A snapshot light field microscope with large depth of field and a preparation method thereof

Abstract

The application discloses a snapshot type light field microscope with large depth of field and a preparation method thereof, and belongs to the technical field of photoelectric devices and microscopic imaging, and comprises the following steps: design of a large-depth curved micro-lens array; femtosecond laser direct writing preparation of the curved micro-lens array; assembly and integration of the curved micro-lens array; preparation and image calibration of the snapshot type light field microscope; the method of the application is to replace the plane configuration with the curved micro-lens array to enhance the parallax, and replace the conventional spherical lens with a specially designed lens surface to expand the depth of focus, thus fundamentally solving the baseline limitation problem of the conventional plane micro-lens array, greatly improving the imaging depth of field and depth perception ability of the optical microscope, and retaining the imaging resolution at the level of the diffraction limit. The high-precision preparation of the special function surface curved micro-lens array can be realized by using the femtosecond laser additive manufacturing technology, and the prepared curved micro-lens array is integrated with a commercial objective through a customized mechanical adapter, so that the preparation of the snapshot type light field microscope with large depth of field is finally realized.

Description

Technical Field

[0001] This invention belongs to the field of optoelectronic devices and microscopic imaging technology, specifically relating to a large depth-of-field snapshot light field microscope and its preparation method. Background Technology

[0002] Optical microscopes are fundamental research tools for magnifying images, playing a crucial role in unraveling the mysteries of the microscopic world and enhancing scientific understanding. However, traditional microscopes, as orthogonal projection devices, can only observe specimens from one direction, leading to the blurring of superimposed features. In contrast, light field microscopy can simultaneously capture spatial and angular information, enabling the generation of high-resolution and precise-depth 3D images through computer algorithms. This technology provides comprehensive and accurate information, facilitating the observation and study of microscopic structural details and dynamic processes, and is of great significance in life sciences, materials science, and nanoscience.

[0003] Currently, light field microscopy is divided into two different types: scanning light field microscopy and snapshot light field microscopy. Scanning light field microscopy detects light field information by scanning the focal plane in 3D space, offering unique advantages such as a large field of view and high spatial resolution. However, the time-consuming nature of the physical scanning process is difficult to overcome, limiting its application to static scenes. Furthermore, the phototoxicity caused by prolonged exposure is not conducive to biological detection. To achieve dynamic target detection, Levoy et al. pioneered the concept of snapshot light field microscopy. This technique captures the spatial and angular information of the light field using a microlens array, allowing for the reconstruction of the sample's three-dimensional information from a single image, thus enabling real-time three-dimensional imaging of microscopic dynamic targets.

[0004] In snapshot light field microscopy, the microlens array is the core component for encoding light field information to achieve multi-aperture imaging, determining the performance of the light field microscope. However, due to the inherent limitations of the baseline size and planar layout of the microlens array, imaging resolution, imaging depth of field, and depth sensing performance are often incompatible in snapshot light field microscopy. Since this inherent balance is difficult to break, researchers have made great efforts to improve the performance of snapshot light field microscopy based on planar microlens configurations, including but not limited to proposing novel light field microscope configurations (such as Galilean schemes, Keplerian schemes, Fourier schemes, and additional relay lenses), structured light field illumination, and more efficient image processing algorithms to improve the spatial resolution of light field detection, reduce artifacts, and enhance image contrast. However, this usually results in complex system configurations, increased computational costs, and reduced imaging speed. Furthermore, due to the limited spatial size, the limited baseline size can usually only obtain reconstructible visual biases within a small depth of field. To achieve large depth-of-field light field detection, a composite imaging scheme combining focal plane scanning and microlens arrays is a common approach for current snapshot light field microscopes. However, this usually sacrifices the speed advantage of snapshot light field microscopes.

