Confocal polarization microscopic imaging device and method based on functional small hole

Abstract

The invention discloses a confocal polarization microscopic imaging device and method based on a functional small hole, and belongs to the technical field of optical microscopic imaging. In the device, an illumination module is used for providing laser in a fixed polarization state; the sample scanning module is used for placing different areas of the to-be-tested sample at the laser focus according to the designed S-shaped moving route; the scattered light collection module is used for collecting scattered light of the to-be-detected sample The functional pinhole module is used for focusing scattered light of the sample to be detected and projecting the scattered light to four spatially separated focuses with different polarization directions in a polarization manner; the light intensity detection module is used for collecting focus light spots polarized and focused by the functional small hole module and calculating intensity to draw a scanning graph. According to the device and the method, the full Stokes parameter metasurface is used as a functional small hole of a confocal imaging system for polarization imaging, and the device and the method have the characteristics of high imaging resolution, high polarization sensitivity, compact structure, good compatibility and the like.

Description

Technical Field

[0001] The invention belongs to the technical field of optical microscopic imaging, and in particular relates to a confocal polarization microscopic imaging device and method based on functional pinholes. Background Art

[0002] Microscopic imaging is a key technology in modern scientific research and plays an important role in biomedicine, precision manufacturing, life sciences and other fields. With the development of science, human beings' various needs for exploring the microscopic world are increasing, which also puts forward new requirements for microscopic imaging technology.

[0003] Traditional microscopes use widefield illumination to expose the entire sample to a light source, which is then imaged by the imaging system into the eye or array detector. Widefield illumination can obtain a relatively large field of view, but because widefield illumination illuminates the entire sample, some light from areas far from the focal plane is also received by the imaging system. These stray lights cause the imaging of the area of ​​interest to become blurred. Confocal technology uses a point conjugate method to add a pinhole in front of the detector. The light far from the focal plane is blocked outside the detector by the pinhole, allowing the confocal system to perform optical sectioning and achieve three-dimensional scanning, thereby improving the resolution of microscopic imaging. Although traditional confocal technology has the capabilities of high resolution, optical sectioning, and three-dimensional imaging, it is essentially intensity imaging and cannot effectively resolve the polarization information of the light field. However, polarization information is crucial for obtaining biological cell tissue components or microstructures. In order to simultaneously obtain polarization information and high-resolution images, many measurement schemes have been developed in recent years, such as amplitude division method, time division method, aperture division method, focal plane division method, etc. These methods can achieve the simultaneous acquisition of the above information, but the optical path is relatively complex and the system stability is not high.

[0004] Therefore, there is an urgent need to develop an optical imaging technology with high resolution, polarization sensitivity and simple optical path. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a confocal polarization microscopy imaging device and method based on functional pinholes, which achieves technical effects such as high resolution, polarization sensitivity, compact structure, and good compatibility.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] A confocal polarization microscopic imaging device based on a functional pinhole comprises an illumination module, a sample scanning module, a scattered light collection module, a functional pinhole module and a light intensity detection module, wherein:

[0008] The illumination module is used to provide laser light with a fixed polarization state;

[0009] The sample scanning module is used to place different areas of the sample to be tested at the laser focus according to the designed S-shaped moving route;

[0010] The scattered light collection module is used to collect scattered light of the sample to be tested;

[0011] The functional pinhole module is used to focus the scattered light of the sample to be tested and polarize it to four focal points with different polarization directions separated in space;

[0012] The light intensity detection module is used to collect the focal spot of the polarized focusing of the functional pinhole module and calculate the intensity to draw a scanning diagram.

[0013] In another aspect, the present invention provides a confocal polarization microscopic imaging method based on a functional pinhole, comprising the following steps:

[0014] The illumination module provides laser light with a fixed polarization state and generates a laser focus;

[0015] The sample scanning module places different areas of the sample to be tested at the laser focus according to the designed S-shaped moving route, and stimulates the sample to be tested to scatter;

[0016] The scattered light collection module collects scattered light of the sample to be tested;

[0017] The functional pinhole module focuses the scattered light of the sample to be tested and projects the light onto four spatially separated focal points with different polarization directions.

[0018] The light intensity detection module collects the focal spot of the polarized focusing of the functional pinhole module and calculates the intensity to draw a scanning diagram.

[0019] The beneficial effects of the present invention are:

[0020] (1) The use of functionalized pinholes enables the present invention to image samples in more dimensions compared to traditional confocal microscopes.

[0021] (2) Compared with measurement methods such as amplitude division, the introduction of full-Stokes parameter metasurface can greatly simplify the optical path and improve the stability of the optical path.

