiple focal points and the propagation characteristics of the excitation light are controlled through phase modulation.

[0029] Next, the excitation light is reduced in size and collimated by lens group 2, and then incident on the two-dimensional scanning galvanometer, and then scanned point by point by the two-dimensional scanning galvanometer. After that, the excitation light passes through the scanning lens, tube lens, and emission filter, and then irradiates the surface of the sample to be imaged through the dichroic plate and objective lens. Then, the excitation light interacts with the fluorescent marker in the sample to be imaged, and excites the fluorescent marker to produce fluorescence;

[0030] The fluorescence generated by the sample to be imaged is split by a two-color filter and transmitted to the area array camera. The area array camera collects fluorescence signals at multiple focal points to form a fluorescence image.

[0031] According to the specific embodiments provided by the present invention, the present invention discloses the following technical effects:

[0032] The present invention proposes a solution for scanning structured light super-resolution microscopy (AMSSIM) based on multi-focus non-diffraction beams. The system introduces a non-diffraction light phase modulation mechanism on the basis of MSSIM, and uses non-diffraction beams such as Airy light and Bessel light to achieve simultaneous imaging at different depth levels. Specifically, AMSSIM can completely capture the multi-layer structure information of the sample in a single scanning process by superimposing non-diffraction beams in multiple foci and synchronously regulating them, thereby significantly improving imaging efficiency and depth resolution. The present invention optimizes the phase distribution, and the method can form a uniform and efficient light focus array, greatly improving the optical power utilization and imaging quality.

[0033] Compared with traditional methods, AMSSIM can improve imaging speed while avoiding the loss of sample information due to limited focus depth. In addition, combined with image reconstruction algorithms, AMSSIM can further enhance the ability to analyze the three-dimensional structure of samples while maintaining high spatial resolution. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0035] Figure 1 A schematic diagram of the structure of a scanning structured light microscopic imaging system based on a multi-focus non-diffraction light beam provided by the present invention;

[0036] Figure 2 The schematic diagram of the multi-focal array of the present invention, wherein (a) is the multi-focal array, the middle focus is the zero-order point, and (b) is the multi-focal simulation diagram finally generated;

[0037] Figure 3 It is a multi-focus array diagram with non-diffraction beam characteristics of the present invention;

[0038] Explanation of the reference numerals: 1. laser light source; 2. first half-wave plate; 3. electro-optic modulator; 4. reflector; 5. first lens; 6. second lens; 7. second half-wave plate; 8. spatial light modulator; 9. third lens; 10. fourth lens; 11. second reflector; 12. two-dimensional scanning galvanometer; 13. scanning lens; 14. tube lens; 15. emission filter; 16. dichroic film; 17. objective lens; 18. sample to be imaged; 19. bandpass filter; 20. converging lens; 21. area array camera. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] The scanning structured light microscopy imaging system and method based on a multi-focus non-diffraction beam provided by the present invention significantly improves the ability to capture the three-dimensional structural information of the sample and the rapid imaging capability by introducing a non-diffraction beam (such as Airy light) and a multi-focus phase modulation mechanism, and solves the bottleneck of traditional super-resolution microscopy imaging technology in depth analysis. This technology provides an efficient and accurate solution for three-dimensional imaging of complex samples in the fields of life sciences, materials sciences, etc.

[0041] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the present invention is further described in detail below with reference to the accompanying drawings and specific embodiments.

[0042] like Figure 1As shown, the scanning structured light microscopy imaging system based on multi-focus non-diffraction beams provided by an embodiment of the present invention includes: a laser light source 1, a first half-wave plate 2, an electro-optic modulator 3, a first reflector 4, a lens group 1, a second half-wave plate 7, a spatial light modulator 8, a lens group 2, a second reflector 11, a two-dimensional scanning galvanometer 12, a scanning lens 13, a tube lens 14, an emission filter 15, a dichroic filter 16, and an objective lens 17, which are sequentially arranged along the excitation light path, and also include a bandpass filter 19, a converging lens 20, and an area array camera 21;

