A focusing light field microscopic imaging system and F number matching method for three-dimensional microparticle image velocimetry system

Abstract

The invention discloses a focusing light field microscopic imaging system for a three-dimensional microscopic particle image velocity measurement system and F Compared with the non-focusing light field microscopy system, the focusing light field microscopy system proposed in the present invention significantly improves the spatial resolution of the area near the focal plane of the system objective lens by changing the relative positions of the microlens array and the CCD sensor and the microscope tube lens, and analyzes and proposes a focusing light field microscopy system based on the structural characteristics of the focusing light field camera and the infinity-corrected microscopy system. F Number matching method; The system and method provided by the present invention overcome the problems of velocity vector measurement error and poor resolution of neighboring focal plane areas introduced by aberrations caused by the measuring body being far away from the focal plane of the objective lens in a three-dimensional microscopic particle image velocimetry system based on non-focusing light field imaging, and provide a technical basis for the assembly and debugging of a focusing light field microscopic imaging system used in a three-dimensional microscopic particle image velocimetry system.

Description

Technical Field

[0001] The invention belongs to the technical field of micro-scale multiphase flow measurement, and specifically relates to a focusing light field microscopic imaging system and an F number matching method for a three-dimensional microscopic particle image velocity measurement system. Background Art

[0002] Microfluidic devices have the advantages of controllable liquid flow, less sample and reagent consumption, and fast analysis speed. In recent years, they have been widely used in biomedicine, chemical engineering, electronic integrated circuits and other fields, such as microfluidic diagnosis, chemical synthesis reactions and chip heat dissipation. The flow inside a microfluidic device is a microscale flow, and the velocity field distribution of the internal microscale flow is the main parameter to describe the flow phenomenon. For example, in a microfluidic heat dissipation chip, the distribution of the flow velocity field is closely related to the efficiency of heat dissipation. Therefore, the realization of three-dimensional instantaneous velocity field measurement of complex flow inside a microfluidic device is of great significance for a deep understanding of the microscale flow dynamics mechanism and the design of microfluidic devices.

[0003] Micro Particle Image Velocimetry (μPIV) is one of the most commonly used non-contact microscale flow field measurement technologies. Many flows in actual microfluidic devices are complex three-dimensional flows. To study these flows in depth, their instantaneous three-dimensional velocity distribution must be measured. For this reason, in recent years, the three-dimensional velocity field measurement technology in microscale flow has attracted more and more attention from researchers. In recent years, light field imaging technology based on microlens arrays can record the light field information of the object space with a single exposure, and the three-dimensional reconstruction of the object space can be achieved through reconstruction technology, thus providing a new way to solve computer vision problems such as three-dimensional reconstruction, significant area detection, and target recognition. The light field imaging technology combined with the traditional microscope can obtain a light field microscope imaging system. The light field microscope imaging system can be used to obtain the position information of tracer particles in the microscale flow field, and combined with the particle image velocimetry technology to establish a light field micro particle image velocimetry system (Light Field Micro Particle Image Velocimetry, referred to as LFμPIV). The advantage of the light field microparticle image velocimetry system is that it uses a similar method to the two-dimensional microparticle image velocimetry system to achieve the measurement of instantaneous three-dimensional flow field. In addition, the light field microparticle image velocimetry technology overcomes the shortcomings of existing traditional three-dimensional microscopic flow field measurement technologies, such as focused laser scanning microparticle image velocimetry technology, stereoscopic microparticle image velocimetry technology, defocused microparticle image velocimetry technology, and digital holographic microparticle image velocimetry technology, which require scanning, multi-view imaging, complex systems, or strict requirements on equipment and environment in obtaining three-dimensional position information.

[0004] All reported light field micro-particle imaging velocimetry systems are based on non-focusing light field microscopy technology ( Figure 2 ). However, the non-focusing light field microscopy system has very poor spatial resolution (lateral resolution and axial resolution) in the area near the focal plane of the objective lens. Therefore, in order to improve the spatial resolution of the imaging system of the non-focusing light field microscopy system, it is necessary to make the image body deviate from the focal plane area of ​​the objective lens. However, the correction of the aberrations (spherical aberration, coma, position dispersion, astigmatism and distortion, etc.) of the objective lens is based on the imaging of the focal plane area. Imaging away from the focal plane area will lead to more serious aberrations. Summary of the invention

[0005] The technical problem to be solved by the present invention is that a three-dimensional micro-particle image velocimetry system based on non-focusing light field microscopy has a very poor spatial resolution in the area near the focal plane of the objective lens, and a three-dimensional micro-particle image velocimetry system based on focusing light field microscopy and an F-number matching method are proposed.

