A fast three-dimensional microscopic imaging method and system based on wavelength multiplexed metasurface

Abstract

The application discloses a kind of based on wavelength multiplexing super surface fast three-dimensional microscopic imaging method and system, system includes: light source module, light modulation module, sample stage and light collection module are arranged along the direction of light beam propagation;The light modulation module is modulated into the sinusoidal stripe distribution structure illumination light required by HiLo microscopic imaging with phase flat uniform illumination light with double-wavelength light beam and projects illumination light to sample stage;The light collection module realizes sample image acquisition on different axial planes and data transmission;According to HiLo fusion algorithm, a plurality of optical section images are combined to form three-dimensional microscopic imaging.The application realizes the snapshot of HiLo optical section microscopic imaging, improves the imaging speed and stability;Realize the three-dimensional microscopic imaging of sample to be measured;With small volume, compact structure and the advantages such as being easy to move, can satisfy the demand of instant detection under complex environment such as field environment, remote area.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of optical microscopy and micro-nano optical technology, in particular to a portable fast three-dimensional microscopic imaging method and system based on wavelength multiplexing metasurface. BACKGROUND

[0002] As a basic technology in biological microscopic imaging, wide-field microscopy is often used for non-contact, fast and low-cost high-resolution imaging of the structural details of biological samples. However, the out-of-focus background noise introduced by the depth of field of the traditional wide-field microscope will reduce the image quality and contrast. Secondly, for thick samples, due to the low light power density of wide-field illumination, it is difficult to excite and penetrate the deep layer of the sample to realize three-dimensional imaging, thus limiting the application of wide-field microscopic imaging in thick samples.

[0003] HiLo microscopic imaging is a simple, fast and effective wide-field optical sectioning imaging technology, which can reconstruct a high-resolution image by shooting a uniform light illumination image and a structured light illumination image. Although the HiLo microscopic technology has optical sectioning capability, in order to obtain the required uniform illumination image and structured light illumination image, the detection camera needs to perform two image shooting and collection, which affects the improvement of imaging speed and imaging quality. In addition, in the traditional HiLo microscope, the structured illumination light is generated by spatial light modulator or digital micromirror device modulation, so the system is large in size, complex in structure and difficult to move. In the field environment or remote area, the detection personnel need to protect and carry the collected sample slices to the laboratory for imaging detection. Therefore, the traditional HiLo microscope architecture cannot meet the preliminary and real-time detection needs in the above complex environment. SUMMARY

[0004] In order to solve the above problems in the traditional HiLo microscopic imaging, the purpose of the present application is to propose a portable fast three-dimensional microscopic imaging method and system based on wavelength multiplexing metasurface, to meet the needs of fast and real-time three-dimensional optical sectioning microscopic imaging.

[0005] In order to achieve the above purpose, the technical scheme of the present application is as follows:

[0006] A rapid three-dimensional microscopic imaging system based on wavelength-multiplexed metasurfaces includes: a light source module, a light modulation module, a sample stage, and a light collection module arranged along the beam propagation direction; the light source module combines the outgoing light from two different operating wavelength light sources and shapes it into a collimated, coaxial dual-wavelength beam; the light modulation module modulates the dual-wavelength beam into illumination light with a sinusoidal fringe distribution structure required for HiLo microscopic imaging and a phase-flat uniform illumination light, and projects the illumination light onto the sample stage; the light collection module realizes sample image acquisition and data transmission on different axial planes; based on the HiLo fusion algorithm, optical slice images on a single axial plane are first reconstructed, and multiple optical slice images are combined to form a three-dimensional microscopic image.

[0007] Optionally, the optical modulation module includes a wavelength multiplexing metasurface device, a first microscope objective, a polarization-insensitive beam splitter, an electrically tunable liquid zoom lens, and a second microscope objective arranged sequentially along the optical path. The wavelength multiplexing metasurface device includes a wavelength multiplexing metasurface and a metasurface mounting base, so that the dual-wavelength beam from the light source module is incident orthogonally onto the wavelength multiplexing metasurface. The wavelength multiplexing metasurface simultaneously generates the structured illumination light and uniform illumination light required for HiLo microscopy imaging. The two modulated illumination lights are collected by the first microscope objective, and after passing through the polarization-insensitive beam splitter, the electrically tunable liquid zoom lens, and the second microscope objective, the illumination light is projected onto the sample stage.

[0008] Optionally, the wavelength-multiplexed metasurface includes a transparent substrate and multiple basic unit structures arranged on the transparent substrate. The basic unit structure is a dielectric nanoantenna, which includes a lattice and a dielectric nanopillar disposed at the center of the upper surface of the lattice. The dimensions of the basic unit structures are all on the subwavelength scale. The transparent substrate is square in shape.

[0009] Optionally, the light collection module includes a second microscope objective, an electrically adjustable liquid zoom lens, a polarization-insensitive beam splitter, a sleeve lens, and a color camera arranged along the optical path direction. Two types of illumination light generated by the wavelength-multiplexed metasurface are projected onto the sample to be tested. The reflected light from the sample is first collected by the second microscope objective and the electrically adjustable liquid zoom lens. The collected light is reflected by the polarization-insensitive beam splitter into the sleeve lens, and finally the sleeve lens images the collected light onto the pixel target surface of the color camera.

[0010] Optionally, the sleeve lens is an aberration-correcting lens, which is matched with the second microscope objective to form an infinity imaging group; the color camera and the electrically adjustable liquid zoom lens are synchronized in zoom time to capture HiLo microscopic images on each scanning plane; the acquired image data is uploaded to the server via Bluetooth.

[0011] Optionally, the light source module comprises two light sources with different operating wavelengths, a fiber combiner and a fiber collimating lens; the tail fiber of the light source is connected to the fiber combiner, and the double-wavelength output light is coupled into the fiber combiner to form a coaxial divergent light beam; the fiber collimating lens collimates the divergent light beam into a coaxial parallel collimated output light beam.

[0012] Optionally, the light source module outputs coaxial collimated double-color light beams with operating wavelengths of λ1 and λ2, which correspond to the two operating wavelengths of the structure illumination light and the uniform illumination light required for HiLo microscopic imaging generated by the wavelength multiplexing super surface.

[0013] Optionally, the sample stage is used for fixing and adjusting the sample to be measured; the sample to be measured is fixed and protected by a glass slide and a cover glass, and the sample stage is provided with a glass slide mounting groove and an adjusting frame, and the adjusting frame can be adjusted to make the illumination light uniformly cover the sample to be measured.