[0022] (3) Single-scan full-Stokes parameter imaging: one scanning point can simultaneously obtain information on four different polarization directions of the scattered light from the sample, and four polarization patterns of different polarizations can be obtained in one processing.

[0023] (4) Compatible with existing confocal microscopy systems, no special customized optical path is required, and confocal polarization microscopy can be achieved by simply replacing the traditional air holes with the metasurface. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1It is a schematic structural diagram of a confocal polarization microscopic imaging device based on a functional pinhole according to the present invention;

[0025] Figure 2 A schematic diagram of the polarization calibration structure of the confocal polarization microscopy imaging device based on a functional pinhole of the present invention;

[0026] Figure 3 Scanning electron microscope image of the full Stokes metasurface used in the embodiments of the present invention;

[0027] Figure 4 The polarization imaging results of the processed two-dimensional microstructure according to the embodiment of the present invention are shown, wherein (a) is a scanning electron microscope image of the two-dimensional microstructure, (b) is a traditional confocal image of the two-dimensional microstructure, (c) is a Stokes parameter S0 image of the two-dimensional microstructure, (d) is a Stokes parameter S1 image of the two-dimensional microstructure, (e) is a Stokes parameter S2 image of the two-dimensional microstructure, and (f) is a Stokes parameter S3 image of the two-dimensional microstructure.

[0028] Reference numerals:

[0029] 1. Laser; 2. First lens; 3. Aperture; 4. Second lens; 5. Polarizer; 6. Piezoelectric displacement platform; 7. Objective lens; 8. Semi-transparent and semi-reflective mirror; 9. Third lens; 10. Full-Stokes parameter metasurface; 11. Fourth lens; 12. Array detector; 13. Calibration sample. DETAILED DESCRIPTION

[0030] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0031] The purpose of the present invention is to provide a confocal polarization microscopic imaging device and method based on a functional pinhole, and the provided full Stokes metasurface can realize polarization projection, so that the polarization information of scattered light at each point of the sample can be polarized and separated by the full Stokes parametric metasurface, and the polarization measurement of the scattered light of the sample is finally realized by collecting the intensity of the polarization separation. The method has the characteristics of high resolution, polarization sensitivity, compact structure, good compatibility, etc., and can realize multi-dimensional microscopic imaging of the sample.

[0032] like Figure 1As shown, the entire device includes an illumination module, a sample scanning module, a scattered light collection module, a functional pinhole module and a light intensity detection module; wherein the illumination module is used to provide a laser with a stable power and a fixed polarization state, the sample scanning module places different areas of the sample to be tested at the laser focus according to the designed S-shaped moving route, the scattered light of the sample to be tested passes through the scattered light collection module and is focused on the full Stokes parameter metasurface of the functional pinhole module, the full Stokes parameter metasurface of the functional pinhole module projects the polarization of the scattered light of the sample onto four spatially separated foci with different polarization directions, the light intensity detection module collects the focal spot, calculates the intensity and draws a scanned image.

[0033] The illumination module includes a laser 1, a first lens 2, an aperture 3, a second lens 4, a polarizer 5, a semi-transparent mirror 8, and an objective lens 7 which are sequentially arranged along the optical path; the light beam emitted by the laser 1 is focused at the aperture 3 through the first lens 2, and after the stray light is filtered out by the aperture 3, a parallel light beam is obtained by the second lens 4, and is converted into a linearly polarized parallel light beam through the polarizer 5, and the linearly polarized parallel light beam is reflected by the semi-transparent mirror 8 into the objective lens 7, and the objective lens 7 focuses the linearly polarized parallel light beam onto the sample to be measured.

[0034] The sample scanning module includes a piezoelectric displacement platform 6; the piezoelectric displacement platform 6 moves the sample to be tested according to the designed step length and direction.

[0035] The scattered light collection module includes an objective lens 7, a semi-transparent and semi-reflective mirror 8, and a third lens 9; the scattered light of the sample to be tested passes through the objective lens 7 and the semi-transparent and semi-reflective mirror 8, and is focused onto the full-Stokes metasurface 10 via the third lens 9; it should be noted that the objective lens 7, the semi-transparent and semi-reflective mirror 8 here and the illumination module in the illumination module can be the same.

[0036] The functional pinhole module includes a full-Stokes parameter metasurface 10; the full-Stokes parameter metasurface 10 projects the polarization of scattered light of the sample to be measured onto four spatially separated foci. Without loss of generality, other metasurfaces may also be used, such as metasurfaces or metastructure gratings that can be used for polarization measurement.