[0043] Wherein, the laser light source 1 is used to emit excitation light; the wavelength of the excitation light is selected according to the properties of the fluorescent marker of the sample to be imaged to ensure the best excitation effect;

[0044] The excitation light emitted by the laser light source 1 passes through the first half-wave plate 2, the electro-optic modulator 3, the first reflector 4, the lens group 1, and the second half-wave plate 7 in sequence and is incident on the spatial light modulator 8. The spatial light modulator 8 is used to synchronously control the multi-focal array and the non-diffraction light beam to generate a multi-focal array with non-diffraction light beam characteristics, and control the generation positions of multiple focal points and the propagation characteristics of the excitation light through phase modulation;

[0045] The excitation light modulated by the spatial light modulator 8 passes through the second lens group, the second reflector 11, the two-dimensional scanning galvanometer 12, the scanning lens 13, the tube lens 14, the emission filter 15, the dichroic filter 16, and the objective lens 17 in sequence, and then irradiates the sample 18 to be imaged. The excitation light interacts with the fluorescent marker in the sample 18 to be imaged, and excites the sample 18 to generate fluorescence.

[0046] The fluorescence generated by the sample 18 to be imaged passes through the objective lens 17 and the dichroic film 16, and after the stray light is filtered out by the bandpass filter 19, it is focused to the area array camera 21 through the converging lens 20. The area array camera 21 collects fluorescence signals at multiple focal points to form a fluorescence image.

[0047] Among them, the image sensor of the area array camera 21 is used to capture the image, and the image sensor type commonly used is CCD (charge coupled device) or CMOS (complementary metal oxide semiconductor). On the surface of the image sensor, each tiny photosensitive element (pixel) captures the light transmitted from the converging lens 20 and converts it into an electrical signal. The electrical signal is then transmitted to the processor of the area array camera 21 for analog-to-digital conversion (ADC), converting the analog signal into a digital signal, and uploading it to a terminal such as a computer for subsequent image reconstruction and other processing. The present application loads a multi-focal array through SLM, and simultaneously captures the fluorescence signal at each focus through the area array camera to form a fluorescent image.

[0048] In the above system structure, the first half-wave plate 2 is used to adjust the polarization state of the excitation light so that the polarization direction of the excitation light matches the polarization direction of the electro-optical modulator 3; the electro-optical modulator 3 modulates the excitation light into a modulated light beam whose light intensity varies periodically with time according to a sinusoidal function; the optically excited light generates a sinusoidal intensity modulated light beam with a significant modulation depth through the first half-wave plate 2 and the electro-optical modulator 3.

[0049] The lens group 1 includes a first lens 5 and a second lens 6. The radius of the first lens 5 is smaller than the radius of the second lens 6. The excitation light incident on the lens group 1 passes through the first lens 5 and the second lens 6 in sequence to be magnified and collimated.

[0050] To ensure the beam modulation effect, the second half-wave plate 7 is arranged between the lens group 1 and the spatial light modulator 8. The second half-wave plate 7 is used to adjust the polarization direction of the excitation light emitted through the lens group 1 so that the polarization direction of the excitation light is parallel to the liquid crystal surface of the spatial light modulator 8. Spatial light modulator (SLM) 8: One of the core components, responsible for synchronously regulating the multi-focal array and the non-diffraction beam to generate a specific light field distribution to achieve multi-level sample imaging.

[0051] After passing through the SLM, the light beam needs to be further reduced in size to ensure that the light beam characteristics are suitable for the subsequent scanning and imaging units. The second lens group includes a third lens 9 and a fourth lens 10, the radius of the third lens 9 is greater than the radius of the fourth lens 10, and the second lens group is used to reduce the size and collimate the excitation light superimposed by the Airy beam and the excitation light, so that the beam size of the excitation light is the same as the size of the excitation light before passing through the first lens group.

