Snapshot type light field microscope with large depth of field and preparation method thereof

Abstract

The invention discloses a large-depth-of-field snapshot type light field microscope and a preparation method thereof, and belongs to the technical field of photoelectric devices and microscopic imaging, and the preparation method comprises the following steps: designing a large-depth-of-field curved surface micro lens array; performing femtosecond laser direct writing preparation on the curved surface micro lens array; assembling and integrating the curved micro-lens array; preparation and image calibration of a snapshot light field microscope; according to the method, plane configuration is replaced by the curved-surface micro-lens array to enhance parallax, a traditional spherical lens is replaced by a specially designed lens surface type to expand the focal depth, the problem of baseline limitation of a traditional plane micro-lens array is fundamentally solved, the imaging depth of field and the depth perception capability of the optical microscope are greatly improved, and the imaging precision of the optical microscope is improved. And the imaging resolution of the diffraction limit level is reserved. The high-precision preparation of the special function surface type curved surface microlens array provided by the invention can be realized by utilizing a femtosecond laser additive manufacturing technology, and the preparation of the large-field-depth snapshot type light field microscope is finally realized by integrating a customized mechanical adapter and a commercial objective lens.

Description

Technical Field

[0001] The invention belongs to the technical field of optoelectronic devices and microscopic imaging, and in particular relates to a snapshot light field microscope with a large depth of field and a preparation method thereof. Background Art

[0002] Optical microscopy is a basic research tool for magnifying images and has played a key role in unlocking the mysteries of the microscopic world and advancing scientific cognition. However, as an orthogonal projection device, traditional microscopes can only observe specimens from one direction, resulting in ambiguity in superimposed features. In contrast, light field microscopy is able to capture both spatial and angular information simultaneously, enabling the generation of 3D images with high resolution and precise depth through computer algorithms. This technology provides comprehensive and accurate information, which is conducive to the observation and study of microstructural details and dynamic processes, and is of great significance in life sciences, materials sciences, and nanosciences.

[0003] Currently, light field microscopes are divided into two different types: scanning light field microscopes and snapshot light field microscopes. Scanning light field microscopes detect light field information through 3D spatial scanning of the focus or focal plane, and have the unique advantages of large field of view and high spatial resolution. However, the time-consuming problem caused by the physical scanning process is difficult to solve, and it is only applicable to static scenes, and the phototoxicity caused by long-term exposure is not friendly to biological detection. In order to achieve dynamic target detection, Levoy et al. first proposed the concept of snapshot light field microscopes, which capture the spatial and angular information of the light field through a microlens array, and reconstruct the three-dimensional information of the sample through a single shot, making real-time three-dimensional imaging of microscopic dynamic targets possible.

[0004] In snapshot light field microscopy, the microlens array is the core component for realizing multi-aperture imaging for encoding light field information, and determines the performance of the light field microscope. However, due to the inherent limitations of the baseline size and planar layout of the microlens array, the imaging resolution, imaging depth of field, and depth perception performance are usually incompatible in snapshot light field microscopy. Since this inherent balance is difficult to break, in order to improve the working performance of snapshot light field microscopy, researchers have made great efforts based on the planar microlens configuration, including but not limited to proposing novel light field microscope configurations (such as Galileo scheme, Kepler scheme, Fourier scheme, and additional relay lenses, etc.), 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 configuration, increased computational costs, and reduced imaging speed. In addition, due to the limited spatial size, the limited baseline size can usually only obtain reconstructible visual deviations within a small depth of field. In order to achieve large depth of field light field detection, a composite imaging scheme combining focal plane scanning with a microlens array is a common scheme for current snapshot light field microscopes to achieve large depth of field detection, but this usually loses the speed advantage of snapshot light field microscopes.