[0014] A wavelength multiplexing super surface-based rapid three-dimensional microscopic imaging method is provided, which is completed by using any of the wavelength multiplexing super surface-based rapid three-dimensional microscopic imaging systems provided in the present application, wherein the light modulation module comprises, in sequence along the light path direction, a wavelength multiplexing super surface, a first microscopic objective, a polarization-insensitive beam splitter, an electrically adjustable liquid zoom lens (8) and a second microscopic objective.

[0015] The specific process is as follows:

[0016] Step 1: Prepare the sample to be measured;

[0017] Prepare the biological sample to be measured into a sample to be measured, and place the sample to be measured in the sample stage, so that the sample to be measured is perpendicular to the optical axis and located on the working distance of the second microscopic objective;

[0018] Step 2: Adjust the illumination light;

[0019] Drive the light source in the light source module to generate coaxial collimated double-color light beams required by the wavelength multiplexing super surface; drive the electrically adjustable liquid zoom lens to make the two illumination lights modulated by the wavelength multiplexing super surface cover the outer surface of the sample to be measured;

[0020] Step 3: Illumination light scanning and image acquisition;

[0021] Set the zoom step ΔL of the electrically adjustable liquid zoom lens, so that the electrically adjustable liquid zoom lens zooms according to the set step ΔL, so that the illumination light scans and covers the sample along the optical axis direction, the acquisition times are N, and the total stroke L is the thickness of the sample to be measured, L=NΔL; in each acquisition process, the electrically adjustable liquid zoom lens needs to zoom by steps and be stable, and the color camera shutter is synchronously driven to acquire double-color illumination images;

[0022] Step 4: Three-dimensional microscopic image reconstruction and storage;

[0023] The image data collected in step 3 is transmitted to the server end through Bluetooth, and the server end first separates the illumination images under two working wavelengths by means of the Bayer filter of the color camera; the two separated images are then fused into one high-resolution optical slice image by means of the HiLo algorithm; finally, N optical slice images are combined to reconstruct the three-dimensional microscopic image of the sample to be measured and stored to the server end.

[0024] Optionally, the step 4 specifically comprises the following steps.

[0025] Step 1: acquiring original image data;

[0026] The original image data received by the server end is N groups, and i is an integer from 1 to N; wherein the image with wavelength λ1 in the i-th group of data is the uniform light illumination image I uniform (x,y,i), and the image with wavelength λ2 is the structured light illumination image I structured (x,y,i);

[0027] Step 2: separating the dual-color illumination images;

[0028] The Bayer filter in the color camera can separate the original image data into two groups of independent illumination image data I uniform (x,y,i) and I structured (x,y,i) according to the wavelength λ1 and the wavelength λ2;

[0029] Step 3: extracting image high-frequency components;

[0030] The high-frequency components I uniform (x,y,i) of the uniform light illumination image I Hi (x,y,i) of the i-th group of data are extracted, and the formula is as follows:

[0031] I Hi (x,y,i) = F -1 {HP f {F[I uniform (x,y,i)]}} (1);

[0032] Wherein, F and F -1 are Fourier transform and inverse transform operations, and HP f is a high-pass filtering operation;

[0033] Step 4: extracting image low-frequency components;

[0034] The local contrast image I uniform (x,y,i) is calculated by using the uniform light illumination image I structured (x,y,i) and the structured light illumination image I C(x, y, i), and extract the low-frequency component I Lo (x, y, i), and extract the low-frequency component I

[0035] I Lo (x, y, i) = F -1 {LP f {F{[(I C (x, y, i) x I uniform (x, y, i)]}} (2);

[0036] I C (x, y, i) = F -1 {F{[(I uniform (x, y, i) - 2 x I structured (x, y, i)] x BFP}} (3);

[0037] where F and F -1 are Fourier transform and inverse transform operations, LP f is a high-pass filtering operation, I C (x, y, i) is a local contrast image, and BFP is a Gaussian band-pass filtering operation.

[0038] Step 5: Fusion of HiLo optical section images

[0039] The focal plane high-frequency component and the low-frequency component in the i-th group of data extracted in steps 3 and 4 are fused into the required HiLo optical section microscopic image I HiLo (x, y, i), and the formula is as follows:

[0040] I HiLo (x, y, i) = I Hi (x, y, i) + η I Lo (x, y, i) (4);

[0041] where η is the weight between the high-frequency component I Hi (x, y, i) and the low-frequency component I Lo (x, y, i).

[0042] Step 6: Acquisition of the i+1-th group of HiLo optical section microscopic images

[0043] For the i+1-th group of data, steps 3-5 are repeated to fuse the HiLo optical section microscopic image I HiLo (x, y, i+1) on the i+1-th axial plane, and thus N groups of HiLo microscopic images I HiLo (x, y, i) on different axial planes are obtained.

[0044] Step 7: Reconstruction of a three-dimensional HiLo microscopic image

[0045] HiLo optical sectioning microscopic images I on different axial planes of N groups are obtained HiLo (x,y,i) are spliced and combined into a three-dimensional HiLo microscopic image I of the sample to be measured HiLo (x,y,z), as follows:

[0046]

[0047] The beneficial effects of the present application are as follows:

[0048] (1) The microscopic imaging system proposed in the present application can simultaneously generate the two illumination lights required for HiLo microscopic imaging compared to traditional spatial light modulators and digital micromirror devices, realize snapshot image acquisition without illumination light switching, and improve the imaging speed of single HiLo optical sectioning image. Secondly, the compact, thin and light characteristics of the super surface make the overall structure of the microscopic imaging system compact, easy to move and suitable for real-time detection.

[0049] (2) The microscopic imaging system proposed in the present application can realize optical sectioning scanning on the axial plane of the sample to be measured by combining the wavelength multiplexing super surface with the electrically adjustable liquid zoom lens, so as to reconstruct the three-dimensional microscopic image of the sample to be measured. At the same time, the Bayer filter of the color camera has independent spectral bands in red, green and blue RGB three colors, which can effectively separate the two working wavelengths, without the need for splitting the light path or the camera to collect images, further reducing the system volume and simplifying the operation steps. BRIEF DESCRIPTION OF DRAWINGS

[0050] Figure 1 The overall structure schematic diagram of the fast three-dimensional microscopic imaging system based on wavelength multiplexing super surface provided for the embodiments of the present application;

[0051] Figure 2 The structure schematic diagram of the light source module provided for the embodiments of the present application;

[0052] Figure 3 The structure schematic diagram of the light modulation module provided for the embodiments of the present application;

[0053] Figure 4 The structure schematic diagram of the light collection module provided for the embodiments of the present application;

[0054] Figure 5 The flow chart of three-dimensional image reconstruction provided for the embodiments of the present application;

[0055] Figure 6 The system architecture operation flow chart provided for the embodiments of the present application;

[0056] Figure 7 The overall schematic diagram of a super surface provided for the embodiments of the present application;

[0057] Figure 8 This is a schematic diagram of the basic unit structure of a metasurface provided in an embodiment of the present invention;

[0058] Figure 9 A phase scan result diagram of a metasurface structure unit provided in an embodiment of the present invention;

[0059] Figure 10 A phase distribution diagram of structured illumination light and uniform illumination light is provided for an embodiment of the present invention;

[0060] Figure 11 This invention provides an embodiment of a metasurface-generated sinusoidal structured light field distribution diagram.