[0005] In summary, the aforementioned research has greatly promoted the improvement of the imaging performance of snapshot light field microscopy and expanded its application market. However, to date, the fundamental constraint between the baseline size of the microlens array and the large depth-of-field parallax has not been resolved. How to achieve snapshot light field microscopy imaging with large depth of field remains an urgent and challenging problem to be solved. Summary of the Invention

[0006] To address the aforementioned problems in existing technologies, this invention provides a large depth-of-field snapshot light field microscope and its fabrication method. This invention utilizes a curved microlens array to replace a planar configuration to enhance parallax and a specially designed lens surface shape to replace traditional spherical lenses to extend the depth of field. This fundamentally solves the baseline limitation problem of traditional planar microlens arrays, significantly improving the imaging depth of field and depth perception capabilities of the optical microscope while retaining diffraction-limited imaging resolution. The high-precision fabrication of the specially designed functional surface curved microlens array proposed in this invention can be achieved using femtosecond laser additive manufacturing technology and integrated with commercial objectives through customized mechanical adapters, ultimately realizing the fabrication of a large depth-of-field snapshot light field microscope.

[0007] This invention is achieved through the following technical solution:

[0008] A method for preparing a large depth-of-field snapshot light field microscope includes the following steps:

[0009] Step 1: Design of a large depth-of-field curved surface microlens array;

[0010] Specific steps: First, based on the optical parameters of the commercial microscope objective to be integrated, determine the sub-lens size radius, focusing range, number of sub-lenses, and bending angle of adjacent lenses for the matching curved microlens array; wherein, the function surface of the sub-lens is designed as a logarithmic surface, and its function surface equation is as follows:

[0011] a=(d2-d1) / R 2 (1)

[0012]

[0013] Where, r, θ, R, n L n0 and λ represent the radius coordinates, azimuth coordinates, sub-lens radius, lens refractive index, ambient refractive index, and lens working center wavelength, respectively; d1 and d2 represent the starting and ending points of lens focusing, respectively; since light travels at different speeds in media with different refractive indices, when the lens works in a non-air medium, optical simulation software is used to further optimize the theoretical focal position of the lens; φ lo.lens The phase cross-sectional profile distribution of a logarithmic lens is represented, and it is converted into the height cross-sectional profile H of the sub-lens based on the relationship between optical path difference and phase difference. lo.lens The formula is as follows:

[0014]

[0015] Finally, based on the number of sub-lenses N and the bending angle of adjacent lenses... This allows us to determine the surface profile height information of the entire curved microlens array;

[0016] Step 2: Fabrication of curved microlens arrays by femtosecond laser direct writing;

[0017] Specific steps: First, the surface contour height information of the curved microlens array calculated in step one is converted into three-dimensional point cloud data that the processing system can recognize and process through a conversion program; then, the substrate is cleaned and optical resin is spin-coated to form a thin layer of optical resin with a thickness of w; the prepared sample is loaded into the femtosecond laser direct writing processing system for sample focusing and leveling; finally, the three-dimensional point cloud data converted from the curved microlens array is imported into the host computer software of the processing system, and point-by-point scanning processing begins. After scanning, the sample is developed and dried to prepare the curved microlens array.

[0018] Step 3: Assembly and integration of the curved microlens array;

[0019] Specific steps: First, the substrate with the curved microlens array prepared in step two is cut into squares of equal length and width, while keeping the center of the curved microlens array on the cut substrate unchanged; then, a custom mechanical adapter with a through hole at the top and internal threads on the sidewalls is designed to mount the curved microlens array; a custom mechanical adapter ring with external threads is fixed to the commercial objective lens for assembling the aforementioned mechanical adapter with the commercial objective lens; the mechanical adapter and the mechanical adapter ring cooperate to achieve the installation and alignment of the curved microlens array and the commercial objective lens; the alignment of the optical axes of the curved microlens array and the commercial objective lens is completed under the microscope system and fixed using optical resin; the distance D between the exit pupil of the curved microlens array and the commercial microscope objective lens is continuously adjusted by the threads of the assembly part; finally, a composite lens integrating the curved microlens array and the commercial microscope objective lens is obtained;

[0020] Step 4: Preparation and image calibration of the snapshot light field microscope;

[0021] Specific steps: First, the composite mirror obtained in step three is installed on a commercial microscope; then, the microscope imaging system is spatially calibrated to establish a mapping relationship between image space and real space. After image calibration, the microscope can perform 3D imaging with a large depth of field, and finally a large depth of field snapshot light field microscope is obtained.

[0022] Furthermore, in step one, the brands of the commercial objectives to be integrated include Olympus, Zeiss, Leica, Nikon, Bruker, or Motic, with a magnification range of 4-100x, a field of view diameter range of 100μm-5mm, a numerical aperture range of 0.1-1.5, and a working distance range of 100μm-20mm.