[0052] A second reflector 11 is arranged between the lens group 2 and the two-dimensional scanning galvanometer 12, and the second reflector 11 is used to change the optical path direction of the excitation light emitted through the lens group 2, so that the optical path direction of the excitation light is spatially conjugate with the two-dimensional scanning galvanometer 12. The optical path of the excitation light forms a conjugate relationship with the scanning plane of the two-dimensional scanning galvanometer. The two-dimensional scanning galvanometer 12 quickly changes the optical path direction of the excitation light so that the excitation light performs a two-dimensional scan on the surface of the sample to be imaged. The two-dimensional scanning galvanometer 12 is usually composed of two mutually perpendicular galvanometers, which control the scanning of the light beam on the X-axis and the Y-axis respectively. Due to the spatial conjugate relationship between the excitation light and the two-dimensional scanning galvanometer 12, high-precision scanning can be achieved, and each scanning point can accurately correspond to a position on the sample to be imaged, realizing the spatial scanning of the light beam, covering different positions of the sample to be imaged, and ensuring the collection of global information.

[0053] Afterwards, the scanning lens 13 and the tube lens 14 are matched after the emission filter 15 to ensure that the entrance pupil of the two-dimensional scanning galvanometer 12 and the objective lens 17 are conjugated to achieve precise scanning imaging. The fluorescence signal is collected by the same objective lens 17 and separated from the main beam path using a dichroic filter 16. The fluorescence signal is focused to the area array camera (PMT) 21 via a converging lens 20 for detection. A bandpass filter 19 is set before the converging lens 2020 to filter out any residual stray light. By controlling the opening and closing of the spatial light modulator 8, seamless switching between beam modes (excitation light, Airy + excitation light) is achieved.

[0054] The scanning lens 13 and the tube lens 14 work together to expand the light beam so that it matches the aperture of the objective lens to ensure a good imaging effect. The emission filter 15 is used to filter stray light to ensure that the excitation light in the system optical path is pure and correct, thereby improving the imaging quality.

[0055] The dichroic filter 16 performs spectral splitting on the light beam, effectively separating the signal light from the excitation light, and forming a light field of the sample at the rear focal plane of the objective lens. The objective lens 17 is responsible for focusing the excitation light beam and collecting the fluorescence signal from the sample, as one of the key components of the imaging system.

[0056] Bandpass filter 19: Filters out excitation light and other stray light, retaining only the fluorescence signal of the sample to ensure the accuracy of the test results. Converging lens 20 converges the collected fluorescence signal into the area array camera to ensure the efficiency and integrity of the light signal transmission. Area array camera 21 records the fluorescence signal from the sample to form image data for subsequent image reconstruction and analysis.

[0057] The system also includes a computer, and the electro-optic modulator, spatial light modulator, two-dimensional scanning galvanometer and array camera are controlled by the computer. All devices are connected to the computer through a high-speed data acquisition card. The computer uses a LabVIEW program to drive the components to work in a coordinated manner, thereby achieving synchronous operation. The control of each device by the computer can refer to the relevant technology in the field, and this application will not be repeated.

[0058] The system achieves fast super-resolution imaging and obtains multi-layer structural information of samples by combining a non-diffraction beam with multiple focal points, significantly improving imaging efficiency and depth resolution capabilities. The present invention achieves fast super-resolution imaging and obtains multi-layer structural information of samples by combining a non-diffraction beam with multiple focal points. In addition, the intensity of the system's excitation light is sinusoidally modulated to ensure high resolution of the obtained image.

[0059] The present invention also provides a scanning structured light microscopic imaging method based on a multi-focus non-diffraction beam, which is applied to the above-mentioned scanning structured light microscopic imaging system based on a multi-focus non-diffraction beam, and includes the following steps:

[0060] The laser light source 1 emits excitation light;

[0061] The excitation light is modulated by the electro-optic modulator 3, and then magnified and collimated by the lens group 1;

[0062] The excitation light passing through the lens group 1 is incident on the spatial light modulator 8 loaded with the Airy beam mask. The spatial light modulator 8 uses zero-order light to synchronously control the multi-focal array and the non-diffraction beam to generate a multi-focal array with non-diffraction beam characteristics. Through phase modulation, the generation positions of multiple focal points and the propagation characteristics of the excitation light are controlled.