[0061] Figure 12 This invention provides a metasurface-generated uniform plane wave optical field distribution diagram.

[0062] Figure 13 Provided for embodiments of the present invention Figure 11 Light intensity distribution at the white dashed line in the middle;

[0063] Figure 14 Provided for embodiments of the present invention Figure 12 Light intensity distribution at the white dashed line in the middle;

[0064] The labels in the diagram represent:

[0065] A-Light source module, B-Light modulation module, C-Light collection module;

[0066] 1-Light source, 2-Fiber optic combiner, 3-Fiber optic collimating lens, 4-Wavelength multiplexing metasurface, 5-Metasurface mounting base, 6-First microscope objective, 7-Polarization-insensitive beam splitter, 8-Electrically adjustable liquid zoom lens, 9-Second microscope objective, 10-Sleeve lens, 11-Color camera, 12-Sample to be tested, 13-Sample stage. Detailed Implementation

[0067] The implementation methods, principle design, and technical effects of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments. The following embodiments are only used to more clearly illustrate the technical solutions of the present invention and should not be used to limit the scope of protection of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort should fall within the scope of protection of the present invention.

[0068] The application discloses a wavelength multiplexing super-surface-based fast three-dimensional microscopic imaging method and system, and the microscopic imaging system mainly comprises a light source module, a light modulation module, a light collection module and a sample stage. The light source module shapes the light source exit light into a collimated coaxial dual-wavelength light beam. The light modulation module is used for modulating the dual-wavelength light beam and projecting the modulated illumination light to a sample to be measured. The light collection module can collect sample images on different axial planes. The sample stage is used for fixing and adjusting the biological sample to be measured. On the basis of the traditional HiLo optical sectioning microscopic imaging, the wavelength multiplexing super-surface and the Bayer filter in the color camera are combined to skillfully avoid the illumination light switching and the light splitting path collection in the traditional HiLo imaging process, so that the snapshot HiLo optical sectioning microscopic imaging is realized, and the imaging speed and stability are improved. Meanwhile, the electrically adjustable liquid zoom lens is responsible for the microscopic image collection on different axial planes, so that the three-dimensional microscopic imaging of the sample to be measured is realized. Compared with the traditional HiLo microscope, the system has the advantages of small size, compact structure and convenience for moving, and can meet the needs of instant detection in complex environments such as field environment and remote area.

[0069] In combination Figures 1-4 , the wavelength multiplexing super-surface-based fast three-dimensional microscopic imaging system comprises a light source module, a light modulation module, a sample stage and a light collection module arranged along the light beam propagation direction; the light source module combines the exit light of two different working wavelength light sources and shapes the combined light into a collimated coaxial dual-wavelength light beam by a fiber collimating lens, serving as an illumination light source. The light modulation module modulates the dual-wavelength incident light beam into a sinusoidal stripe distributed structure illumination light and a phase flat uniform illumination light required by the HiLo microscopic imaging and projects the illumination light to the sample to be measured. The light collection module realizes sample image collection on different axial planes and data transmission. According to the HiLo fusion algorithm, the optical sectioning images on a single axial plane are first reconstructed, and finally a plurality of optical sectioning images are combined to form three-dimensional microscopic imaging. The sample stage is used for fixing and adjusting the biological sample to be measured. In some embodiments, the light source module comprises two different working wavelength fiber output light sources, a fiber combiner and a fiber collimating lens. The tail fiber of the light source is connected with the fiber combiner, and the dual-wavelength output light is coupled into the combiner to form a coaxial divergent light beam. The fiber collimating lens collimates the divergent light beam into a coaxial parallel collimated output light beam. The light source used in the application can generally be selected from an LED light source, a laser light source and the like, and the light source is conveniently given in the form of fiber transmission, so that the tail fiber of the light source is connected with the fiber combiner.

[0070] In some embodiments, the light source module outputs a coaxial and collimated dual-color light beam, with working wavelengths of λ1 and λ2, corresponding to the two working wavelengths of the structural illumination light and the uniform illumination light required for HiLo microscopic imaging by the wavelength multiplexing metasurface. In particular, at the two working wavelengths, the fiber combiner has low insertion loss, and the fiber collimating lens has achromatic characteristics to ensure that the dual-wavelength light beam is collimated and coaxial output with good beam quality.

[0071] In some embodiments, the light modulation module includes, in sequence along the light path direction, a wavelength multiplexing metasurface device, a first microscopic objective, a beam splitter, an electrically adjustable liquid zoom lens, and a second microscopic objective; the wavelength multiplexing metasurface device includes a wavelength multiplexing metasurface and a metasurface mounting seat, so that the dual-wavelength light beam of the light source module is normally incident to the wavelength multiplexing metasurface; the wavelength multiplexing metasurface simultaneously generates the structural illumination light and the uniform illumination light required for HiLo microscopic imaging, the two modulated illumination lights are collected by the first microscopic objective, pass through the beam splitter, the electrically adjustable liquid zoom lens, and the second microscopic objective, and are projected to the sample stage; the metasurface device is fixed on a small five-axis displacement stage, and the dual-color output light of the light source module is normally incident to the metasurface by adjusting the spatial position and the pitch angle of the displacement stage.

[0072] In some embodiments, in combination Figure 7 and 8 , the wavelength multiplexing metasurface 4 includes a transparent substrate and a plurality of basic unit structures arranged on the transparent substrate, the basic unit structures are dielectric nanoantennas, the dielectric nanoantenna includes a lattice and a dielectric nanocolumn arranged at the center of the upper surface of the lattice; the size of the basic unit structure is of a subwavelength order; and the shape of the transparent substrate is a square. The design method of the structure of the wavelength multiplexing metasurface for snapshot HiLo optical sectioning microscopic imaging specifically includes the following steps:

[0073] Step 1: Based on the transmission phase control principle, the response mechanism between the basic unit structure and the incident light is analyzed, the relationship curves between the transmission phase, the transmission phase difference and the structural parameters of the dielectric nanocolumn at the two working wavelengths are determined, and a basic unit structure phase library is established. The following substeps are included:

[0074] Step 11: The relationship curve between the transmission phase of the dielectric nanocolumn and the bottom diameter D at the two working wavelengths is obtained by using an electromagnetic simulation software.