[0006] Basic idea: A focusing microscopic light field camera system is assembled using an inverted fluorescence microscope (including a green fluorescence excitation module), a high-speed CCD camera, a 1:1 relay system (composed of two Nikon 50mm 1.4G fixed-focus lenses), a microlens array (orthogonal square, 100% filling rate) and connectors (cage plate, cage rod and connecting ring). A three-dimensional microparticle image velocity measurement system based on focusing light field microscopic imaging is built using this camera system and an illumination system (a 532nm continuous laser, epi-illumination optical path). The distance between the tube lens and the microlens array and between the microlens array and the high-speed CCD camera sensor in the system is determined according to the proposed F-number matching method. By acquiring two consecutive exposure images at a certain time interval, the three-dimensional velocity field of the measured fluid is obtained after image processing and three-dimensional cross-correlation calculation.

[0007] A focusing light field microscopic imaging system for a three-dimensional microparticle image velocimetry system, comprising:

[0008] An illumination light source is used to provide an excitation light source for tracer particles in the flow field; it includes a continuous laser with a wavelength of 532nm and an incident illumination light path;

[0009] Focusing light field microscopy system, used to capture and record the light field information of tracer particles in microscale flow fields; including an inverted fluorescence microscope (infinity-corrected optical system, including green fluorescence excitation module), a high-speed camera, a microlens array (orthogonal square, 100% filling rate), a relay system (composed of two Nikon 50mm 1.4G lenses) and several fixed components (cage rods, cage plates and connecting rings);

[0010] A computer and a test program are used to store light field images and three-dimensional velocity field calculations obtained by a high-speed CCD camera; select two frames of light field photos with a time interval of Δt; use image processing technology, such as three-dimensional deconvolution reconstruction, to obtain three-dimensional position information of tracer particles; and obtain and display the three-dimensional velocity field information of the microfluidic field through a three-dimensional cross-correlation algorithm;

[0011] A method for matching the F number of a focusing light field microscopy system is provided. According to the structural characteristics of the focusing light field microscopy system, a matching relationship between important geometric parameters of the focusing light field microscopy system is analyzed by a method of geometric optics. The method comprises the following steps:

[0012] Step 1: Determine the distance between the tube lens and the virtual image plane; in the focusing light field microscopy imaging system, the microscope adopts an infinity-corrected optical system, and the virtual image plane is located at the focal plane of the tube lens. Therefore, the distance between the tube lens and the virtual image plane is the focal length of the tube lens (f t );

[0013] Step 2: Determine the distance between the tube lens and the microlens array:

[0014] When L tm >f t hour,

[0015]

[0016] When L tm <f t hour,

[0017] L tm =(k-1)f μ

[0018] Step 3: Determine the distance between the microlens array and the CCD sensor:

[0019] When L tm >f t hour,

[0020]

[0021] When L tm <f t hour,

[0022]

[0023] Where: L tm is the distance between the tube lens and the microlens array; f μ is the focal length of the microlens; L μc is the distance between the microlens array and the CCD sensor;

[0024] Step 4: Determine the distance between the tube lens and the microlens array and the distance between the microlens array and the CCD sensor. Through a 1:1 relay system, project the imaging surface 1:1 onto the CCD surface of the camera to obtain the focal plane and defocus calibration pictures.

[0025] Compared with the prior art, the present invention has the following beneficial effects: the present invention overcomes the problems of velocity vector measurement error and poor resolution of neighboring focal plane areas introduced by aberrations caused by the measuring body being away from the focal plane of the objective lens in a three-dimensional microscopic particle image velocimetry system based on non-focusing light field imaging, and provides a technical basis for the assembly and debugging of a focusing light field microscopic imaging system used in a three-dimensional microscopic particle image velocimetry system. Compared with traditional methods, the results obtained by the method provided by the present invention are stable and reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the technical description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0027] Figure 1 Focusing three-dimensional microparticle image velocimetry system.