[0063] Next, the excitation light is reduced in size and collimated by lens group 2, and then incident on the two-dimensional scanning galvanometer 12, and then scanned point by point by the two-dimensional scanning galvanometer 12. After that, the excitation light passes through the scanning lens 13, the tube lens 14 and the emission filter 15, and then irradiates the surface of the sample to be imaged 18 through the dichroic film 16 and the objective lens 17. Then, the excitation light interacts with the fluorescent marker in the sample to be imaged 18, and excites the fluorescent marker to produce fluorescence;

[0064] The fluorescence generated by the sample 18 to be imaged is split by the dichroic filter 16 and transmitted to the area array camera 21. The area array camera 21 collects fluorescence signals at multiple focal points to form a fluorescence image.

[0065] Specifically, the present invention provides a method for synchronously controlling a multi-focal array and a non-diffraction beam, comprising:

[0066] 1. The phase modulation method of SLM realizes the generation of uniform light focus array while improving the light energy utilization and image quality.

[0067] The conventional SLM phase modulation method is to filter out the zero-order light and retain the first-order light for modulation. The main reason is that when the SLM performs phase modulation, the zero-order light usually represents a light beam that has not been effectively modulated. It is the part of the light beam that still retains the original phase information after passing through the SLM, and usually does not participate in the construction of the desired modulated light field or image information. Therefore, the zero-order light will cause interference, reduce the imaging quality, and blur the modulated optical signal. In order to ensure a pure modulated light field, the zero-order light is usually filtered out, and only the first-order light or higher-order diffracted light is retained to achieve better modulation effects and higher imaging resolution. However, the zero-order light is the part with the highest power in the SLM. If it is filtered out directly, the utilization rate of light energy will be significantly reduced, resulting in a decrease in imaging efficiency. Therefore, the present invention proposes a new phase modulation strategy, which optimizes and reuses the zero-order light to avoid direct filtering, thereby improving the utilization rate of light energy. This method can not only ensure the modulation accuracy, but also achieve a more efficient imaging process while maintaining a high light intensity. It is particularly suitable for super-resolution microscopy imaging systems that require high power and high resolution. Specific method:

[0068] The present invention provides a method for generating a 3×3 light focus array, and the specific steps are as follows: First, generate a 3×3 light focus array according to the size of the SLM. Next, select the zero-frequency point in the array, and weaken the light intensity of the point to a preset value, for example, weaken it to 50% of the initial light intensity. Subsequently, the weakened light intensity is evenly distributed to other light focal points in the array to ensure the effective utilization of the total light energy. After the light intensity is redistributed, enter the WGS (wavefront Gerchberg-Saxton) iterative algorithm, and optimize the phase distribution of the light field through multiple phase modulations and Fourier transforms to finally achieve the desired light focus array intensity distribution. The method of the present invention can improve the light energy utilization rate of other light focal points while weakening the zero-frequency point light intensity, thereby improving the overall efficiency of the system. The principle of the generated point array is as follows Figure 2 As shown, the red focus in the middle of Figure (a) is the zero-order point, and Figure (b) is the final generated multi-focus simulation image.

[0069] 2. The present invention further proposes a method for generating a multi-focal array with non-diffraction beam characteristics.

[0070] Taking the non-diffraction Airy beam as an example, we first use MATLAB simulation to generate the non-diffraction Airy beam in the computer. The electric field distribution of the Airy beam is as follows:

[0071]

[0072] The symbol Ai stands for the Airy function, which is a special function for solving nonlinear differential equations and usually appears in the description of accelerated beams and diffraction phenomena. a is the attenuation coefficient, which controls the attenuation and amplitude of the beam, and i represents the imaginary term, which is related to the phase and is used to describe the phase change of the light wave. This formula describes an Airy beam that propagates at an accelerated speed along the x and y axes. The intensity and phase of the beam are affected by the parameters a, e, the spatial position x, y, and the imaginary phase term i. By adjusting these parameters, the propagation behavior of the beam and the light field distribution can be precisely controlled and described.