[0075] The specific operation is as follows: when the incident light is 532 nm and the height H of the dielectric nanocolumn is fixed at 700 nm, the bottom diameter D of the dielectric nanocolumn is scanned in the range of 0.2P-0.8P, P refers to the side length of the lattice, and the scanning interval is 1 nm, so as to establish the first transmission phase The relationship curve between the diameter D of the bottom surface of the dielectric nanocolumn and the transmission phase; likewise, when the incident light is 633 nm, the height H of the dielectric nanocolumn is fixed at 700 nm, the diameter D of the bottom surface of the dielectric nanocolumn is scanned in the range of 0.2P-0.8P, the scanning interval is 1 nm, and the second transmission phase is established point by point The relationship curve between the diameter D of the bottom surface of the dielectric nanocolumn and the transmission phase. In the electromagnetic simulation software, the transmission phase can be obtained according to the following transmission phase control principle:

[0076]

[0077] wherein, is the transmission phase; n eff is the effective refractive index, which is determined by the diameter D of the bottom surface of the dielectric nanocolumn. By changing the size of the diameter D of the bottom surface of the dielectric nanocolumn, the transmission phase covers 0-2π to meet the phase control requirements; the height H of the dielectric nanocolumn is 700 nm; λ i is the wavelength of the incident light, λ1 and λ2 are 532 nm and 633 nm respectively (λ1 and λ2 are selected according to the requirements of the incident light).

[0078] Step 12, calculate the transmission phase difference under two working wavelengths and draw the relationship curve between the diameter D of the bottom surface of the dielectric nanocolumn and the transmission phase difference, and the calculation formula is:

[0079]

[0080] Figure 9 The super surface structure unit phase scanning result provided by the embodiment is shown in FIG. 1, which shows the relationship curve between the diameter D of the bottom surface of the dielectric nanocolumn and the transmission phase, and the basic unit structure phase library is established based on the data of the curve. Figure 9 The relationship curve between the diameter D of the bottom surface of the dielectric nanocolumn and the transmission phase, and the basic unit structure phase library is established based on the data of the curve.

[0081] Step 2: determine the design phase of the uniform illumination light and the structured illumination light, and the target phase difference between the uniform illumination light and the structured illumination light. Specifically as follows:

[0082] Figure 10 The design phase distribution diagram of the structured illumination light and the uniform illumination light provided by the embodiment. For the required sinusoidal fringe of the structured illumination light, the design phase of the super surface under this working wavelength is determined based on the phase distribution of the phase type sinusoidal grating. The design phase distribution of the wavelength multiplexing super surface to realize the sinusoidal fringe structured illumination light is:

[0083]

[0084] ​The basic unit structure has an abscissa x-range of -10 to 10 μm; the sinusoidal modulation parameter m = π / 2; the period interval L = 500 nm; and the reference phase.

[0085] As stated in formula (3), it can be obtained that for λ1 at different x-coordinates, For λ2,

[0086] For the plane wave required for uniform light, the design phase distribution of the wavelength-reused metasurface to achieve uniform illumination is as follows:

[0087]

[0088] Where, for λ1, For λ2,

[0089] Calculate the target phase difference at two operating wavelengths:

[0090]

[0091] We can obtain that for λ1, For λ2,

[0092] Step 3: Calculate the target phase difference based on the result of Step 2. In the basic unit structure phase library established in step 1, select those phases equal to the target phase difference at both working wavelengths. The selected medium nanopillars have a height H of 700 nm, a bottom diameter D of 109 nm and 118 nm, and a lattice side length P of 350 nm. The two selected medium nanopillars with different bottom diameters D are arranged according to the design phase distribution equation described in formula (3) to establish a wavelength multiplexing metasurface structure, wherein the side length of the transparent substrate is 20 μm.

[0093] Since the basic unit structure dimensions are all on the subwavelength scale, the metasurface of this invention features planarity, lightweight design, and ease of photonic integration, making it suitable for miniaturization of optical systems. The optical field distribution of the metasurface at different operating wavelengths was calculated using electromagnetic simulation software. Figure 11 An embodiment of the present invention provides a metasurface-generated sinusoidal structured light field distribution map, such as... Figure 11 As shown, under the condition of incident light wavelength of 633nm, the metasurface generates sinusoidal fringe structured light with a periodic interval of 500nm and a fringe size of 700nm. Figure 13 This invention provides a metasurface-generated uniform plane wave optical field distribution map, as shown in the embodiment of the invention. Figure 13As shown, when the incident light wavelength is 532 nm, the super surface generates a plane wave with uniform intensity. Figure 13 The light intensity distribution at the white dotted line. Figure 11 The light intensity distribution at the white dotted line. Figure 14 The light intensity distribution at the white dotted line. Figure 12 The light intensity distribution at the white dotted line. The light field distribution and light intensity distribution results shown in the above figures show that the wavelength multiplexing super surface proposed in the technical scheme of the present application can realize wavelength multiplexing function for two different wavelengths of incident light. When light with wavelength λ1 is incident on the super surface, a uniform illumination light with flat phase is generated. When light with wavelength λ2 is incident on the super surface, a structured illumination light with sinusoidal stripe distribution is generated. When a dual-wavelength mixed light source with wavelength λ1 and wavelength λ2 is used as incident light, the super surface can simultaneously generate the two kinds of illumination light required for HiLo microscopic imaging, which can meet the requirements of the two kinds of illumination light for HiLo optical sectioning microscopic imaging. Therefore, the super surface can realize the function of reconstructing a high-resolution image through a single shooting and collection, and compared with the conventional HiLo microscopic imaging technology, the method of the present application can effectively improve the imaging speed and imaging stability, and reduce the complexity of the imaging and reconstruction process.

[0094] In some embodiments, the first and second microscopic objectives, the polarization-insensitive beam splitter, and the electrically tunable liquid variable-focus lens in the light modulation module have achromatic characteristics at two working wavelengths. The working parameters of the first and second microscopic objectives are completely consistent. By adjusting the loading voltage of the electrically tunable liquid variable-focus lens, the illumination light field is scanned along the optical axis direction according to the set step to cover the entire sample to be measured. During the zooming process, the magnification of the liquid lens needs to be matched with the microscopic objective to ensure that the illumination light covers the sample to be measured while eliminating the distortion of the illumination light.