[0028] Figure 2 Non-focusing light-field microscopy.

[0029] Figure 3 Imaging relationship (L tm >f t ).

[0030] Figure 4 Microlens and CCD sensor position calibration relationship (L tm >f t ).

[0031] Figure 5 Imaging relationship (L tm <f t ).

[0032] Figure 6 Microlens and CCD sensor position calibration relationship (L tm <f t ).

[0033] Figure 7 Schematic diagram of the imaging system after calibration (L tm >f t ).

[0034] Figure 8 Schematic diagram of the imaging system after calibration (Ltm <f t ).

[0035] Fig. 9 Actual picture of the imaging system after calibration.

[0036] Fig.10 The image of the surface luminous object on the CCD camera when it is in the focal plane of the objective lens (left) and out of focus (right).

[0037] Among them, 1-micro syringe pump, 2-microscale channel, 3-microscope, 301-objective lens, 302-dichroic mirror, 303-tube lens, 4-laser, 5-light field camera system, 501-microlens array, 502-1:1 relay lens, 503-CCD sensor, 6-computer, 7-virtual image plane, 8-objective lens focal plane, 9-cage plate, 10-cage rods, 11-microscope interface. DETAILED DESCRIPTION

[0038] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0039] Embodiment 1:

[0040] Focusing light field microscopy system is used to capture and record the light field information of tracer particles in microscale flow fields, such as Figure 1 As shown, it includes: an inverted fluorescence microscope 3 (infinity-corrected optical system, including a dichroic mirror (302)), a CCD sensor 503, a microlens array 501 (orthogonal square, 100% filling rate), a relay system 502 (composed of two Nikon 50mm1.4G lenses) and a number of fixed elements (cage rods 10 and cage plates 9);

[0041] Computer 6 and test program are used to store light field pictures and three-dimensional velocity field calculations obtained by high-speed CCD camera. Select two frames of light field pictures with a time interval of Δt; use image processing technology, such as three-dimensional deconvolution reconstruction to obtain the three-dimensional position information of tracer particles; obtain the three-dimensional velocity field information of the microfluidic field through a three-dimensional cross-correlation algorithm and display it;

[0042] The imaging system composed of the tube lens 303, the micro lens array 501 and the CCD sensor 503, the distances between the tube lens 303, the micro lens array 501 and the CCD sensor 503 meet the principle of a focusing light field camera;

[0043] As the focusing light field microscopic imaging system of the three-dimensional microscopic particle image velocimetry system proposed by the present invention, in order to ensure that the addition of the microlens array does not limit the resolution capability of the microscope imaging system, and at the same time ensure that the sub-images of adjacent microlenses on the CCD sensor 503 can cover the most pixels under the microlenses and do not overlap (adjacent sub-images are tangent), it is necessary to match the F number of the focusing light field microscopic imaging system, so as to determine the parameters such as the distance between the tube lens 303 and the microlens array 501, the distance between the microlens array 501 and the CCD sensor 503, the size of the microlens and the size of the CCD sensor. According to the numerical aperture (NA) of the objective lens, the aperture (D) of the microlens and the focal length (f μ ) determines the distance (L) between the tube lens and the microlens array 501 tm ), the distance between the microlens array 501 and the CCD sensor 503 (L μc ); specifically:

[0044] When L tm >f t When f t is the focal length of the microscope tube lens, according to the imaging relationship (such as Figure 3 and Figure 4 ), satisfying the following relationship:

[0045]

[0046] L tm =f t +L vm (4)

[0047]

[0048] According to formula (1)-(4), we can get:

[0049]

[0050] Where: L vm D is the distance from the microlens array 501 to the virtual image plane 7; v is the side length of the virtual image plane corresponding to each microlens; D t is the aperture of the microscope tube lens 303.