[0073] is the spatial scale parameter, and Z represents the propagation distance.

[0074] Then the Airy beam is precisely superimposed on each focal position. By superimposing the Airy beam on each focal point, each focal position has the characteristics of a non-diffraction beam, and finally a multi-focal array with non-diffraction beam characteristics is generated, such as Figure 3As shown. Then, the multi-focus Airy beam phase pattern designed in MATLAB is loaded into the SLM, which modulates the light wavefront. Through precise phase modulation, the generation positions of multiple focal points and the propagation characteristics of the beam can be controlled. This method can effectively enhance the non-diffraction characteristics of the focal array, ensure the intensity and shape stability of the beam over a long propagation distance, and further improve the imaging quality and resolution of the system.

[0075] The innovation of the present invention lies in the use of a multi-focus non-diffraction beam scanning structured light super-resolution microscopy imaging method and system. By combining the non-diffraction beam and the multi-focus, fast super-resolution imaging is achieved and the multi-layer structure information of the sample is obtained. In addition, the intensity of the excitation light is sinusoidally modulated to ensure the high resolution of the obtained image.

[0076] The principles and implementation methods of the present invention are described in this article using specific examples. The description of the above embodiments is only used to help understand the method and core idea of ​​the present invention. At the same time, for those skilled in the art, according to the idea of ​​the present invention, there will be changes in the specific implementation methods and application scope. In summary, the content of this specification should not be understood as limiting the present invention.

Claims

1. A scanning structured light microscopy imaging system based on a multi-focus non-diffraction beam, characterized in that: include: A laser light source (1), an electro-optic modulator (3), a lens group 1, a spatial light modulator (8), a lens group 2, a two-dimensional scanning galvanometer (12), a scanning lens (13), a tube lens (14), an emission filter (15), a dichroic filter (16), and an objective lens (17) are sequentially arranged along an excitation light path, and also includes an area array camera (21); The laser light source (1) is used to emit excitation light; the excitation light emitted by the laser light source (1) passes through the electro-optic modulator (3) and the lens group 1 in sequence and is incident on the spatial light modulator (8); the spatial light modulator (8) is used to synchronously control the multi-focal array and the non-diffraction light beam to generate a multi-focal array with non-diffraction light beam characteristics, and to control the generation positions of the multiple focal points and the propagation characteristics of the excitation light through phase modulation; The excitation light modulated by the spatial light modulator (8) passes through lens group 2, a two-dimensional scanning galvanometer (12), a scanning lens (13), a tube lens (14), an emission filter (15), a dichroic plate (16), and an objective lens (17) in sequence, and then irradiates the sample to be imaged (18), wherein the excitation light interacts with the fluorescent marker in the sample to be imaged (18), thereby exciting the sample to be imaged (18) to generate fluorescence; The fluorescence generated by the sample to be imaged (18) passes through the objective lens (17) and the dichroic film (16) and is then transmitted to the area array camera (21). The area array camera (21) collects fluorescence signals at multiple focal points to form a fluorescence image.

2. The scanning structured light microscopy imaging system based on multi-focus non-diffraction beams according to claim 1, characterized in that: The method for generating the multi-focal array is as follows: First, a light focus array of a set size is generated according to the size of the spatial light modulator (8); Next, a zero-frequency point in the light focus array is selected, and the light intensity at the point is weakened to a preset value; Then, the weakened light intensity is evenly distributed to other light focal points in the light focal point array. After the light intensity is redistributed, the WGS iterative algorithm is entered to optimize the phase distribution of the light field through multiple phase modulations and Fourier transforms, and finally the required intensity distribution of the light focus array is achieved to obtain a multi-focus array.

3. The scanning structured light microscopy imaging system based on multi-focus non-diffraction beams according to claim 2, characterized in that: The generating of the multi-focal array with non-diffraction beam characteristics specifically comprises the following steps: First, a diffraction-free beam is generated based on a set phase pattern; Secondly, through phase modulation, the diffraction-free beam is superimposed on each focal position in the multi-focal array, so that each focal position has the characteristics of a diffraction-free beam, and finally a multi-focal array with the characteristics of a diffraction-free beam is generated.