[0005] In summary, the above research work has a great driving force in improving the imaging performance of snapshot light field microscopes and expanding their application market. However, up to now, the restrictive relationship between the baseline size of the microlens array and the large depth of field parallax has not been fundamentally solved. How to achieve snapshot light field microscopy imaging under large depth of field is still an urgent and challenging problem to be solved. Summary of the invention

[0006] In view of the above problems existing in the prior art, the present invention provides a snapshot light field microscope with a large depth of field and a preparation method thereof. The present invention uses a curved microlens array to replace a plane configuration to enhance parallax, and a specially designed lens surface to replace a traditional spherical lens to extend the depth of focus, which fundamentally solves the baseline limitation problem of the traditional plane microlens array, greatly improves the imaging depth of field and depth perception ability of the optical microscope, and retains the imaging resolution at the diffraction limit level. The high-precision preparation of the special function surface curved microlens array proposed in the present invention can be achieved using the "femtosecond laser additive manufacturing" technology, and integrated with a commercial objective lens through a customized mechanical adapter, ultimately realizing the preparation of a snapshot light field microscope with a large depth of field.

[0007] The present invention is achieved through the following technical solutions:

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

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

[0010] Specific steps: First, according to the optical parameters of the commercial microscope objective to be integrated, the sub-lens size radius, focusing range, number of sub-lenses and bending angle of adjacent lenses of the matching curved microlens array are determined; 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] Among them, r, θ, R, n L , n0, λ represent the radius coordinate, azimuth coordinate, sub-lens radius, lens refractive index, ambient refractive index and lens working center wavelength respectively; d1 and d2 represent the starting point and end point of lens focusing respectively; due to the different propagation speeds of light in media with different refractive indices, when the lens works in a non-air medium, the optical simulation software is used to further optimize the theoretical focal position of the lens; φ lo.lens It represents the phase cross-sectional profile distribution of the logarithmic lens, which is converted into the height cross-sectional profile H of the sub-lens according to the relationship between the optical path difference and the phase difference. lo.lens , the formula is as follows:

[0014]

[0015] Finally, according to the number of sub-lenses N and the bending angle of adjacent lenses The surface profile height information of the entire curved microlens array can be determined;

[0016] Step 2: preparation of curved microlens array by femtosecond laser direct writing;

[0017] Specific steps: first, the surface profile height information of the curved microlens array calculated in step 1 is converted into three-dimensional point cloud data that can be recognized and processed by the processing system through a conversion program; then, the substrate is cleaned and an 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 to perform sample focusing and leveling; finally, the three-dimensional point cloud data converted from the curved microlens array is imported into the upper computer software of the processing system, and point-by-point scanning processing is started. The scanned sample is developed and dried to prepare the curved microlens array;

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

[0019] Specific steps: first, cut the substrate with the curved microlens array prepared in step 2 into squares with equal length and width, and keep the center of the curved microlens array on the cut substrate unchanged; then, customize a mechanical adapter with a through hole on the top and internal threads on the side wall for loading the curved microlens array; customize a mechanical adapter ring with external threads and fix it on the commercial objective lens for assembling the aforementioned mechanical adapter with the commercial objective lens; the mechanical adapter cooperates with the mechanical adapter ring to achieve the installation and alignment of the curved microlens array and the commercial objective lens; wherein, the alignment of the optical axes of the curved microlens array and the commercial objective lens is completed under the microscope system and fixed with optical resin; the distance D between the curved microlens array and the exit pupil of the commercial microscope objective lens is continuously adjusted by the threads of the assembly parts; finally, a composite objective lens in which the curved microlens array and the commercial microscope objective lens are assembled and integrated is obtained;

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

[0021] Specific steps: First, the composite objective lens obtained in step three is installed on a commercial microscope; then, the microscope imaging system is spatially calibrated to establish a mapping relationship from image space to 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] Further, in step 1, the brands of the commercial objective lenses to be integrated include Olympus, Zeiss, Leica, Nikon, Bruker or Motic, with a magnification range of 4-100 times, a field of view diameter range of 100 μm-5 mm, a numerical aperture range of 0.1-1.5, and a working distance range of 100 μm-20 mm;