[0023] In step one, the radius R of the sub-lens ranges from 5μm to 1cm; the focal positions d1 and d2 of the sub-lens range from 20μm to 10mm; and the refractive index n of the lens material... L The value range of n is 1.3-3.0, and the value range of the refractive index n0 of the working environment is 1.0-2.3; the working center wavelength λ of the sub-lens ranges from 350-1600nm; the number R of the curved microlens array ranges from 7-2000, and the bending angle of adjacent lenses is... The range is 0-45°.

[0024] Furthermore, in step two, the substrate is made of glass, quartz, sapphire, or other transparent substrate materials in the visible or near-infrared bands; the length and width of the substrate range from 5mm to 10cm, and the thickness ranges from 50μm to 2mm.

[0025] In step two, the optical resin can be any ultraviolet optical resin that can be processed by femtosecond laser two-photon polymerization, mainly including: epoxy resin SU-8, organic-inorganic hybrid photoresist IP-Dip, IP-S, IP-L, SZ2080, ultraviolet optical curing adhesive NOA61, NOA63, etc.; the thickness w of the optical resin thin layer after spin coating is in the range of 10μm-2mm.

[0026] Furthermore, in step two, the femtosecond laser in the femtosecond laser direct writing system has a center wavelength of 343-1030nm, a pulse width of 50-300fs, and a repetition frequency of 1KHz-100MHz; the processing objective has a magnification of 4-100x and a numerical aperture of 0.1-1.5; the laser processing power is 5-50mw, and the single-point exposure time is 20-5000μs; the photoresist development time is 10-150min.

[0027] Furthermore, in step two, the sample sheet after spin coating with optical resin is selected for pre-baking based on the photosensitivity of the resin. The pre-baking process is carried out on a hot stage, and the sample after pre-baking is naturally cooled in the air environment for later use. The pre-baking heating instrument used is a constant temperature heating stage or a constant temperature heating chamber, etc. The pre-baking temperature range is 50-150℃, and the pre-baking time range is 10min-3h.

[0028] Furthermore, in step three, the length and width of the cut square curved microlens substrate range from 500μm to 2cm; the through-hole diameter of the mechanical adapter used to mount the curved microlens array ranges from 50μm to 5mm, the internal thread diameter ranges from 20mm to 60mm, and the length ranges from 1mm to 50mm; the external thread diameter of the mechanical adapter ring ranges from 20mm to 60mm, and the length ranges from 1mm to 5mm; the mechanical assembly is made of metals such as aluminum, iron, copper, and steel.

[0029] Furthermore, in step three, the optical resin used for fixation includes: epoxy resin SU-8, organic-inorganic hybrid photoresist IP-Dip, IP-S, IP-L, SZ2080, or UV optical curing adhesive NOA61, NOA63.

[0030] In step three, the distance D between the curved microlens array and the exit pupil of the commercial microscope objective is continuously adjustable within a range of 100μm-25mm.

[0031] Furthermore, in step four, the commercial microscopes mentioned include various models of transmission optical microscopes from brands such as Olympus, Zeiss, Leica, Nikon, Bruker, and Motic.

[0032] In step four, the spatial calibration methods include direct linear transformation, polynomial calibration, structured calibration, neural network calibration, and other calibration methods.

[0033] Compared with the prior art, the advantages of the present invention are as follows:

[0034] (1) A large depth-of-field snapshot light field microscope and its preparation method of the present invention uses a curved microlens array to replace the traditional planar configuration. Under the same baseline size, enhanced parallax can be obtained to support 3D imaging over a larger distance range.

[0035] (2) By using the special design of the logarithmic surface of the sub-lens to obtain an extended depth of field, the non-planar imaging challenge caused by the curved arrangement of microlenses is solved, and a wider range of imaging depth of field is obtained.

[0036] (3) High-precision and rapid fabrication of special surface curved microlens arrays is achieved through femtosecond laser two-photon polymerization technology. Stable integration of curved microlens arrays with commercial objectives is achieved through mechanical adapters. Finally, a large depth-of-field snapshot light field microscope is fabricated to achieve real-time 3D imaging under large depth of field.

[0037] (4) The proposed solution of external curved surface microlens array can realize large depth of field real-time 3D imaging without destroying the existing commercial microscope structure. It has the characteristics of simple configuration, compact structure and strong portability. It is also compatible with existing performance improvement solutions such as algorithm assistance, structured light illumination and composite scanning. It has broad prospects in scientific research and practical applications. Attached Figure Description

[0038] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts are not necessarily drawn to scale.