4. The scanning structured light microscopy imaging system based on multi-focus non-diffraction beams according to claim 1, characterized in that: A first half-wave plate (2) is also provided between the laser light source (1) and the electro-optic modulator (3), and the first half-wave plate (2) is used to adjust the polarization state of the excitation light; a first reflector (4) is also provided between the electro-optic modulator (3) and the first lens group, and the excitation light adjusted by the electro-optic modulator (3) is reflected to the first lens group via the first reflector (4).

5. The scanning structured light microscopy imaging system based on multi-focus non-diffraction beams according to claim 1, characterized in that: The lens group 1 comprises a first lens (5) and a second lens (6); the radius of the first lens (5) is smaller than the radius of the second lens (6); the excitation light incident on the lens group 1 passes through the first lens (5) and the second lens (6) in sequence to be magnified and collimated.

6. The scanning structured light microscopy imaging system based on multi-focus non-diffraction beams according to claim 1, characterized in that: The second lens group comprises a third lens (9) and a fourth lens (10), the radius of the third lens (9) being greater than the radius of the fourth lens (10), and the second lens group is used to reduce the size of the excitation light superimposed on the Airy beam and the excitation light and to collimate the excitation light so that the beam size of the excitation light is the same as the size of the excitation light before passing through the first lens group.

7. The scanning structured light microscopy imaging system based on multi-focus non-diffraction beams according to claim 1, characterized in that: A second half-wave plate (7) is also provided between the lens group 1 and the spatial light modulator (8), and the second half-wave plate (7) is used to adjust the polarization direction of the excitation light emitted through the lens group 1 so that the polarization direction of the excitation light is parallel to the liquid crystal surface of the spatial light modulator (8).

8. The scanning structured light microscopy imaging system based on multi-focus non-diffraction beams according to claim 1, characterized in that: A second reflector (11) is provided between the second lens group and the two-dimensional scanning galvanometer (12), and the second reflector (11) is used to change the optical path direction of the excitation light emitted through the second lens group so that the optical path direction of the excitation light is spatially conjugate with the two-dimensional scanning galvanometer (12).

9. The scanning structured light microscopy imaging system based on multi-focus non-diffraction beams according to claim 1, characterized in that: A bandpass filter (19) and a converging lens (20) are provided between the dichroic film (16) and the area array camera (21); the fluorescent signal emitted by the dichroic film (16) is focused to the area array camera (21) through the converging lens (20) after stray light is filtered out by the bandpass filter (19).

10. A scanning structured light microscopy imaging method based on a multi-focus non-diffraction beam, characterized in that: The scanning structured light microscopic imaging system based on a multi-focus non-diffraction beam as described in any one of claims 1 to 9 comprises the following steps: The laser light source (1) emits excitation light; The excitation light is modulated by an electro-optical modulator (3), and then magnified and collimated by a lens group 1; The excitation light passing through the first lens group is incident on a spatial light modulator (8) loaded with an Airy beam mask, and is modulated by the spatial light modulator (8) using zero-order light to generate a multi-focal array with non-diffraction beam characteristics, and the generation positions of the multiple focal points and the propagation characteristics of the excitation light are controlled through phase modulation; Next, the excitation light is reduced in size and collimated by lens group 2, and then incident on a two-dimensional scanning galvanometer (12), and then scanned point by point by the two-dimensional scanning galvanometer (12). After that, the excitation light passes through a scanning lens (13), a tube lens (14), and an emission filter (15), and then irradiates the surface of a sample to be imaged (18) via a dichroic plate (16) and an objective lens (17). Then, the excitation light interacts with a fluorescent marker in the sample to be imaged (18), and excites the fluorescent marker to generate fluorescence. The fluorescence generated by the sample to be imaged (18) is split by the two-color film (16) and transmitted to the area array camera (21). The area array camera (21) collects fluorescence signals at multiple focal points to form a fluorescence image.

Images