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

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

[0025] In step 2, 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 photoresists IP-Dip, IP-S, IP-L, SZ2080, ultraviolet optical curing glue 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 2, the central wavelength of the femtosecond laser in the femtosecond laser direct writing processing system is 343-1030nm, the pulse width is 50-300fs, and the repetition frequency is 1KHz-100MHz; the magnification of the processing objective lens is 4-100 times, and the numerical aperture is 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 2, after the optical resin is spin-coated, the sample sheet is pre-baked according to the photosensitivity of the resin. The pre-baking process is carried out on a hot table, and the sample after pre-baking is naturally cooled in an air environment for use. The pre-baking heating instrument used is a constant temperature heating table or a constant temperature heating box and other heating equipment. The pre-baking temperature range is 50-150°C, 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 500 μm to 2 cm; the through hole diameter of the mechanical adapter for loading the curved microlens array ranges from 50 μm to 5 mm, the internal thread diameter ranges from 20 mm to 60 mm, and the length ranges from 1 mm to 50 mm; the external thread diameter of the mechanical adapter ring ranges from 20 mm to 60 mm, and the length ranges from 1 mm to 5 mm; the material of the mechanical assembly parts is metal materials such as aluminum, iron, copper, and steel.

[0029] Furthermore, in step 3, the optical resin used for fixing includes: epoxy resin SU-8, organic-inorganic hybrid photoresist IP-Dip, IP-S, IP-L, SZ2080 or UV optical curing glue NOA61, NOA63;

[0030] In step 3, the continuously adjustable range of the distance D between the curved microlens array and the exit pupil of the commercial microscope objective is 100 μm-25 mm.

[0031] Furthermore, in step 4, the commercial microscopes include transmission optical microscopes of various models of brands such as Olympus, Zeiss, Leica, Nikon, Bruker, and Motic;

[0032] In step 4, the spatial calibration method includes calibration methods such as direct linear transformation, polynomial calibration, structured light calibration, and neural network calibration.

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

[0034] (1) A snapshot light field microscope with a large depth of field and a method for preparing the same of the present invention uses a curved microlens array to replace the traditional planar configuration, and can obtain enhanced parallax at the same baseline size to support 3D imaging over a larger distance range;

[0035] (2) The extended depth of focus is achieved through the special design of the logarithmic surface of the sub-lens, which solves the non-planar imaging challenge caused by the curved surface arrangement of the micro-lenses and obtains a wider range of imaging depth of field;

[0036] (3) Through femtosecond laser two-photon polymerization technology, high-precision and rapid preparation of special surface-shaped curved microlens arrays is achieved, and through mechanical adapters, stable integration of curved microlens arrays and commercial objective lenses is achieved, and finally a large depth of field snapshot light field microscope is prepared to achieve real-time 3D imaging under a large depth of field;

[0037] (4) The proposed external curved microlens array solution can achieve real-time 3D imaging with a large depth of field without destroying the structure of existing commercial microscopes. 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 compound scanning. It has broad prospects in scientific research and practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the specific embodiments or the description of the prior art. 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 according to the actual scale.

[0039] Figure 1 It is a schematic diagram of the structure of the large depth of field and snapshot light field microscope of the present invention;

[0040] Figure 2 It is a schematic diagram of 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 by the present invention;

[0042] Figure 4 It is a comparison diagram of the simulation and experimental structures of the intensity distribution of the focused light field of the spherical and logarithmic surface microlenses of the present invention;

[0043] Figure 5 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 It is a schematic diagram of the multi-aperture parallax enhancement principle of the curved microlens array of the present invention;

[0045] Figure 7 This is a comparison diagram of imaging results of target objects at different distances using curved surface microlens arrays with different bending angles of the present invention;

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

[0047] Fig. 9 A physical diagram of the process and results of assembling and integrating the curved microlens array of the present invention with a commercial microscope objective lens;

[0048] Fig.10 A real picture of the curved microlens array base, large depth of field, snapshot-type light field microscope of the present invention;

[0049] Fig.11 A schematic diagram of the three-dimensional imaging result of a triangle of a two-dimensional plane object at a certain distance using a fast, illumination-type light field microscope with a large depth of field prepared by the present invention;

[0050] Fig.12 A schematic diagram of the three-dimensional imaging result of a three-dimensional object tetrahedron at a certain distance using a large depth of field, snapshot-type light field microscope prepared by the present invention;