[0039] Figure 1 This is a schematic diagram of the structural composition of the large depth-of-field, snapshot-type light field microscope of the present invention.

[0040] Figure 2 This is a schematic diagram illustrating the 3D imaging principle of the large depth-of-field snapshot light field microscope of the present invention.

[0041] Figure 3 The morphology characterization results of the logarithmic surface curved microlens array prepared in this invention are shown.

[0042] Figure 4 The diagram shows a comparison of the simulation and experimental structures of the focused light field intensity distribution of the spherical and logarithmic surface microlenses of this invention.

[0043] Figure 5 This is a schematic diagram and simulation results of the focused light field intensity distribution of the logarithmic surface curved microlens array of the present invention;

[0044] Figure 6 This is a schematic diagram illustrating the multi-aperture parallax enhancement principle of the curved microlens array of the present invention;

[0045] Figure 7 This is a comparison of the imaging results of the curved surface microlens array with different curvature angles of the present invention on target objects at different distances;

[0046] Figure 8 This is a schematic diagram of the assembly and integration process of the curved microlens array of the present invention with a commercial microscope objective;

[0047] Figure 9 This is a physical image showing the assembly and integration process and results of the curved microlens array of the present invention with a commercial microscope objective;

[0048] Figure 10 This is a physical image of the curved microlens array base, large depth of field, snapshot light field microscope of the present invention;

[0049] Figure 11 This is a schematic diagram of the three-dimensional imaging result of a two-dimensional planar object triangle at a certain distance using the large depth-of-field fast-illumination light field microscope prepared according to the present invention;

[0050] Figure 12 This is a schematic diagram of the three-dimensional imaging result of a three-dimensional object, a quadrangular pyramid, at a certain distance using the large depth-of-field, snapshot-type light field microscope prepared according to the present invention.

[0051] Figure 13 The present invention provides a photograph and schematic diagram of the experimental apparatus for observing the motion of microparticles in a microfluidic chip using a large depth-of-field, snapshot-type light field microscope.

[0052] Figure 14 The images show multi-aperture images and reconstructed spatial positions of moving microparticles within a microfluidic chip at different times, acquired using a large depth-of-field, snapshot-type light field microscope prepared according to the present invention; from left to right, the times are t=0s, t=0.6s, t=1.2s, t=1.8s, t=2.4s, and t=3.0s. Detailed Implementation

[0053] To clearly and completely describe the technical solution and its specific working process of the present invention, the specific embodiments of the present invention are as follows, in conjunction with the accompanying drawings:

[0054] Example 1

[0055] This invention utilizes curved microlens arrays to replace planar configurations to enhance parallax and specially designed lens surface shapes to replace traditional spherical lenses to extend the depth of field. It fundamentally solves the baseline limitation problem of traditional planar microlens arrays, significantly improving the imaging depth of field and depth perception capabilities of optical microscopes while retaining diffraction-limited imaging resolution. The high-precision fabrication of the proposed special functional surface curved microlens array can be achieved using femtosecond laser additive manufacturing technology, possessing a precise functional surface profile that conforms to the design, enabling multi-aperture imaging with large depth of field and enhanced parallax.

[0056] This embodiment provides a method for fabricating a curved microlens array with large depth of field and enhanced parallax, the specific steps of which are as follows:

[0057] (1) Design of a large depth-of-field curved surface microlens array;

[0058] Specific steps: First, based on the optical parameters of the commercial objective to be integrated (including magnification, field of view, numerical aperture, working distance, etc.), determine the sub-lens size radius, focusing range, number of sub-lenses, and bending angle of adjacent lenses for the matching curved microlens array; wherein, the function surface of the sub-lens is designed as a logarithmic surface, and its function surface equation is as follows:

[0059] a=(d2-d1) / R 2 (1)

[0060]

[0061] Where, r, θ, R, n L n0 and λ represent the radius coordinates, azimuth coordinates, lens radius, lens refractive index, ambient refractive index, and lens working center wavelength, respectively; d1 and d2 represent the starting and ending points of lens focusing, respectively; since light travels at different speeds in media with different refractive indices, when the lens is working in a non-air medium, optical simulation software can be used to further optimize the theoretical focal position of the lens; φ lo . lens To represent the phase profile distribution of a logarithmic lens, it is necessary to convert it into a height profile H based on the relationship between the optical path difference and the phase difference. lo.lens The formula is as follows:

[0062]

[0063] Specifically, in this embodiment, based on the optical parameters of the commercial microscope objective to be integrated (magnification 20x, field of view diameter 1.3mm, numerical aperture 0.4, working distance 7mm), the sub-lens radius R is determined to be 40μm, and the refractive index n of the lens material is determined to be... LGiven a refractive index of 1.5, an operating environment refractive index n0 of 1.33, sub-lens focal positions d1 and d2 of 200 μm and 600 μm respectively, and a lens operating center wavelength of 633 nm, the height profile of the sub-lens is determined. Next, the number of sub-lenses N is determined to be 19, and the bending angle of adjacent lenses is determined. A height of 19.8° is sufficient to determine the surface profile height information of the entire curved microlens array;

[0064] (2) Fabrication of curved microlens arrays by femtosecond laser direct writing;

[0065] The specific steps for fabricating a special functional surface microlens array using femtosecond laser direct writing mainly consist of four parts: acquisition of 3D processing point cloud data, sample preparation, femtosecond laser additive manufacturing, and device development and post-processing; as detailed below:

[0066] First, based on the surface contour height information of the curved microlens array calculated in step (1), the surface contour information of the device is converted into three-dimensional point cloud data that can be recognized and processed by the processing system through a conversion program. The three-dimensional point cloud processing data format is (X,Y,Z,L) with an additional shutter switch. Second, the sample preparation: a glass substrate sheet with a length of 50mm, a width of 25mm, and a thickness of 0.17mm is placed in an ultrasonic cleaner and ultrasonically cleaned for 20 minutes to remove large glass fragments and dust particles from the substrate surface, so as to avoid light scattering during processing and affecting the processing quality. Optical resin SZ2080 is spin-coated on the cleaned substrate sheet surface. The thickness w of the optical resin thin layer after spin-coating is 200μm. The sample sheet is placed on a heating stage for pre-baking. The pre-baking temperature is 100℃ and the pre-baking time is 30 minutes. After pre-baking, it is cooled at room temperature.

[0067] Then, femtosecond laser additive manufacturing: The prepared sample sheet is placed into a galvanometer-based femtosecond laser direct-write processing system. In this system, a femtosecond pulsed laser (center wavelength 780nm, pulse width 100fs, repetition frequency 80MHz) generated by a fiber femtosecond laser oscillator passes sequentially through a power adjustment system, a dispersion compensation system, a high-speed optical shutter, and a beam expansion and collimation system. The beam deflection angle is controlled by a scanning galvanometer, and then the 4F optical system projects the angled laser beam onto the entrance pupil of the objective lens. An oil immersion objective lens with 60x magnification and a numerical aperture of 1.42, manufactured by Olympus, focuses the incident light tightly onto the interior of the sample. Combined with the movement of a three-dimensional motion platform, high-speed, large-area three-dimensional processing is achieved. Simultaneously, a real-time monitoring system consisting of an illumination source, a long-pass filter, and an imaging CCD observes the sample's status during processing. Before formal processing, the position where the fluorescence spot critically appears is located by focusing, and the laser is focused at the interface between the polymer film and the substrate, which serves as the starting interface for processing. Subsequently, the spatial plane of the sample was corrected and the sample was leveled by performing a focusing process at different positions on the sample sheet. Then, the point cloud text file of the acquired curved microlens array was imported into the processing software, and the laser power measured in front of the objective lens was set to 18mW using the power adjustment system. Finally, the single-point exposure time was set to 200μs in the processing software, and point-by-point scanning processing began.

[0068] Finally, the processed sample was immersed in the photoresist developer solution (n-propanol) for 40 minutes. After development, the sample was removed from the developer solution and allowed to air dry naturally, thus obtaining a large depth-of-field curved surface microlens array.

[0069] like Figure 1 It can be seen that the curved microlens array is placed between the microscope objective and the 3D imaging target object, and can collect multi-aperture imaging results with large depth of field and enhanced parallax on the detector, thereby realizing snapshot light field imaging under large depth of field.

