阅读说明：本技术 一种改进的光场显微成像装置及构建方法 (Improved light field microscopic imaging device and construction method ) 是由 许传龙 顾梦涛 宋祥磊 于 2020-04-21 设计创作，主要内容包括：本发明公开了一种改进的光场显微成像装置及构建方法,其中光场显微成像装置包括显微镜物镜、显微镜筒镜、微透镜阵列以及相机传感器,所述相机传感器采集显微镜物镜成像于显微镜筒镜上的图像；其特征在于：所述微透镜阵列位于显微镜像平面与相机传感器之间,显微镜筒镜成像于显微镜像平面上,再经过微透镜阵列的作用后,再次成像于相机传感器上。本发明根据离焦距离定义求得微透镜阵列和显微镜像平面的距离；根据F数匹配原则求得相机传感器与微透镜阵列距离。本发明所提供的改进的光场显微成像装置可在物镜焦平面附近获得高重建分辨率,可作为光场显微粒子图像测速系统的成像装置。(The invention discloses an improved light field microscopic imaging device and a construction method thereof, wherein the light field microscopic imaging device comprises a microscope objective lens, a microscope tube lens, a micro lens array and a camera sensor, wherein the camera sensor collects an image formed by the microscope objective lens on the microscope tube lens; the method is characterized in that: the micro lens array is positioned between the micro mirror image plane and the camera sensor, the microscope tube lens is imaged on the micro mirror image plane, and the micro lens array is imaged on the camera sensor again after the micro lens array. The distance between the micro lens array and a microscope image plane is obtained according to the defocus distance definition; and (4) solving the distance between the camera sensor and the micro-lens array according to an F number matching principle. The improved light field microscopic imaging device provided by the invention can obtain high reconstruction resolution near the focal plane of the objective lens, and can be used as an imaging device of a light field microscopic particle image velocimetry system.)

1. An improved light field microscopic imaging device comprises a microscope objective lens, a microscope tube lens, a micro lens array and a camera sensor, wherein the camera sensor collects an image formed on the microscope tube lens by the microscope objective lens; the method is characterized in that: the micro lens array is positioned between the micro mirror image plane and the camera sensor, the microscope tube lens is imaged on the micro mirror image plane, and the micro lens array is imaged on the camera sensor again after the micro lens array.

2. An improved light field microimaging apparatus as claimed in claim 1, wherein:

distance a between the microlens array and the microscope mirror plane:

in the formula, z_maxIs the maximum value of the measurement depth; m is the magnification of the objective lens; NA is the numerical aperture of the objective lens; d is the microlens aperture;

distance b of camera sensor from microlens array:

in the formula, D_tIs the bore diameter of the microscope tube lens, f_tIs the focal length of the microscope tube lens.

3. A light field microscopic imaging apparatus according to claim 2, characterized in that: and a reflecting mirror for forming a reflecting light path by the microscope objective lens and the microscope column mirror is arranged between the microscope objective lens and the microscope column mirror.

4. A light field microscopic imaging apparatus according to claim 2, characterized in that: the light field microscopic imaging device also comprises a light source and a dichroic mirror which is used for reflecting the light source and then passing through the microscope objective lens.

5. A method of constructing an improved light field microscopy imaging device as defined in any one of claims 1 to 4 comprising the steps of:

the method comprises the following steps: the method comprises the following steps of calculating the diameters of diffraction spots of a micro-lens array surface under different defocus distances in the traditional light field micro-imaging device and a focusing light field micro-imaging device through numerical simulation, and fitting the functional relation between the diameters of the diffraction spots and the defocus distances:

DD＝2M*NA*Δz

in the formula, DD is the diameter of diffraction spot of the micro-lens array surface; m is the magnification of the objective lens; NA is the numerical aperture of the objective lens; Δ z is the defocus distance;

step two: substituting the function obtained in the step one into DD not more than d to obtain the defocusing distance range of the light emitted by the sampling point limited in a micro lens:

wherein d is the microlens aperture;

step three: after determining the range of the defocus distance in which the reconstruction resolution is sharply reduced, obtaining the distance a between the microlens array and the microscope image plane when the optimal reconstruction resolution is obtained in the measurement depth range according to the definition of the defocus distance:

in the formula, z_maxIs the maximum value of the measurement depth;

step four: calculating the distance b between the camera sensor and the micro lens array according to the optical parameters and the distance a between the micro lens array and the microscope mirror image plane:

in the formula, D_tIs the bore diameter of the microscope tube lens, f_tIs the focal length of the microscope tube lens;

step five: and constructing an improved light field microscopic imaging device according to the parameters obtained in the first step to the fourth step.

6. The construction method according to claim 5, wherein in the first step, the defocus distance Δ z is defined as the distance between the sampling point and the focal plane of the light field micro-imaging device, and the focal plane of the device is conjugate to the plane of the microlens array; in a conventional light field microscopic imaging device, Δ z ═ z |, and in a focusing light field microscopic imaging device, Δ z ═ z-a_f/M²L, where z is the depth of the sampling point, a_fThe distance between the micro lens array and the microscope mirror plane in the focusing light field microscopic imaging device.

7. The method according to claim 5, wherein in the second step, whether or not the reconstruction resolution is sharply decreased is determined by comparing the diffraction spot diameter on the microlens array surface with the size of the microlens aperture.

8. The construction method according to claim 5, wherein in the third step, the measurement depth range is set to-50 μm, which can satisfy the micro-channel measurement depth requirement on the premise of obtaining high-quality light field picture, and z is_max＝50μm。

Technical Field

The invention relates to an improved Light field microscopic imaging device, which can be applied to a Light field microscopic particle image velocimetry (Light-field Micro-PIV) system and belongs to the technical field of Micro-scale fluid measurement.

Background

The Light field microscopic imaging device can realize the recording of three-dimensional Light field information of the tracer particles by one-time exposure, and a Light field microscopic particle image velocimetry (Light-field Micro-PIV) system based on the Light field microscopic imaging device can effectively improve the time resolution and is beneficial to the instantaneous measurement and characteristic research of Micro-scale flow. The light field microscopic imaging device consists of a laser light source, an inverted fluorescence microscope and a cage type light field camera. In an inverted fluorescence microscope, the light source formed by laser lights the chip to be measured after being refracted by the objective lens, resulting in a funnel-shaped body light source with maximum light intensity at the focal plane of the objective lens, and thus the optimal imaging area is located near the focal plane of the objective lens.

Conventional light field microimaging devices require the microlens array to be located at the microscope image plane and the camera sensor to be located at the back focal plane of the microlens array. In the traditional light field microscopic imaging device, when the tracer particles are positioned near the focal plane of the objective lens, the emitted light is limited in one micro lens, so that redundant sampling is formed, and the reconstruction resolution is reduced sharply. The micro-channel to be detected containing the tracer particles is placed in the area far away from the focal plane of the objective lens, so that the reconstruction resolution of the traditional light field micro-imaging device can be improved, but the laser intensity of the area is weakened, the intensity of fluorescence generated by the stimulated tracer particles is further reduced, and finally the brightness and the contrast of a light field image are reduced. Therefore, the conventional light field microscopic imaging apparatus needs to obtain high reconstruction resolution by sacrificing brightness and contrast of the light field image.

In order to meet the requirements of high image quality and high reconstruction resolution, the light field microscopic imaging device needs to be improved, and a set of light field microscopic imaging device capable of obtaining the optimal reconstruction resolution near the focal plane of the objective lens is constructed.

Disclosure of Invention

The invention aims to solve the technical problem that the traditional light field microscopic imaging device cannot meet the requirements of high image quality and high reconstruction resolution at the same time, and provides an improved light field microscopic imaging device and a construction method thereof so as to obtain the optimal reconstruction resolution near the focal plane of an objective lens.