[0051] When L tm <f t When, according to the imaging relationship (such as Figure 5 and Figure 6 ), satisfying the following relationship:

[0052]

[0053]

[0054] L tm =f t -L vm (11)

[0055]

[0056] According to formula (4)-(6), we can get:

[0057] L tm =(k-1)f μ (13)

[0058]

[0059] Embodiment 2:

[0060] The system uses a Nikon fluorescence microscope (model: Ti2-U), the microscope optical path system is an inverted infinity optical path system, the objective lens uses an infinity-corrected fluorescence objective lens, the microlens array uses a microlens array with square microlenses and 100% coverage produced by AMS (Advanced Microoptic Systems) of Germany, and the CCD camera uses a B2320 CCD camera produced by Imperx of the United States. The parameters are shown in Table 1.

[0061]

[0062] Determine the distance between the tube lens 303 and the microlens array 501:

[0063] When L tm >f t hour,

[0064]

[0065] When L tm <f t hour,

[0066] L tm =(k-1)f μ =197.697mm

[0067] Determine the distance between the microlens array 501 and the CCD sensor 503:

[0068] When L tm >f t hour,

[0069]

[0070] When L tm <f t hour,

[0071]

[0072] After determining the distance between the tube lens 303 and the microlens array 501 and the distance between the microlens array 501 and the CCD sensor 503, the imaging surface is projected 1:1 onto the CCD surface of the camera through a 1:1 relay system 502, as shown in FIG. Figure 7-Figure 10 As shown in the figure, they are respectively the principle diagram of the calibration system, the actual picture, and the calibration pictures of the focal plane and defocus obtained on the CCD camera after calibration.

[0073] The above description is only a specific implementation mode of the present invention, but the protection scope of the invention is not limited thereto. Any changes or substitutions that can be easily thought of by a technician familiar with the field within the technical scope disclosed by the present invention should be covered within the protection scope of the invention.

Claims

1. A focusing light field microscopic imaging system for a three-dimensional microparticle image velocimetry system, characterized in that: include: An illumination light source, used to provide an excitation light source for tracer particles in the flow field, including a continuous laser with a wavelength of 532nm and an incident illumination light path; A focusing light field microscopic imaging system is used to capture and record the light field information of tracer particles in a microscale flow field, including an inverted fluorescence microscope, a high-speed camera, a microlens array, a relay system, and several fixed components; Computer and test program for storing light field images obtained by high-speed CCD camera and three-dimensional velocity field calculation; The F-number matching method of the focusing light field microscopy imaging system is based on the structural characteristics of the focusing light field microscopy imaging system and uses the method of geometric optics to analyze the matching relationship between the important geometric parameters of the focusing light field microscopy imaging system, and includes the following steps: Step 1: Determine the distance between the tube lens and the virtual image plane; The microscope in the focusing light field microscopy system adopts an infinity-corrected optical system. The virtual image plane is located at the focal plane of the tube lens. Therefore, the distance between the tube lens and the virtual image plane is the focal length of the tube lens ( f t ); Step 2: Determine the distance between the tube lens and the microlens array: when L tm > f t hour, When L tm < f t hour, Step 3: Determine the distance between the microlens array and the CCD sensor: when L tm > f t hour, When L tm < f t hour, Where: L tm is the distance between the tube lens and the microlens array; f μ is the focal length of the microlens; L μc is the distance between the microlens array and the CCD sensor, where D is the aperture of the microlens, D t is the aperture of the microscope tube lens; Step 4: Determine the distance between the tube lens and the microlens array and the distance between the microlens array and the CCD sensor. Through a 1:1 relay system, project the imaging surface 1:1 onto the CCD surface of the camera to obtain the focal plane and defocus calibration pictures.

2. A focusing light field microscopic imaging system for a three-dimensional microparticle image velocimetry system according to claim 1, characterized in that: The fluorescence microscope comprises a green fluorescence excitation module.

3. The focusing light field microscopic imaging system for a three-dimensional microparticle image velocimetry system according to claim 1, characterized in that: The microlens array is an orthogonal square with a 100% filling rate.

4. The focusing light field microscopic imaging system for a three-dimensional microparticle image velocimetry system according to claim 1, characterized in that: The relay system consists of two Nikon 50 mm 1.4G lenses.

5. The focusing light field microscopic imaging system for a three-dimensional microparticle image velocimetry system according to claim 1, characterized in that: The fixing element is composed of cage rods, cage plates and connecting rings.

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