[0051] Fig.13 Actual photos and schematic diagrams of an experimental device for observing the movement of microscopic particles in a microfluidic chip using a large depth of field and snapshot light field microscope prepared for use in the present invention;

[0052] Fig.14 Multi-aperture images and reconstructed spatial position results of microscopic particles moving in a microfluidic chip at different times collected by a large depth of field, snapshot light field microscope prepared for use of 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 DESCRIPTION

[0053] In order to clearly and completely describe the technical solution and its specific working process of the present invention, the specific implementation methods of the present invention are as follows in conjunction with the accompanying drawings of the specification:

[0054] Example 1

[0055] The present invention uses a curved microlens array to replace a flat configuration to enhance parallax, and a specially designed lens surface to replace a traditional spherical lens to extend the depth of focus, which fundamentally solves the baseline limitation problem of the traditional flat microlens array, greatly improves the imaging depth of field and depth perception of the optical microscope, and retains the imaging resolution at the diffraction limit level. The high-precision preparation of the proposed special function surface type curved microlens array can be achieved using the "femtosecond laser additive manufacturing" technology, with a precise function surface profile that meets the design, and can achieve multi-aperture imaging with a large depth of field and parallax enhancement.

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

[0057] (1) Design of curved microlens array with large depth of field;

[0058] Specific steps: First, according to the optical parameters of the commercial objective lens to be integrated (including magnification, field of view size, numerical aperture, working distance, etc.), the sub-lens size radius, focusing range, number of sub-lenses and bending angle of adjacent lenses of the matching curved microlens array are determined; 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] Among them, r, θ, R, n L , n0, λ represent the radius coordinate, azimuth coordinate, lens radius, lens refractive index, ambient refractive index and lens working center wavelength respectively; d1 and d2 represent the starting point and end point of lens focusing respectively; due to the different propagation speeds of light in media with different refractive indices, when the lens works in a non-air medium, the optical simulation software can be used to further optimize the theoretical focal position of the lens; φ lo . lens The phase cross-sectional profile distribution of the logarithmic lens needs to be converted into a height cross-sectional profile H according to the relationship between the optical path difference and the phase difference. lo.lens , the formula is as follows:

[0062]

[0063] Specifically, in this embodiment, the radius R of the sub-lens is 40 μm, and the refractive index n of the lens material is 40 μm, according to the optical parameters of the commercial microscope objective lens to be integrated (magnification 20 times, field of view diameter 1.3 mm, numerical aperture 0.4, working distance 7 mm). LThe height profile of the sub-lens is determined by taking the working environment refractive index n0 as 1.5, the working environment refractive index n0 as 1.33, the sub-lens focal positions d1 and d2 as 200μm and 600μm respectively, and the working center wavelength of the lens as 633nm. Then the height profile of the sub-lens is determined; the number of sub-lenses N is determined to be 19 and the bending angle of the adjacent lens is determined to be The surface profile height information of the entire curved microlens array can be determined when the angle is 19.8°.

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

[0065] Specific steps: The preparation of special function surface curved microlens array by femtosecond laser direct writing mainly includes four parts, namely the acquisition of 3D processing point cloud data, preliminary preparation of samples, femtosecond laser additive manufacturing, and device development and post-processing; the details are as follows:

[0066] First, according to the surface profile height information of the curved microlens array calculated in step (1), the surface profile 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 format of the three-dimensional point cloud processing data is the (X, Y, Z, L) form with an additional optical gate switch; second, the preliminary preparation of the sample: a glass-based substrate sheet with a length of 50 mm, a width of 25 mm, and a thickness of 0.17 mm is placed in an ultrasonic machine and ultrasonically cleaned for 20 minutes to remove large-sized glass debris and dust particles on the surface of the substrate to avoid light scattering during the processing process and affecting the processing quality; optical resin SZ2080 is spin-coated on the surface of the cleaned substrate sheet, and the thickness w of the optical resin thin layer after spin coating is 200 μm; the sample sheet is placed on a heating table for pre-baking, the pre-baking temperature is 100° C., the pre-baking time is 30 minutes, and the sample is cooled at room temperature after pre-baking.