In order to solve the technical problems, the invention adopts the technical scheme that:

an improved light field microscopic imaging device comprises a microscope objective lens, a microscope tube lens, a micro lens array and a camera sensor, wherein the camera sensor collects an image formed on the microscope tube lens by the microscope objective lens; the method is characterized in that: the micro lens array is positioned between the micro mirror image plane and the camera sensor, the microscope tube lens is imaged on the micro mirror image plane, and the micro lens array is imaged on the camera sensor again after the micro lens array.

Distance a between the microlens array and the microscope mirror plane:

in the formula, z_maxIs the maximum value of the measurement depth; m is the magnification of the objective lens; NA is the numerical aperture of the objective lens; d is the microlens aperture;

distance b of camera sensor from microlens array:

in the formula, D_tIs the bore diameter of the microscope tube lens, f_tIs the focal length of the microscope tube lens.

And a reflecting mirror for forming a reflecting light path by the microscope objective lens and the microscope column mirror is arranged between the microscope objective lens and the microscope column mirror.

The light field microscopic imaging device also comprises a light source and a dichroic mirror which is used for reflecting the light source and then passing through the microscope objective lens.

A construction method of an improved light field microscopic imaging device is characterized by comprising the following steps:

the method comprises the following steps: the method comprises the following steps of calculating the diameters of diffraction spots of a micro-lens array surface under different defocus distances in the traditional light field micro-imaging device and a focusing light field micro-imaging device through numerical simulation, and fitting the functional relation between the diameters of the diffraction spots and the defocus distances:

DD＝2M*NA*Δz

in the formula, DD is the diameter of diffraction spot of the micro-lens array surface; m is the magnification of the objective lens; NA is the numerical aperture of the objective lens; Δ z is the defocus distance.

Step two: and substituting the function obtained in the step one into the value DD not more than d (d is the aperture of the micro lens), and obtaining the defocusing distance range within which the light emitted by the sampling point is limited in one micro lens (namely the reconstruction resolution of the light field microscopic imaging device is sharply reduced):

wherein d is the microlens aperture.

Step three: after determining the range of the defocus distance in which the reconstruction resolution is sharply reduced, obtaining the distance a between the microlens array and the microscope image plane when the optimal reconstruction resolution is obtained in the measurement depth range according to the definition of the defocus distance:

in the formula, z_maxIs the maximum value of the measurement depth.

Step four: calculating the distance b between the camera sensor and the micro lens array according to the optical parameters and the distance a between the micro lens array and the microscopic image plane by an F number matching principle:

in the formula, D_tIs the aperture of a microscope tube lens; f. of_tIs the focal length of the microscope tube lens.

And constructing an improved light field microscopic imaging device according to the parameters obtained in the first step to the fourth step.

In the first step, the defocusing distance delta z is defined as the distance between the sampling point and the focal plane of the light field microscopic imaging device, and the focal plane of the device is conjugated with the plane of the micro-lens array. In a conventional light field microscopic imaging device, Δ z ═ z |, and in a focusing light field microscopic imaging device, Δ z ═ z-a_f/M²Where z is the depth of the sampling point (the focal plane depth of the objective lens is 0), a_fThe distance between the micro lens array and the microscope mirror plane in the focusing light field microscopic imaging device.

And in the second step, judging whether the reconstruction resolution ratio is sharply reduced or not by comparing the diameter of the diffraction spot on the microlens array surface with the aperture size of the microlens.

In the third step, the measurement depth range is set to be-50 μm, so that the requirement of micro-channel measurement depth can be met on the premise of obtaining a high-quality light field picture, and z is_max＝50μm。

Compared with the prior light field microscopic imaging device, the invention has the following advantages: by changing the positions of the micro-lens array and the camera sensor, the improved light field microscopic imaging device can obtain higher reconstruction resolution within a measurement depth range of-50 mu m, so that the requirements of high reconstruction resolution and high light field image quality are met at the same time. And in the measuring depth range of-50 mu m, the reconstruction resolution of the improved light field microscopic imaging device is superior to that of a focusing device. The light field microscopic particle image velocimetry system assembled based on the improved light field microscopic imaging device can complete the measurement of the three-dimensional velocity field in the micro-channel.