[0095] In some embodiments, the light collection module includes the second microscopic objective, the electrically tunable liquid variable-focus lens, the polarization-insensitive beam splitter, the sleeve lens, and the color camera in the light modulation module. The two kinds of illumination light generated by the super surface are projected to the sample to be measured, and the reflected light from the sample is first collected by the second microscopic objective and the electrically tunable liquid variable-focus lens. The collected light is reflected to the sleeve lens through the beam splitter, and the sleeve lens finally images the collected light to the pixel target surface of the color camera.

[0096] In some embodiments, the sleeve lens in the light collection module is an aberration-corrected lens matched with the second microscopic objective to form an infinite imaging group. The color camera and the liquid lens zooming are time-synchronized, and the HiLo microscopic images on each scanning plane are shot and collected. The collected image data is uploaded to the server end through Bluetooth.

[0097] In combination with Figure 5The wavelength multiplexing metasurface-based fast three-dimensional microscopic imaging method of the application is completed by using the wavelength multiplexing metasurface-based fast three-dimensional microscopic imaging system of any one of the application, and the specific process is as follows:

[0098] Step 1: preparing a sample to be tested;

[0099] The biological sample to be tested is placed on a glass slide, and a protective solution is dropped to fix and protect the integrity of the sample using a cover glass; the prepared sample to be tested is installed into the installation slot in the sample stage, and the adjusting frame of the stage is adjusted to make the sample to be tested perpendicular to the optical axis and located on the working distance of the second microscope objective;

[0100] Step 2: adjusting the illumination light;

[0101] (1) driving the LED light source in the light source module to generate the coaxial collimated double-color light beam required by the wavelength multiplexing metasurface;

[0102] (2) driving the electrically tunable liquid zoom lens to make the two kinds of illumination light generated by the metasurface modulation cover the outer surface of the sample to be tested;

[0103] Step 3: illumination light scanning and image acquisition;

[0104] The zoom step ΔL of the electrically tunable liquid zoom lens is set, so that the liquid lens zooms according to the set step ΔL, and the illumination light scans and covers the sample along the optical axis direction, the acquisition times are N, and the total stroke L is the thickness of the sample to be tested, L=NΔL; in each acquisition process, the liquid lens needs to zoom by steps and be stable, and the color camera shutter is synchronously driven to acquire the double-color illumination image;

[0105] Step 4: three-dimensional microscopic image reconstruction and storage;

[0106] The image data acquired in step 3 is transmitted to the server end through Bluetooth, the server end first separates the illumination images under two working wavelengths by means of the Bayer filter of the color camera; the two separated images are fused into a high-resolution optical section image by means of the HiLo algorithm; finally, N optical section images are combined to reconstruct the three-dimensional microscopic image of the sample to be tested and stored in the server end.

[0107] In combination Figure 6 , step 4 specifically comprises the following steps:

[0108] Step 1: acquiring original image data;

[0109] The original image data received by the server end is N groups, and i is an integer from 1 to N; wherein the image with wavelength λ1 in the i-th group of data is the uniform light illumination image I uniform (x,y,i), and the image with wavelength λ2 is the structured light illumination image I structured (x,y,i).

[0110] Step 2: Separate the dual-color illumination images;

[0111] The Bayer filter in the color camera can separate the raw image data into two sets of independent illumination image data I uniform (x,y,i) and I structured (x,y,i);

[0112] Step 3: Extract the high-frequency component of the image;

[0113] The high-frequency component of the image is extracted from the uniform light illumination image I uniform (x,y,i) of the i-th set of data, as follows: Hi

[0114] I Hi (x,y,i) = F 1 {HP f {F[I uniform (x,y,i)]}} (1);

[0115] where F and F -1 are Fourier transform and inverse transform operations, and HP f is a high-pass filter operation;

[0116] Step 4: Extract the low-frequency component of the image;

[0117] The low-frequency component of the image is extracted from the structured light illumination image I uniform (x,y,i) and the uniform light illumination image I structured (x,y,i) of the i-th set of data, as follows: C Lo (x,y,i) = F

[0118] I Lo (x,y,i) = F -1 {LP f {F[I C (x,y,i) x I uniform (x,y,i)]}} (2);

[0119] I C (x,y,i) = F -1 {F{[(I uniform (x,y,i) - 2 x I structured (x,y,i))] x BFP}} (3);

[0120] where F and F -1 are Fourier transform and inverse transform operations, and LP​​f I C (x,y,i) is a local contrast image, and BFP is a Gaussian band-pass filtering operation.

[0121] Step 5: Fusion of HiLo optical section images

[0122] The focal plane high-frequency component and the low-frequency component in the i-th group of data extracted in steps 3 and 4 are fused into the required HiLo optical section microscopic image I HiLo (x,y,i), and the formula is as follows:

[0123] I HiLo (x,y,i) = I Hi (x,y,i) + ηI Lo (x,y,i) (4).

[0124] wherein η is the weight between the high-frequency component I Hi (x,y,i) and the low-frequency component I Lo (x,y,i).

[0125] Step 6: Acquisition of the i+1-th group of HiLo optical section microscopic images

[0126] For the i+1-th group of data, steps 3-5 are repeated to fuse the HiLo optical section microscopic image I HiLo (x,y,i+1) on the i+1-th axial plane; thus, N groups of HiLo microscopic images I HiLo (x,y,i) on different axial planes are obtained.

[0127] Step 7: Reconstruction of a three-dimensional HiLo microscopic image

[0128] The N groups of HiLo optical section microscopic images I HiLo (x,y,i) on different axial planes are spliced and combined into a three-dimensional HiLo microscopic image I HiLo (x,y,z) of the sample to be measured, and the formula is as follows:

[0129]

[0130] Example 1

[0131] Figure 1 The overall structure schematic diagram of the fast three-dimensional microscopic imaging system based on wavelength multiplexing super surface provided by the embodiment of the present application is as shown in Figure 1As shown, the microscopic imaging system mainly includes a light source module A, a light modulation module B, a light collection module C and a sample stage 13. Among them, the main function of the light source module A is that the optical fiber combiner 2 combines the output light of the optical fiber LED light source 1 with two different working wavelengths, and the optical fiber collimating lens 3 shapes it into a collimated coaxial double-wavelength light beam as an illumination light source. The light modulation module B is used to modulate the double-wavelength incident light beam into the sinusoidal stripe structure illumination light and the plane wave uniform illumination light required by the HiLo microscopic imaging, and project the illumination light to the sample to be measured. The light collection module C realizes the sample image collection on different axes and the data transmission function to complete the three-dimensional microscopic imaging. The sample stage 13 is used to fix and adjust the sample to be measured. The sample to be measured is fixed and protected by a glass slide and a cover glass. The stage is provided with a glass slide mounting groove and an adjusting frame, and the position and pitch angle of the adjusting frame can be adjusted to make the illumination light uniformly cover the sample to be measured.