[0037] The light intensity detection module includes a fourth lens 11 and an array detector 12 which are sequentially arranged along the optical path; after the four focal light spots of the polarized projection of the full-Stokes parameter metasurface 10 pass through the fourth lens 11, the array detector 12 collects the intensities of the four focal light spots.

[0038] like Figure 2As shown, since there are many optical elements in the optical path of the entire confocal polarization microscopy imaging device, which will affect the polarization of the light beam, this embodiment uses the characteristic value correction method to perform polarization correction on the optical path. During the correction, it is assumed that the low numerical aperture objective lens 7 used in this example will not affect the polarization. The objective lens 7 is directly removed from the optical path, and three characteristic calibration samples 13 are placed in the position of the objective lens 7 in sequence: a polarizer ( )、Polarizer( )、quarter wave plate( ), and after collecting the data, correction is performed according to the eigenvalue correction procedure.

[0039] Figure 3 This is a scanning electron microscope image of a full-Stokes parameter metasurface. The full-Stokes parameter metasurface 10 uses electron beam exposure and other technologies to use silicon to process an elliptical columnar particle array on a translucent glass substrate. The material of the particle array can be metal or a high-refractive index medium, including but not limited to gold, aluminum, silicon, silicon nitride, titanium dioxide, etc. The full-Stokes parameter metasurface 10 focuses the incident light polarization to different positions.

[0040] Based on the above device, the present invention also provides a confocal polarization microscopic imaging method based on a functional pinhole. The light emitted by the laser 1 is focused at the aperture 3 through the first lens 2. After the stray light is filtered out by the aperture 3, a parallel light beam is obtained by the second lens 4, which is converted into linear polarization light through the polarizer 5 and reflected by the semi-transparent and semi-reflective mirror 8 into the objective lens 7. The objective lens 7 focuses the incident parallel light onto the sample to be tested;

[0041] The sample to be tested is placed on the piezoelectric displacement platform 6 in the sample scanning module, and its characteristic is that the sample to be tested moves along the designed S-shaped route. The scattered signal light of the sample to be tested passes through the objective lens 7 and the semi-transparent and semi-reflective mirror 8, and is focused by the third lens 9 to the full Stokes metasurface 10;

[0042] The full-Stokes parameter metasurface 10 projects the incident uniformly polarized light to four focal points, and the polarization directions of the four focal points are: (horizontally), (Diagonal, with the horizontal direction angular direction), (vertical direction), (right-hand circular polarization direction); the four foci are separated in space, and the intensity is the projection of the incident light under the corresponding polarization basis vector;

[0043] The fourth lens 11 in the light intensity detection module images the focal spot obtained by polarization projection of the full Stokes parameter metasurface 10 onto the area array detector 12. The area array detector 12 collects multiple images obtained by point-by-point scanning of the sample to be tested. The images of the four focal points in each image are symmetrically distributed, and the intensity is determined by the polarization of the incident light. The intensity position is accurately located from each image collected by the area array detector to extract the intensity information of the focus;

[0044] The entire device is provided with parallel polarized light required for illumination by the illumination module. The sample scanning module places different areas on the sample to be tested (the number of scanning points in the x and y directions are N and M respectively) at the laser focus. The scattered light of the sample to be tested is polarized and focused by the full Stokes parameter metasurface 10, and is imaged by the fourth lens 11 to the area array detector 12 to obtain N×M images. The intensity information in these N×M images is extracted and the scanned image of the sample to be tested is drawn by a computer. The electron microscope image of the two-dimensional microstructure sample is shown in the figure. Figure 4 As shown in (a), the size of a unit structure is , the entire scanning field of view is The images collected by a conventional confocal microscopy system using the same objective lens and a matching pinhole size (approximately the size of an Airy disk) are shown below. Figure 4 As shown in (b), no other information can be found in each unit structure. The present invention characterizes the amount of information contained in the image by calculating the information entropy of the image. The results show that the full Stokes parameter image obtained by the confocal polarization microscopy imaging device contains more information, such as Figure 4 (c) Stokes parameters S0 of the two-dimensional microstructure sample, Figure 4 (d) Stokes parameters S1, Figure 4 (e) Stokes parameter S2 of the two-dimensional microstructure sample, Figure 4 As shown in (f) Stokes parameter S3 of the two-dimensional microstructure sample, for the two-dimensional microstructure sample, the information entropy obtained by the confocal polarization microscopy imaging device is about twice that of the traditional confocal.