Drawings

FIG. 1 is a block diagram of a light field microscopic imaging apparatus;

FIG. 2 is a graph of the variation of the diameter of the diffraction spot on the surface of the microlens array with the defocusing distance;

FIG. 3F number matching schematic;

FIG. 4 is a reconstructed resolution comparison map;

FIG. 5 is a three-dimensional velocity field plot of a microchannel.

Wherein: 1-laser light source, 2-objective focal plane, 3-microscope objective, 4-dichroic mirror, 5-reflector, 6-microscope tube mirror, 7-microscope image plane, 8-microlens array, 9-camera sensor.

Detailed Description

The present invention will be described in detail below with reference to the accompanying drawings.

As shown in fig. 1, a laser light source 1 is reflected by a dichroic mirror 4 and then forms a bulk light source through a microscope objective lens 3. Due to the microscope objective 3, the bulk light source is funnel-shaped and the maximum light intensity is located at the objective focal plane 2. The fluorescent light emitted by the trace particles after being excited by the light source is imaged on a microscopic image plane 7 after being acted by a microscope (comprising a microscope objective lens 3, a dichroic mirror 4, a reflecting mirror 5 and a microscope tube lens 6), and is imaged on a camera sensor 9 again after being acted by a micro lens array 8.

In fig. 1, a is the distance between the microlens array 8 and the microscope mirror plane 7, b is the distance between the camera sensor 9 and the microlens array 8, and the subscripts denote the device type, where c denotes the conventional type, f denotes the focusing type, and n denotes the improved light field microimaging device.

Different types of light field microimaging devices can be composed by adjusting the positions of the microlens array 8 and the camera sensor 9. In a conventional light field microimaging device, the plane of the microlens array 8 coincides with the microscope image plane 7, i.e. a_cDistance b between camera sensor 9 and microlens array 8 of 0_cAnd focal length f of the microlens_mlEqual; in a focusing type light field microscopic imaging device, a_f，b_f，f_mlNeed to satisfy the Gaussian imaging relation 1/a_f+1/b_f＝1/f_ml。

In this embodiment, the optical parameters are shown in table 1.

TABLE 1 optical parameter table

Symbol	Physical significance	Value of
			M	Magnification of objective lens	10
NA	Numerical aperture of objective lens	0.3
			Dtl	Bore of cylindrical lens	12mm
ftl	Focal length of cylindrical mirror	200mm
			d	Microlens aperture	136μm
fml	Focal length of microlens	2060μm
			af	Distance between microlens array and microscope image plane in focusing device	22mm
bf	Distance between camera sensor and microlens array in focusing device	2276μm

The invention constructs an improved light field microscopic imaging device capable of obtaining the best reconstruction resolution near the focal plane of an objective lens by selecting the proper positions of a micro lens array 8 and a camera sensor 9, and the method comprises the following specific steps:

the method comprises the following steps: and acquiring the functional relation between the diameter of the diffraction spot and the defocusing distance.

Based on scalar diffraction theory, according to the parameters listed in table 1, the diffraction spot diameters of the microlens array surface at different defocus distances in the conventional light field microscopic imaging device and focused light field microscopic imaging device are calculated through numerical simulation, and the result is shown in fig. 2. It can be found that the diameter of the diffraction spot on the surface of the microlens array 8 is approximately proportional to the defocus distance, and the slope is 6, which is equal to the value of 2M × NA, in both the conventional light field microimaging device and the focusing light field microimaging device. Thus, the diffraction spot diameter DD is a function of defocus distance as follows:

DD＝2M*NA*Δz

in the formula, DD is the diameter of diffraction spot of the micro-lens array surface; m is the magnification of the objective lens; NA is the numerical aperture of the objective lens; Δ z is the defocus distance.