[0070] like Figure 2 As shown, under the action of curved microlens array, the multi-aperture image collected by the imaging detector is imported into the calibrated neural network reconstruction method, which can reconstruct the three-dimensional information of the target object and realize real-time 3D imaging under large depth of field.

[0071] Depend on Figure 3 As shown, the logarithmic surface curved microlens array prepared by femtosecond laser direct writing technology has a smooth surface, complete structure, and good morphology, and the overall preparation effect is good.

[0072] like Figure 4 As shown, the logarithmic microlens obtained in this embodiment has a longer focal depth and more uniform energy distribution compared to traditional spherical microlenses, thus enabling the acquisition of a greater imaging depth of field.

[0073] like Figure 5 As shown, although curved planar microlens arrays present challenges for non-planar imaging, with the front and rear focal planes of the microlens array being curved surfaces, there is an effective detection range between the front and rear focal planes, within which the imaging results of all sub-lenses can be acquired.

[0074] like Figure 6 As shown, the imaging parallax between different sub-apertures in a traditional planar microlens array is limited. However, when the planar microlens array is bent, the multi-aperture imaging parallax is significantly enhanced.

[0075] like Figure 7 As shown, the parallax of multi-aperture imaging decreases as the imaging distance increases, and disappears after a certain imaging distance. On the other hand, the parallax of multi-aperture imaging increases as the bending angle increases. Therefore, to obtain sufficient parallax under a large depth of field, the bending angle needs to be increased.

[0076] Example 2

[0077] This embodiment integrates the prepared curved microlens array with a commercial objective lens through a customized mechanical adapter, realizing the fabrication of a large depth-of-field snapshot light field microscope. The specific steps are as follows:

[0078] Steps (1) and (2) are the same as in Example 1.

[0079] (3) Assembly and integration of curved microlens arrays;

[0080] Specific steps: First, use a diamond wire cutter to cut the substrate of the curved microlens prepared in step (2) into squares with a length and width of 8mm, and keep the center of the curved microlens unchanged after cutting the substrate; Next, customize an aluminum-based mechanical adapter with a through hole (through hole diameter 2mm) on the top and an internal thread on the side wall (thread diameter 28mm, length 15mm) for mounting the curved microlens array; customize a mechanical adapter ring with an external thread (thread diameter 28mm, length 5mm) and fix it on the commercial objective lens for assembling the aforementioned mechanical adapter with the commercial objective lens. The mechanical adapter and mechanical adapter ring work together to achieve the installation and alignment of the curved microlens array and the commercial objective lens. The alignment of the optical axes of the curved microlens array and the commercial objective lens is completed under the microscope system and fixed using optical resin NOA63. The distance D between the exit pupils of the curved microlens array and the commercial microscope objective lens is adjusted to be greater than 7mm by adjusting the threads, so that the focal plane of the curved microlens array coincides with the focal plane of the commercial objective lens. Finally, a composite lens integrating the curved microlens array and the commercial microscope objective lens is obtained.

[0081] (4) Preparation and image calibration of snapshot light field microscope;

[0082] Specific steps: First, install the composite mirror with the curved microlens array assembled in step (3) onto the commercial microscope CX41 produced by Olympus; next, use the neural network calibration method to spatially calibrate the microscope imaging system, establish the mapping relationship from image space to real space, and the microscope after image calibration can perform 3D imaging under large depth of field, realizing the preparation of a large depth of field snapshot light field microscope; the prepared large depth of field snapshot light field microscope can perform real-time 3D imaging of microscopic targets.

[0083] like Figure 8 As shown, the logarithmic surface microlens array is assembled and integrated with commercial microscope objectives through a mechanical adapter with a through hole and internal thread and an external thread mechanical adapter ring.

[0084] like Figure 9 As shown, the fabricated curved microlens array can be stably assembled together using a customized mechanical adapter, and the distance between the microlens array and the objective lens exit pupil can be continuously adjusted via threads.

[0085] like Figure 10 As shown, a composite mirror assembled from a curved microlens array and a commercial objective lens can be mounted on a commercial transmission optical microscope, thereby completing the fabrication of a large depth-of-field, snapshot-type light field microscope.

[0086] like Figure 11 It can be seen that the prepared large depth-of-field, snapshot-type light field microscope can realize three-dimensional imaging of two-dimensional planar objects at different distances. The size and distance reconstruction information of the two-dimensional object triangles have a high degree of consistency with the real information, and the three-dimensional imaging capability is strong.