[0132] Figure 2 The light source module structure schematic diagram provided by the embodiment of the application is shown as follows, Figure 2 As shown, the light source module mainly includes the optical fiber output LED light source 1 with two different working wavelengths, the optical fiber combiner 2 and the optical fiber collimating lens 3. The tail fiber of the optical fiber output LED light source 1 is first connected with the optical fiber combiner 2, and the double-color output light is coupled into the optical fiber combiner 2 to form a coaxial divergent light beam. The function of the optical fiber collimating lens 3 is to collimate the divergent light beam into a coaxial parallel light beam to meet the normal incidence condition of the super surface. The output light is a coaxial collimated double-color light beam with wavelengths of 532 nm and 633 nm, which respectively correspond to the working wavelengths of the two illumination lights required by the HiLo microscopic imaging of the wavelength multiplexing super surface 4. The two illumination lights are structured light with sinusoidal stripe distribution and uniform plane wave with flat phase. It is worth noting that under the two working wavelengths, the optical fiber combiner 2 requires low insertion loss, and the optical fiber collimating lens 3 has the property of achromatism to ensure the beam quality of the coaxial collimated output double-wavelength light beam.

[0133] Figure 3 The light modulation module structure schematic diagram provided by the embodiment of the application is shown as follows, Figure 3As shown; the light modulation module B includes: wavelength multiplexing super surface 4, first microscope objective 6, polarization insensitive beam splitter 7, electrically adjustable liquid zoom lens 8 and second microscope objective 9. Among them, the wavelength multiplexing super surface 4 is composed of a transparent substrate and a periodic medium basic unit structure. The basic unit structure is a cylindrical nano antenna, the periodic lattice is square, and the nanometer column is located at the center of the lattice, and the size is all subwavelength. By reasonably designing the basic unit structure parameters and the phase distribution, the wavelength multiplexing super surface 4 can realize the function: when the double-wavelength light beams of common optical path are normally incident to the super surface, the device can simultaneously generate the phase flat uniform plane wave illumination light and the sinusoidal stripe distribution structure illumination light required by HiLo microscopic imaging. The super surface device is fixed on a small five-axis displacement table, and the position and pitch angle of the displacement table are adjusted to make the double-color output light of the light source module normally incident to the super surface. The two kinds of illumination light fields generated by the super surface modulation are collected by the first microscope objective 6, and the illumination light is transmitted to the sample to be measured after passing through the polarization insensitive beam splitter 7, the electrically adjustable liquid zoom lens 8 and the second microscope objective 9. It is worth noting that the first microscope objective 6, the polarization insensitive beam splitter 7, the electrically adjustable liquid zoom lens 8 and the second microscope objective 9 in the light modulation module B have achromatic characteristics at two working wavelengths, so as to ensure that the two illumination lights are located in the same axial plane. The parameters of the two microscope objectives are consistent. The loading voltage of the electrically adjustable liquid zoom lens is adjusted to make the liquid lens gradually zoom according to the set step. At the same time, the magnification of the liquid lens is matched with the microscope objective during zooming, so as to ensure that the illumination light fully covers the sample to be measured.

[0134] Figure 4 The light collection module structure schematic diagram provided for the embodiment of the application is shown as Figure 4 The light collection module C includes a polarization insensitive beam splitter 7, an electrically adjustable liquid zoom lens 8, a second microscope objective 9, a sleeve lens 10 and a color camera 11. The light reflected by the sample to be measured is first collected by the second microscope objective 9 and the electrically adjustable liquid zoom lens 8, and then reflected to the sleeve lens 10 through the beam splitter 7, and finally imaged to the pixel target surface of the color camera 11 by the sleeve lens 10. The sleeve lens 10 and the second microscope objective 9 in the light collection module C form an infinite distance imaging group, and the main function of the sleeve lens 10 is to eliminate system aberration. The loading voltage of the electrically adjustable liquid zoom lens 8 is adjusted to make the illumination light field scan and cover the entire sample to be measured along the optical axis direction according to the set step. The color camera 11 synchronously captures the HiLo microscopic images on each scanning plane, and uploads the data to the server end.

[0135] Figure 5 The system architecture operation flow chart provided for the embodiment of the application is shown as Figure 5 The specific process is as follows:

[0136] Step 1: Prepare the sample to be tested.

[0137] Place the biological sample to be tested on a glass slide, drop in the protective solution, and use a cover glass to fix and protect the integrity of the sample. Install the prepared sample to be tested into the installation slot in the sample stage, adjust the adjustment frame of the stage to make the sample to be tested perpendicular to the optical axis and located at the working distance of the second microscope objective.

[0138] Step 2: Adjust the illumination light.

[0139] (1) Drive the LED light source in the light source module to generate the coaxial collimated dual-color light beam required by the wavelength multiplexing metasurface.

[0140] (2) Drive the electrically tunable liquid zoom lens to make the two kinds of illumination light generated by the metasurface modulation cover the outer surface of the sample to be tested.

[0141] Step 3: Illumination light scanning and image acquisition.

[0142] Set the zoom step size ΔL of the electrically tunable liquid zoom lens, so that the liquid lens zooms according to the set step size ΔL, making the illumination light scan and cover the sample along the optical axis direction, the acquisition times are N, and the total travel distance L is the thickness of the sample to be tested, L=NΔL. During each acquisition process, the liquid lens needs to zoom by steps and be stable, and the color camera shutter is driven synchronously to acquire the dual-color illumination image.

[0143] Step 4: Three-dimensional microscopic image reconstruction and storage.

[0144] The image data acquired in step 3 is transmitted to the server end through Bluetooth, and the server end first separates the illumination images under two working wavelengths by means of the Bayer filter of the color camera. The two separated images are then fused into a high-resolution optical section image by means of the HiLo algorithm. Finally, N optical section images are combined to reconstruct the three-dimensional microscopic image of the sample to be tested and stored in the server end.