[0067] Then, femtosecond laser additive manufacturing: the prepared sample is placed in a galvanometer-based femtosecond laser direct writing processing system; in this processing system, the femtosecond pulse laser (central wavelength of 780nm, pulse width of 100fs, repetition frequency of 80MHz) generated by the fiber femtosecond laser oscillator passes through the power regulation system, dispersion compensation system, high-speed optical gate and beam expansion collimation system in turn, and the beam deflection angle is controlled by the scanning galvanometer, and then the angle of the laser beam is deflected and projected to the entrance pupil of the objective lens by the 4F optical system. The oil-immersed objective lens with a magnification of 60 times and a numerical aperture of 1.42 produced by Olympus focuses the incident light tightly into the processing sample, and cooperates with the movement of the three-dimensional motion platform to achieve high-speed, large-area three-dimensional processing; at the same time, a real-time monitoring system composed of an illumination light source, a long-wave pass filter, and an imaging CCD is used to observe the state of the sample during processing in real time. Before formal processing, the critical position of the fluorescent spot is found by focusing, and the laser is focused to the interface between the polymer film and the substrate sheet, which is used as the starting interface for processing. Then, by performing the focusing process at different positions of the sample, the spatial plane of the sample is corrected and the sample is leveled. Then, the point cloud text file of the acquired curved microlens array is imported into the processing software, and the laser power measured in front of the objective lens is set to 18mw through the power adjustment system. Finally, the single point exposure time is set to 200μs on the processing software, and the point-by-point scanning processing is started.

[0068] Finally, the processed sample was immersed in a photoresist developer, n-propanol, for 40 minutes. After the development was completed, the sample was taken out of the developer and dried naturally in an air environment, and finally a large depth of field curved microlens array was prepared.

[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 multi-aperture imaging results with large depth of field and parallax enhancement can be collected on the detector, thereby realizing snapshot light field imaging with a large depth of field.

[0070] like Figure 2 As shown, under the action of the curved microlens array, the multi-aperture images collected by the imaging detector are imported into the calibrated neural network reconstruction method to reconstruct the three-dimensional information of the target object and realize real-time 3D imaging with a 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, a complete structure, a good morphology, and a good overall preparation effect.

[0072] like Figure 4 As shown, the logarithmic surface microlens obtained in this embodiment has a longer focal depth and uniform energy distribution compared to the traditional spherical microlens, so a larger imaging depth of field can be obtained.

[0073] like Figure 5 As shown, although the curved planar microlens array brings non-planar imaging challenges, the front focal plane and the back focal plane of the microlens array are curved surfaces, but there is an effective detection range between the front and back focal planes, within which the imaging results of all sub-lenses can be collected.

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

[0075] like Figure 7 As shown in the figure, the multi-aperture imaging parallax decreases with the increase of imaging distance, and the imaging parallax disappears when the imaging distance exceeds a certain value. On the other hand, the multi-aperture imaging parallax increases with the increase of bending angle. Therefore, in order to obtain sufficient parallax at a large depth of field, the bending angle needs to be increased.

[0076] Example 2

[0077] In this embodiment, the prepared curved microlens array is integrated with a commercial objective lens through a customized mechanical adapter to achieve the preparation 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 those in Example 1.

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

[0080] The specific steps are as follows: first, using a diamond wire cutting machine to cut the substrate of the curved microlens prepared in step (2) into a square with a length and width of 8 mm, and keep the curved microlens unchanged at the center of the substrate after cutting; next, a mechanical adapter with an aluminum base, a through hole on the top (through hole diameter 2 mm), and an internal thread on the side wall (thread diameter 28 mm, length 15 mm) is customized for loading the curved microlens array; a mechanical adapter ring with an external thread (thread diameter 28 mm, length 5 mm) is customized and fixed on the commercial objective lens for assembling the aforementioned mechanical adapter and the commercial objective lens. The mechanical adapter cooperates with the mechanical adapter ring to realize 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 with optical resin NOA63; the distance D between the curved microlens array and the exit pupil of the commercial microscope objective lens is adjusted to be greater than 7 mm by screw thread, so that the focal plane of the curved microlens coincides with the focal plane of the commercial objective lens; finally, a composite objective lens in which the curved microlens array and the commercial microscope objective lens are assembled and integrated is obtained.