[0087] like Figure 12 It can be seen that the prepared large depth-of-field, snapshot-type light field microscope can realize three-dimensional imaging of micro-sized three-dimensional objects at different distances. The three-dimensional size information of the three-dimensional object, the quadrangular pyramid, has a high degree of consistency with the real information, and the three-dimensional imaging capability is strong.

[0088] The large depth-of-field snapshot light field microscope prepared above is used to monitor three-dimensional flow fields, breaking the limitation of traditional microscopes that can only observe 2D flow fields. It enables real-time three-dimensional reconstruction of the motion trajectory of non-fluorescent particles in the flow field. The specific steps are as follows:

[0089] Steps (1), (2), (3), and (4) are the same as in Example 2.

[0090] Among them, the large depth-of-field snapshot light field microscope observes non-fluorescent particles of microfluidic chips that move with changes in the flow field.

[0091] Depend on Figure 13It can be seen that the large depth-of-field, snapshot-type light field microscope prepared in this embodiment can be used to observe the movement of micro-sized particles within a microfluidic chip.

[0092] Depend on Figure 14 It can be seen that the large depth-of-field, snapshot-type light field microscope prepared in this embodiment can acquire motion images of micro-sized particles in the microfluidic chip at different times, and realize real-time tracking and detection of dynamic targets under large depth of field.

[0093] The preferred embodiments of the present invention have been described in detail above with reference to the accompanying drawings. However, the present invention is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0094] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0095] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A method for preparing a large depth-of-field snapshot light field microscope, characterized in that, Specifically, the steps include the following: Step 1: Design of a large depth-of-field curved surface microlens array; Specific steps: First, based on the optical parameters of the commercial microscope objective to be integrated, determine the sub-lens size radius, focusing range, number of sub-lenses, and bending angle of adjacent lenses for the matching curved microlens array; wherein, the function surface of the sub-lens is designed as a logarithmic surface, and its function surface equation is as follows: (1) (2) in, , , , , , These represent the radius coordinates, azimuth coordinates, sub-lens radius, lens refractive index, ambient refractive index, and lens working center wavelength, respectively. and These represent the starting and ending points of the lens focusing, respectively. Since light travels at different speeds in media with different refractive indices, when the lens is working in a non-air medium, optical simulation software is used to further optimize the theoretical focal position of the lens. The phase cross-sectional profile distribution of a logarithmic lens is represented, and it is converted into the height cross-sectional profile of a sub-lens based on the relationship between optical path difference and phase difference. The formula is as follows: (3) Finally, based on the number of sub-lenses The bending angle of adjacent lenses This allows us to determine the surface profile height information of the entire curved microlens array; Step 2: Fabrication of curved microlens arrays by femtosecond laser direct writing; Specific steps: First, the surface contour height information of the curved microlens array calculated in step one is converted into three-dimensional point cloud data that the processing system can recognize and process using a conversion program; then, the substrate is cleaned and spin-coated with optical resin to form a thickness of [missing information]. The optical resin thin layer is prepared; the prepared sample sheet is loaded into the femtosecond laser direct writing processing system for sample focusing and leveling; finally, the three-dimensional point cloud data converted from the curved microlens array is imported into the host computer software of the processing system, and point-by-point scanning processing begins. After scanning, the sample is developed and dried to prepare the curved microlens array. Step 3: Assembly and integration of the curved microlens array; Specific steps: First, the substrate with the curved microlens array prepared in step two is cut into squares of equal length and width, while keeping the center of the curved microlens unchanged after cutting. Then, a mechanical adapter with a through hole at the top and internal threads on the sidewalls is customized for mounting the curved microlens array. A mechanical adapter ring with external threads is customized and fixed to the commercial objective for assembling the aforementioned mechanical adapter with the commercial objective. The mechanical adapter and the mechanical adapter ring cooperate to realize the installation and alignment of the curved microlens array and the commercial objective. The alignment of the optical axes of the curved microlens array and the commercial objective is completed under the microscope system and fixed with optical resin. The distance D between the exit pupil of the curved microlens array and the commercial microscope objective is continuously adjusted by the threads of the assembly. Finally, a composite lens integrating the curved microlens array and the commercial microscope objective is obtained. Step 4: Preparation and image calibration of the snapshot light field microscope; Specific steps: First, the composite mirror obtained in step three is installed on a commercial microscope; then, the microscope imaging system is spatially calibrated to establish a mapping relationship between image space and real space. After image calibration, the microscope can perform 3D imaging with a large depth of field, and finally a large depth of field snapshot light field microscope is obtained.