[0145] Figure 6 The three-dimensional microscopic image reconstruction flowchart provided by the embodiment of the present application. As shown in Figure 6 , the specific process is as follows:

[0146] Step 1: Obtain the original image data

[0147] The original image data I(x,y) received by the server end is a total of 2N groups, of which the image with wavelength λ1=532nm is the uniform light illumination image I uniform (x,y,i), a total of N groups; the image with wavelength λ2=633nm is the structured light illumination image I structured (x,y,i), a total of N groups; N is the image acquisition times of the color camera, and i is an integer from 1 to N;

[0148] Step 2: Separate the dual-color illumination images

[0149] The Bayer filter in the color camera can cover three spectral bands of red, green and blue RGB three colors, and each spectral band is independent of each other, so the Bayer filter can separate the original image data into two groups of independent illumination image data I uniform (x,y,i) and I structured (x,y,i);

[0150] Step 3: Extract the high-frequency component of the image

[0151] The uniform light illumination image I uniform (x,y,i) of the i-th group of data is subjected to a high-pass filtering operation to extract the high-frequency component I Hi (x,y,i) on the image focal plane, and the formula is as follows:

[0152] I Hi (x,y,i) = F -1 {HP f {F[I uniform (x,y,i)]}} (1);

[0153] Where F and F -1 are Fourier transform and inverse transform operations, and HP f is a high-pass filtering operation.

[0154] Step 4: Extract the low-frequency component of the image

[0155] The local contrast image I uniform (x,y,i) is calculated using the uniform light illumination image I structured (x,y,i) and the structured light illumination image I C (x,y,i) of the i-th group of data, and the local contrast image I C (x,y,i) is subjected to a low-pass filtering operation to extract the low-frequency component I Lo (x,y,i) on the image focal plane, and the formula is as follows:

[0156] I Lo (x,y,i) = F -1 {LP f {F[I C (x,y,i) × I uniform (x,y,i)]}} (2);

[0157] I C (x,y,i) = F -1 {F{[(I uniform (x,y,i) - 2·I structured(x, y, i) = I (x, y, i) x BFP (3) ;

[0158] where F and F -1 are Fourier transform and inverse transform operations, LP f is a high-pass filter operation, I C (x, y, i) is a local contrast image, and BFP is a Gaussian band-pass filter operation.

[0159] Step 5: Fusion of HiLo optical section images

[0160] The high-frequency component and the low-frequency component of the i-th group of data extracted in steps 3 and 4 are fused into the required HiLo optical section microscopic image I HiLo (x, y, i), as follows:

[0161] I HiLo (x, y, i) = I Hi (x, y, i) + ηI Lo (x, y, i) (4) ;

[0162] where η is the weight between the high-frequency component I Hi (x, y, i) and the low-frequency component I Lo (x, y, i).

[0163] Step 6: Acquisition of the i+1-th group of HiLo optical section microscopic images

[0164] For the i+1-th group of data, steps 3-5 are repeated to fuse the HiLo optical section microscopic image I HiLo (x, y, i+1) on the i+1-th axial plane. Thus, N groups of HiLo microscopic images I HiLo (x, y, i) on different axial planes are obtained.

[0165] Step 7: Reconstruction of a three-dimensional HiLo microscopic image

[0166] The N groups of HiLo optical section microscopic images I HiLo (x, y, i) on different axial planes are spliced to combine into a three-dimensional HiLo microscopic image I HiLo (x, y, z) of the sample to be measured, as follows:

[0167]

[0168] The preferred embodiments of the present disclosure are described in detail above, but the present disclosure is not limited to the specific details in the above-described embodiments. Within the technical concept of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications all belong to the protection scope of the present disclosure.

[0169] It should be further noted that various technical features described in the above specific embodiments can be combined in any suitable manner, and the disclosure is not limited to the combinations explicitly described herein, unless otherwise indicated.

[0170] Furthermore, various different embodiments of the disclosure can be combined in any suitable manner, as long as it does not deviate from the spirit of the disclosure, and it should be considered as disclosed by the disclosure.

Claims

1. A fast three-dimensional microscopic imaging system based on wavelength multiplexed metasurface, characterized in that, The application relates to a HiLo microscope imaging system. The application comprises a light source module (A), a light modulation module (B), a sample stage (13) and a light collection module (C) arranged along the light beam propagation direction. The light source module (A) combines the light emitted by two light sources with different working wavelengths and shapes the combined light into a collimated coaxial dual-wavelength light beam. The light modulation module (B) modulates the dual-wavelength light beam into a sinusoidal stripe distributed structured illumination light required by HiLo microscopic imaging and a phase flat uniform illumination light and projects the illumination light to the sample stage (13). The light collection module (C) realizes sample image collection on different axial planes and data transmission; according to a HiLo fusion algorithm, optical slice images on single axial planes are firstly reconstructed, and multiple optical slice images are combined to form three-dimensional microscopic imaging. The light modulation module (B) comprises, sequentially arranged along the light path direction, a wavelength multiplexing super surface device, a first microscopic objective (6), a polarization insensitive beam splitter (7), an electrically adjustable liquid zoom lens (8) and a second microscopic objective (9); the wavelength multiplexing super surface device comprises a wavelength multiplexing super surface (4) and a super surface mounting seat (5), so that the dual-wavelength light beam of the light source module is normally incident to the wavelength multiplexing super surface (4); the wavelength multiplexing super surface (4) simultaneously generates the structured illumination light and the uniform illumination light required by HiLo microscopic imaging, the two kinds of modulated illumination light are collected by the first microscopic objective (6), and after passing through the polarization insensitive beam splitter (7), the electrically adjustable liquid zoom lens (8) and the second microscopic objective (9), the illumination light is projected to the sample stage (13). The wavelength multiplexing super surface (4) comprises a transparent substrate and a plurality of basic unit structures arranged on the transparent substrate, the basic unit structure is a dielectric nano antenna, the dielectric nano antenna comprises a lattice and a dielectric nano column arranged at the center of the upper surface of the lattice; the size of the basic unit structure is subwavelength; and the shape of the transparent substrate is square. The light collection module (C) comprises, sequentially arranged along the light path direction, the second microscopic objective (9), the electrically adjustable liquid zoom lens (8), the polarization insensitive beam splitter (7), a sleeve lens (10) and a color camera (11); the two kinds of illumination light generated by the wavelength multiplexing super surface (4) are projected to a sample to be measured, the reflected light of the sample is firstly collected by the second microscopic objective (9) and the electrically adjustable liquid zoom lens (8), the collected light is reflected to the sleeve lens (10) through the polarization insensitive beam splitter (7), and the sleeve lens (10) finally images the collected light to the pixel target surface of the color camera (11).