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

[0082] The specific steps are as follows: first, the composite microscope equipped with the curved microlens array in step (3) is installed on a commercial microscope CX41 produced by Olympus; next, the microscope imaging system is spatially calibrated using a neural network calibration method to establish a mapping relationship from image space to real space. After image calibration, the microscope can perform 3D imaging with a large depth of field, thereby realizing the preparation of a large depth of field snapshot light field microscope; the prepared large depth of field snapshot light field microscope can be used to perform real-time 3D imaging of microscopic targets.

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

[0084] like Fig. 9 As shown, the prepared curved microlens array can be stably assembled together through a customized mechanical adapter, and the distance between the microlens array and the objective exit pupil can be continuously adjusted through a thread.

[0085] like Fig.10 As shown, the composite objective lens composed of a curved microlens array and a commercial objective lens can be installed on a commercial transmission optical microscope to complete the preparation of a large depth of field, snapshot light field microscope.

[0086] like Fig.11 It can be seen that the prepared large depth of field, snapshot light field microscope can realize three-dimensional imaging of two-dimensional plane objects at different distances. The size and distance reconstructed information of the two-dimensional object triangle is highly consistent with the real information, and the three-dimensional imaging capability is strong.

[0087] like Fig.12 It can be seen that the prepared large depth of field, snapshot 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 tetrahedron is highly consistent 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 the three-dimensional flow field, breaking the limitation that traditional microscopes can only observe the 2D flow field, and realizing the 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 those in Example 2.

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

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

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

[0093] The preferred embodiments of the present invention are described in detail above in conjunction with the accompanying drawings. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to 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 further describe various possible combinations.

[0095] In addition, various embodiments of the present invention may be arbitrarily combined, and as long as they do not violate the concept of the present invention, they should also be regarded as the contents disclosed by the present invention.