The defocus distance Δ z is defined as the distance between the sampling point and the focal plane of the light field microimaging device, which is conjugate to the plane of the microlens array. In a conventional light field microscopic imaging device, Δ z ═ z |, and in a focusing light field microscopic imaging device, Δ z ═ z-a_f/M²Where z is the depth of the sampling point (the focal plane depth of the objective lens is 0), a_fThe distance between the micro lens array and the microscope mirror image plane in the focusing light field microscopic imaging device.

Step two: and calculating the defocus distance range with sharply reduced reconstruction resolution.

When the light emitted by the sampling point is limited in one micro lens, the reconstruction resolution of the light field micro imaging device is sharply reduced, and whether the light emitted by the sampling point is limited in one micro lens can be judged by comparing the diameter of the diffraction spot of the micro lens array surface with the aperture size of the micro lens. Therefore, substituting the function obtained in the step one into DD ≦ d can calculate the defocus distance range in which the reconstruction resolution is sharply reduced, that is:

wherein d is the microlens aperture.

In this embodiment, d/(2M NA) has a value of 23 μ M. Therefore, according to the definition of the defocus distance, in the conventional light field microscopic imaging device, the depth range of the sharp reduction of the reconstruction resolution is that z is-23 μm; in the focusing light field microscopic imaging device, the depth range is 197-243 mu m.

Step three: determining the distance a of the microlens array from the microscope mirror plane_n。

The depth of the micro-channel of the micro-fluidic chip is mostly 50-100 μm, and in order to obtain a high-quality and high-contrast light field image, the micro-fluidic chip should be symmetrically arranged along the objective focal plane 2. Therefore, the depth is set to a range of-50 to 50 μm. In order to prevent the reconstruction resolution from being sharply reduced within the set depth range, the distance a between the microlens array 8 and the microscope mirror plane 7 is increased in the improved light field microimaging device_nIt should satisfy:

since the reconstruction resolution decreases as the defocus distance increases, in order to obtain the optimal reconstruction resolution, a_nShould take a minimum value, the distance a of the microlens array 8 from the microscope mirror plane 7 should therefore be taken_nComprises the following steps:

in the formula, z_maxIs the maximum value of the measurement depth.

Since the depth measurement is subject to error during the experiment, the value of d/(2M NA) is enlarged to 30 μ M in order to reduce the influence of this error. Calculated, in this embodiment, a_n＝8mm。

Step four: determining the distance b of a camera sensor from a microlens array_n。

The F-number matching requires that the sub-aperture image formed by a single microlens be tangent on the camera sensor 9, the principle of which is shown in fig. 3. According to the similar triangle principle, the following relationships exist among the parameters:

in the formula, Li represents the side length of a microscope mirror plane corresponding to a single microlens; d_tIs the aperture of a microscope tube lens; f. of_tIs the focal length of the microscope tube lens.

By simultaneous solving the two formulas, the distance b between the camera sensor 9 and the microlens array 8 in the improved light field microimaging device can be obtained_nComprises the following steps:

calculated, in this embodiment, b_n＝1837μm。

To this end, can be according to a_nAnd b_nAnd constructing an improved light field microscopic imaging device.

FIG. 4 is a comparison graph of the reconstruction resolution of three types of light field microscopic imaging devices at a depth z of-50 μm. Wherein the reconstruction resolution is expressed in terms of Full-width-at-half-maximum (FWHM), and the FWHM is inversely proportional to the reconstruction resolution. In fig. 4, the subscript x indicates the transverse reconstruction resolution, the subscript z indicates the axial reconstruction resolution, and the parenthesis indicates the types of light field microscopic imaging devices, which are respectively the conventional type, the focusing type and the improved type proposed by the present invention. It can be found that the traditional type has a sharp reduction of the reconstruction resolution, but the improved light field microscopic imaging device and the focusing type of the invention have no phenomenon, and the reconstruction resolution of the improved light field microscopic imaging device of the invention is superior to that of the focusing type.

FIG. 5 is a result of measuring a three-dimensional velocity field of a Micro-channel based on the improved light field Micro-imaging device, which illustrates that the improved light field Micro-imaging device can be used for constructing a light field Micro-PIV system to measure the three-dimensional velocity field of the Micro-channel.