2. The wavelength-multiplexed metasurface-based fast three-dimensional microscopic imaging system of claim 1, wherein, The sleeve lens (10) is an aberration-corrected lens matched with the second microscopic objective (9) to form an infinite distance imaging group. The color camera (11) and the electrically adjustable liquid zoom lens (8) are time-synchronized during zooming, and HiLo microscopic images on each scanning plane are shot and collected; the collected image data is uploaded to a server end through Bluetooth.

3. The wavelength-multiplexed metasurface-based fast three-dimensional microscopic imaging system according to claim 1 or 2, characterized in that, The light source module (A) comprises two light sources (1) with different working wavelengths, a fiber combiner (2) and a fiber collimating lens (3). The tail fiber of the light source (1) is connected with the optical fiber combiner (2), and the double-wavelength output light is coupled into the optical fiber combiner (2) to form a coaxial divergent light beam; The optical fiber collimating lens (3) collimates the divergent light beam into a coaxial parallel collimated output light beam.

4. The wavelength-multiplexed metasurface-based fast three-dimensional microscopic imaging system according to claim 1 or 2, characterized in that, The light source module (A) outputs coaxial collimated dual-color light beams, and the working wavelengths are λ 1 and λ 2, respectively, corresponding to two working wavelengths of the structure illumination light and the uniform illumination light required for HiLo microscopic imaging generated by the wavelength multiplexing metasurface (4).

5. The wavelength-multiplexed metasurface-based fast three-dimensional microscopic imaging system according to claim 1 or 2, wherein, The sample stage (13) is used for fixing and adjusting the sample to be measured; The sample to be measured is fixed and protected by a glass slide and a cover glass, and the sample stage (13) is provided with a glass slide mounting groove and an adjusting frame.

6. A method for fast three-dimensional microscopic imaging based on wavelength multiplexed metasurface, characterized in that, The fast three-dimensional microscopic imaging system based on the wavelength multiplexing metasurface is completed by using the wavelength multiplexing metasurface based on the wavelength multiplexing metasurface according to any one of claims 1-5, wherein the light modulation module (B) comprises a wavelength multiplexing metasurface (4), a first microscopic objective lens (6), a polarization-insensitive beam splitter (7), an electrically adjustable liquid zoom lens (8) and a second microscopic objective lens (9) arranged in sequence along the light path direction; The specific process is as follows: Step 1: Prepare the sample to be measured; The biological sample to be measured is prepared by using a glass slide and placed in the sample stage (13), so that the sample to be measured is perpendicular to the optical axis and located on the working distance of the second microscopic objective lens (9); Step 2: Adjust the illumination light; Drive the light source (1) in the light source module (A) to generate the coaxial collimated double-color light beam required by the wavelength multiplexing metasurface (4); drive the electrically adjustable liquid zoom lens (8) to make the two illumination lights modulated by the wavelength multiplexing metasurface (4) cover the outer surface of the sample to be measured; Step 3: Illumination light scanning and image acquisition; Setting the zoom step size Δ of the electrically tunable liquid zoom lens (8) L , making the electrically tunable liquid zoom lens (8) zoom according to the set step size Δ L , making the illumination light scan the sample along the optical axis direction, and the number of acquisition times is N , the total stroke L is the thickness of the sample to be measured, L=N Δ L ; in each acquisition process, the electrically tunable liquid zoom lens (8) needs to zoom by step size and be stable, and synchronously drive the color camera shutter to acquire the double-color illumination image; Step 4: Three-dimensional microscopic image reconstruction and storage; The image data collected in step 3 is transmitted to the server end through Bluetooth, and the server end first separates the illumination images under two working wavelengths by means of the Bayer filter of the color camera; the two separated images are then fused into a high-resolution optical section image by means of the HiLo algorithm; finally, the two high-resolution optical section images are combined N The three-dimensional microscopic image of the sample to be measured is reconstructed from the two optical section images, and is stored in the server end.

7. The wavelength-multiplexed metasurface-based fast three-dimensional microscopic imaging method according to claim 6, wherein, The step 4 specifically comprises the following steps: Step 4.1: Obtain the original image data; The original image data received by the server side is divided into two groups N , i wherein the first group data is the uniform light illumination image data N , and the second group data is the structured light illumination image data i ; wherein the first group data is the uniform light illumination image data λ with a wavelength of 1 I uniform (x,y,i) , and the second group data is the structured light illumination image data λ with a wavelength of 2 I structured (x,y, i) ; Step 4.2: Separate the double-color illumination image; Bayer filters in color cameras are able to separate raw image data into two sets of independent illumination image data by wavelength λ 1 and wavelength λ 2 I uniform (x,y,i) with I structured (x,y,i) ; Step 4.3: Extract the high-frequency component of the image; Uniformly illuminated images of the first i Uniformly illuminated images of the first I uniform (x,y,i) Extracting high frequency components of the image I Hi (x,y,i) The formula is as follows: （1）； wherein F and F -1 are Fourier transform and inverse transform operations, HP f is a high-pass filtering operation; Step 4.4: Extract the low-frequency component of the image; Uniformly illuminated images of the group data are utilized i I uniform (x,y,i) And structured light illuminated images I structured (x,y,i) Compute a local contrast image I C (x,y,i) And extract low frequency components on the image focal plane I Lo (x,y,i) , as follows:​ （2）； （3）； wherein F and F -1 are Fourier transform and inverse transform operations, LP f is a high-pass filtering operation, I C (x,y,i) is a local contrast image, BFP is a Gaussian band-pass filtering operation; Step 4.5: Fuse the HiLo optical section image; The focal plane high frequency component and the focal plane low frequency component in the group data are fused into the required HiLo optical section microscopic image i The focal plane high frequency component and the focal plane low frequency component in the group data are fused into the required HiLo optical section microscopic image I HiLo (x,y,i) , as follows: （4）； wherein η is a high frequency component I Hi (x,y,i) is a low frequency component I Lo (x,y,i) is a weight between the high frequency component Step 4.6: Day 1 i+ 1 Acquisition of HiLo optical sectioning microscopy images for Group 1 For the 1st i+1 set of data, repeat steps 4.3-4.5 to fuse the HiLo optical sectioning micrograph images on the 1st i+1 set of axial planes I HiLo (x,y,i+1) ; thereby obtaining the HiLo micrograph images on the 1st N set of different axial planes I HiLo (x,y,i) ; Step 4.7: Reconstruct the three-dimensional HiLo microscopic image; Will N HiLo optical slice micrographs on different axial planes I HiLo (x,y,i) Three-dimensional HiLo microscopic images of the sample to be tested, stitched together I HiLo (x,y,z) The formula is as follows: （5）。

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