Claims

1. A method for preparing a snapshot light field microscope with a large depth of field, characterized in that: The specific steps include: Step 1: Design of large depth of field curved microlens array; Specific steps: First, according to the optical parameters of the commercial microscope objective to be integrated, the sub-lens size radius, focusing range, number of sub-lenses and bending angle of adjacent lenses of the matching curved microlens array are determined; wherein the function surface of the sub-lens is designed as a logarithmic surface, and its function surface equation is as follows: a=(d2-d1) / R 2 (1) Among them, r, θ, R, n L , n0, λ represent the radius coordinate, azimuth coordinate, sub-lens radius, lens refractive index, ambient refractive index and lens working center wavelength respectively; d1 and d2 represent the starting point and end point of lens focusing respectively; due to the different propagation speeds of light in media with different refractive indices, when the lens works in a non-air medium, the optical simulation software is used to further optimize the theoretical focal position of the lens; φ lo，lens It represents the phase cross-sectional profile distribution of the logarithmic lens, which is converted into the height cross-sectional profile H of the sub-lens according to the relationship between the optical path difference and the phase difference. lo.lens , the formula is as follows: Finally, according to the number of sub-lenses N and the bending angle of adjacent lenses The surface profile height information of the entire curved microlens array can be determined; Step 2: preparation of curved microlens array by femtosecond laser direct writing; Specific steps: first, the surface profile height information of the curved microlens array calculated in step 1 is converted into three-dimensional point cloud data that can be recognized and processed by the processing system through a conversion program; then, the substrate is cleaned and an 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 to perform sample focusing and leveling; finally, the three-dimensional point cloud data converted from the curved microlens array is imported into the upper computer software of the processing system, and point-by-point scanning processing is started. The scanned sample is developed and dried to prepare the curved microlens array; Step 3: Assembly and integration of curved microlens array; Specific steps: first, cut the substrate with the curved microlens array prepared in step 2 into squares with equal length and width, and keep the curved microlens unchanged at the center of the substrate after cutting; then, customize a mechanical adapter with a through hole on the top and internal threads on the side wall for loading the curved microlens array; customize a mechanical adapter ring with external threads and fix it on the commercial objective lens for assembling the aforementioned mechanical adapter and the commercial objective lens; the mechanical adapter cooperates with the mechanical adapter ring to achieve the installation and alignment of the curved microlens array and the commercial objective lens; wherein, the alignment of the optical axes of the curved microlens array and the commercial objective lens is completed under the microscope system and fixed with optical resin; the distance D between the curved microlens array and the exit pupil of the commercial microscope objective lens is continuously adjusted by the threads of the assembly parts; finally, a composite objective lens in which the curved microlens array and the commercial microscope objective lens are assembled and integrated is obtained; Step 4: Preparation and image calibration of snapshot light field microscope; Specific steps: First, the composite objective lens obtained in step three is installed on a commercial microscope; then, the microscope imaging system is spatially calibrated to establish a mapping relationship from image space to 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 snapshot light field microscope with a large depth of field as claimed in claim 1, characterized in that: In step 1, the brands of the commercial objective lens to be integrated include Olympus, Zeiss, Leica, Nikon, Bruker or Motic, with a magnification range of 4-100 times, a field of view diameter range of 100 μm-5 mm, a numerical aperture range of 0.1-1.5, and a working distance range of 100 μm-20 mm; In step 1, the sub-lens radius A ranges from 5 μm to 1 cm; the sub-lens focal positions d1 and d2 range from 20 μm to 10 mm; the lens material refractive index n L The value range of is 1.3-3.0, the value range of the working environment refractive index n0 is 1.0-2.3; the working center wavelength λ of the sub-lens is in the range of 350-1600nm; the number of sub-lenses R of the curved microlens array is in the range of 7-2000, and the bending angle of adjacent lenses is The range is 0-45°.

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

4. The method for preparing a snapshot light field microscope with a large depth of field as claimed in claim 1, characterized in that: In step 2, the central wavelength of the femtosecond laser in the femtosecond laser direct writing processing system is 343-1030nm, the pulse width is 50-300fs, and the repetition frequency is 1KHz-100MHz; the magnification of the processing objective lens is 4-100 times, and the numerical aperture is 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.

5. The method for preparing a snapshot light field microscope with a large depth of field as claimed in claim 1, characterized in that: In step 2, after spin coating the optical resin, the sample sheet is pre-baked according to the photosensitivity of the resin. The pre-baking process is carried out on a hot table, and the sample after pre-baking is naturally cooled in an air environment for use. The pre-baking heating instrument used is a constant temperature heating table or a constant temperature heating box, the pre-baking temperature range is 50-150°C, and the pre-baking time range is 10min-3h.

6. The method for preparing a snapshot light field microscope with a large depth of field as claimed 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 range of the mechanical adapter for loading the curved microlens array is 50μm-5mm, the internal thread diameter range is 20mm-60mm, and the length range is 1mm-50mm; the external thread diameter range of the mechanical adapter ring is 20mm-60mm, and the length range is 1mm-5mm; the material of the mechanical assembly parts is aluminum, iron, copper, and steel.

7. The method for preparing a snapshot light field microscope with a large depth of field as claimed in claim 1, characterized in that: In step 3, the optical resin used for fixing includes: epoxy resin SU-8, organic-inorganic hybrid photoresist IP-Dip, IP-S, IP-L, SZ2080 or UV optical curing glue NOA61, NOA63; In step 3, the continuously adjustable range of the distance D between the curved microlens array and the exit pupil of the commercial microscope objective is 100 μm-25 mm.

8. The method for preparing a snapshot light field microscope with a large depth of field as claimed in claim 1, characterized in that: In step 4, the commercial microscopes include transmission optical microscopes of various models from Olympus, Zeiss, Leica, Nikon, Bruker, and Motic; In step 4, the spatial calibration method includes direct linear transformation, polynomial calibration, structural optical calibration, and neural network calibration.

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