2. The method for preparing a large depth-of-field snapshot light field microscope as described in claim 1, characterized in that, In step one, the brands of the commercial objectives to be integrated include Olympus, Zeiss, Leica, Nikon, Bruker or Motic, with a magnification range of 4-100x, a field diameter range of 100μm-5mm, a numerical aperture range of 0.1-1.5, and a working distance range of 100μm-20mm. In step one, the radius of the sub-lens The range is 5μm-1cm; the focal position of the sub-lens and The value range is 20μm-10mm; the refractive index of the lens material is... The value range is 1.3-3.0, and the refractive index of the working environment is... The value range is 1.0-2.3; the working center wavelength of the sub-lens is... The range is 350-1600nm; the number of sub-lenses in the curved microlens array The range is 7-2000, and the bending angle of adjacent lenses is... The range is 0-45°.

3. The method for preparing a large depth-of-field snapshot light field microscope as described in claim 1, characterized in that, In step two, the substrate is made of glass, quartz, sapphire, or other transparent substrate materials in the visible or near-infrared bands; the length and width of the substrate range from 5mm to 10cm, and the thickness ranges from 50μm to 2mm. In step two, the optical resin can be any ultraviolet optical resin that can be processed using femtosecond laser two-photon polymerization, including: epoxy resin SU-8, organic-inorganic hybrid photoresists IP-Dip, IP-S, IP-L, and SZ2080, and ultraviolet optical curing adhesives NOA61 and NOA63; the thickness of the optical resin thin layer after spin coating... The range is 10μm-2mm.

4. The method for preparing a large depth-of-field snapshot light field microscope as described in claim 1, characterized in that, In step two, the femtosecond laser in the femtosecond laser direct writing system has a center wavelength of 343-1030nm, a pulse width of 50-300fs, and a repetition frequency of 1KHz-100MHz; the processing objective has a magnification of 4-100x and a numerical aperture of 0.1-1.5; the laser processing power is 5-50mw; the single-point exposure time is 20-5000μs; and the photoresist development time is 10-150min.

5. The method for preparing a large depth-of-field snapshot light field microscope as described in claim 1, characterized in that, In step two, the sample sheet after spin coating with optical resin is selected for pre-baking based on the photosensitivity of the resin. The pre-baking process is carried out on a hot stage, and the sample after pre-baking is allowed to cool naturally in the air environment for later use. The pre-baking heating instrument used is a constant temperature heating stage or a constant temperature heating chamber, with a pre-baking temperature range of 50-150℃ and a pre-baking time range of 10min-3h.

6. The method for preparing a large depth-of-field snapshot light field microscope as described in claim 1, characterized in that, In step three, the length and width of the cut square curved microlens substrate range from 500μm to 2cm; the through-hole diameter of the mechanical adapter used to mount the curved microlens array ranges from 50μm to 5mm, the internal thread diameter ranges from 20mm to 60mm, and the length ranges from 1mm to 50mm; the external thread diameter of the mechanical adapter ring ranges from 20mm to 60mm, and the length ranges from 1mm to 5mm; the materials of the mechanical components are aluminum, iron, copper, and steel.

7. The method for preparing a large depth-of-field snapshot light field microscope as described in claim 1, characterized in that, In step three, the optical resin used for fixation includes: epoxy resin SU-8, organic-inorganic hybrid photoresist IP-Dip, IP-S, IP-L, SZ2080, or UV-curable photoresist NOA61, NOA63. In step three, the distance D between the curved microlens array and the exit pupil of the commercial microscope objective is continuously adjustable within a range of 100μm-25mm.

8. The method for preparing a large depth-of-field snapshot light field microscope as described in claim 1, characterized in that, In step four, the commercial microscopes mentioned include various models of transmission optical microscopes from Olympus, Zeiss, Leica, Nikon, Bruker, and Motic. In step four, the spatial calibration methods include direct linear transformation, polynomial calibration, structured calibration, and neural network calibration.

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