[0045] The confocal polarization microscopy device and method based on functional pinholes of the present invention utilizes the full Stokes parameter projection characteristics to design a full Stokes parameter metasurface of appropriate size to replace the traditional confocal pinhole, thereby realizing high-resolution polarization imaging of the sample. The method has the characteristics of high resolution, polarization sensitivity, compact structure, and good compatibility, and can realize high-resolution multi-dimensional microscopy imaging of the sample.

[0046] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A confocal polarization microscopic imaging device based on a functional pinhole, characterized in that: It includes an illumination module, a sample scanning module, a scattered light collection module, a functional pinhole module and a light intensity detection module, wherein: The illumination module is used to provide laser light with a fixed polarization state; The sample scanning module is used to place different areas of the sample to be tested at the laser focus according to the designed S-shaped moving route; The scattered light collection module is used to collect scattered light of the sample to be tested; The functional pinhole module is used to focus the scattered light of the sample to be tested and polarize it to four focal points with different polarization directions separated in space; The light intensity detection module is used to collect the focal spot of the polarized focusing of the functional pinhole module and calculate the intensity to draw a scanning diagram.

2. A confocal polarization microscopic imaging device based on a functional pinhole according to claim 1, characterized in that: The functional pinhole module includes a full-Stokes parameter metasurface, which projects the polarized scattered light of the sample to be measured incident on the metasurface onto four spatially separated foci with different polarization directions.

3. A confocal polarization microscopic imaging device based on a functional pinhole according to claim 2, characterized in that: The lighting module includes a laser, a first lens, an aperture, a second lens, a polarizer, a semi-transparent mirror, and an objective lens, wherein the light beam emitted by the laser is focused at the aperture through the first lens, and after the stray light is filtered out by the aperture, a parallel light beam is obtained by the second lens, which is converted into a linearly polarized parallel light beam through the polarizer, and then reflected by the semi-transparent mirror into the objective lens, and the objective lens focuses the linearly polarized parallel light beam onto the sample to be measured.

4. The confocal polarization microscopic imaging device based on a functional pinhole according to claim 3, characterized in that: The sample scanning module includes a piezoelectric displacement platform, which moves the sample to be tested according to the designed step length and direction.

5. The confocal polarization microscopic imaging device based on a functional pinhole according to claim 4, characterized in that: The scattered light collection module includes an objective lens, a semi-transparent and semi-reflective mirror, and a third lens. The scattered light of the sample to be tested passes through the objective lens and the semi-transparent and semi-reflective mirror and is focused by the third lens onto the full-Stokes parameter metasurface.

6. The confocal polarization microscopic imaging device based on a functional pinhole according to claim 5, characterized in that: The light intensity detection module includes a fourth lens and an array detector. The intensity of four focal spots with different polarization directions of the full-Stokes parameter metasurface polarization projection passes through the fourth lens and is collected by the array detector.

7. The confocal polarization microscopic imaging device based on a functional pinhole according to claim 1, characterized in that: The full-Stokes parameter metasurface of the functional pinhole module can also be replaced by any metasurface or metagrating capable of polarization measurement.

8. The confocal polarization microscopic imaging device based on a functional pinhole according to claim 2, characterized in that: The full-Stokes parameter metasurface is an elliptical columnar particle array processed by using silicon on a light-transmitting glass substrate, and the material of the particle array is metal or a high refractive index medium.

9. The confocal polarization microscopic imaging device based on a functional pinhole according to claim 1, characterized in that: The polarization directions of the four focal points with different polarization directions are horizontal, Angular direction, vertical direction, right-hand circular polarization direction.

10. A confocal polarization microscopy imaging method based on a functional pinhole, characterized in that: The following steps are involved: The illumination module provides laser light with a fixed polarization state and generates a laser focus; The sample scanning module places different areas of the sample to be tested at the laser focus according to the designed S-shaped moving route, and stimulates the sample to be tested to scatter; The scattered light collection module collects scattered light of the sample to be tested; The functional pinhole module focuses the scattered light of the sample to be tested and projects the light onto four spatially separated focal points with different polarization directions. The light intensity detection module collects the focal spot of the polarized focusing of the functional pinhole module and calculates the intensity to draw a scanning diagram.

11. The confocal polarization microscopy imaging method based on a functional pinhole according to claim 10, characterized in that: The sample scanning module moves the sample to be tested so that the sample to be tested is illuminated at different positions in the x and y directions. The scattered light of the sample to be tested is focused by the full-Stokes parameter metasurface polarization projection of the functional pinhole module, and is imaged by the planar array detector of the light intensity detection module to obtain N×M images and calculate the intensity respectively, thereby obtaining a confocal scanning image of the sample to be tested with resolutions of N and M in the x and y directions respectively.

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