Confocal Polarization Microscopic Imaging Device and Method Based on Functional Small Holes

Abstract

The present invention discloses a confocal polarization microscopy imaging device and method based on functional small holes, belonging to the technical field of optical microscopy imaging. In the device, an illumination module is used to provide a laser with a fixed polarization state; a sample scanning module is used to place different regions of a sample to be measured at the laser focus according to a designed S-shaped moving route; a scattered light collection module is used to collect the scattered light of the sample to be measured; a functional small hole module is used to focus the scattered light of the sample to be measured and project it polarized onto four foci with different polarization directions separated in space; a light intensity detection module is used to collect the focal spots polarized and focused by the functional small hole module and calculate the intensity to draw a scanned image. The device and method use a full Stokes parameter metasurface as the functional small hole of the confocal imaging system for polarization imaging, and have the characteristics of high imaging resolution, high polarization sensitivity, compact structure, good compatibility, etc.

Description

Technical Field

[0001] The present invention belongs to the technical field of optical microscopy imaging, and particularly relates to a confocal polarization microscopy imaging device and method based on a functional small hole. Background Art

[0002] Microscopy imaging is a key technology in modern scientific research and plays an important role in fields such as biomedicine, precision manufacturing, and life sciences. With the development of science, various demands for exploring the microscopic world by humans have increased day by day, which also puts forward new requirements for microscopy imaging technology.

[0003] Traditional microscopes use wide-field illumination to expose the entire sample to the light source, and then the imaging system images it into the eye or a area array detector. Wide-field illumination can obtain a relatively large field of view. However, since wide-field illumination illuminates the entire sample, the light from some regions far from the focal plane is also received by the imaging system, and these stray lights cause the imaging of the region of interest to become blurred. The confocal technique uses a point conjugate method, and a pinhole is added in front of the detector. The light far from the focal plane is blocked by the pinhole outside the detector, enabling the confocal system to perform optical sectioning, realize three-dimensional scanning, and improve the microscopy imaging resolution. Although traditional confocal techniques have the capabilities of high resolution, optical sectioning, and three-dimensional imaging, they are essentially intensity imaging and cannot effectively analyze the polarization information of the light field. However, the polarization information is crucial for obtaining the components or microscopic structures of biological cells and tissues. To simultaneously obtain polarization information and high-resolution images, many measurement schemes have emerged in recent years, such as amplitude splitting method, time sharing method, aperture splitting method, focal plane splitting 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] To solve the above technical problems, the present invention provides a confocal polarization microscopy imaging device and method based on a functional small hole, achieving 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 microscopy imaging device based on a functional small hole, comprising an illumination module, a sample scanning module, a scattered light collection module, a functional small hole module, and a light intensity detection module, wherein,

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

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

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

[0011] The functional small hole module is used to focus the scattered light of the sample to be measured and project it polarized onto four foci with different polarization directions separated in space;

[0012] The light intensity detection module is used to collect the focal spot of the polarized focus of the functional small hole module, and calculate the intensity to draw a scan image.

[0013] On the other hand, the present invention provides a confocal polarization microscopy imaging method based on a functional small hole, including the following steps:

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

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

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

[0017] The functional small hole module focuses the scattered light of the sample to be measured and projects it polarized onto four foci with different polarization directions separated in space;

[0018] The light intensity detection module collects the focal spot of the polarized focus of the functional small hole module, and calculates the intensity to draw a scan image.

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

[0020] (1) The use of functional small holes enables the present invention to image the sample in more dimensions compared with traditional confocal microscopes.

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

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

[0023] (4) It is compatible with existing confocal microscopy imaging systems. Without special customization of the optical path, confocal polarization microscopy imaging can be achieved only by replacing the traditional air small hole with a metasurface. Description of the Drawings

[0024] Figure 1Schematic structural diagram of the confocal polarization microscopy imaging device based on functional small holes according to the present invention;

[0025] Figure 2 Schematic structural diagram of the polarization calibration of the confocal polarization microscopy imaging device based on functional small holes according to the present invention;

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

[0027] Figure 4 Polarization imaging results of the processed two-dimensional microstructures in the embodiment of the present invention. Among them, (a) is the scanning electron microscope image of the two-dimensional microstructures, (b) is the traditional confocal image of the two-dimensional microstructures, (c) is the Stokes parameter S0 image of the two-dimensional microstructures, (d) is the Stokes parameter S1 image of the two-dimensional microstructures, (e) is the Stokes parameter S2 image of the two-dimensional microstructures, and (f) is the Stokes parameter S3 image of the two-dimensional microstructures.

[0028] Reference numerals:

[0029] 1. Laser; 2. First lens; 3. Diaphragm; 4. Second lens; 5. Polarizer; 6. Piezoelectric displacement platform; 7. Objective lens; 8. Beam splitter; 9. Third lens; 10. Full Stokes parameter metasurface; 11. Fourth lens; 12. Area array detector; 13. Calibration sample. Detailed implementation manners

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

[0031] The object of the present invention is to provide a confocal polarization microscopy imaging device and method based on functional small holes. The provided full Stokes metasurface can realize polarization projection, so that the polarization information of the scattered light at each point of the sample can be polarization-separated by the full Stokes parameter metasurface. By collecting the intensity of the polarization separation, the polarization measurement of the scattered light of the sample is finally realized. This 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] As Figure 1As shown in the figure, the entire device includes an illumination module, a sample scanning module, a scattered light collection module, a functional small hole module, and a light intensity detection module. Among them, the illumination module is used to provide a laser with a stable power and a fixed polarization state. The sample scanning module places different regions of the sample to be measured at the laser focus according to the designed S-shaped movement route. The scattered light of the sample to be measured passes through the scattered light collection module and is focused on the full Stokes parameter metasurface of the functional small hole module. The full Stokes parameter metasurface of the functional small hole module projects the polarization of the scattered light of the sample onto four foci with different polarization directions separated in space. The light intensity detection module collects the focal spot and calculates the intensity to draw a scanned image.

[0033] Among them, the illumination module includes a laser 1, a first lens 2, a diaphragm 3, a second lens 4, a polarizer 5, a semi-transparent semi-reflective mirror 8, and an objective lens 7 arranged in sequence along the optical path. The beam emitted by the laser 1 is focused on the diaphragm 3 through the first lens 2. After passing through the diaphragm 3 to filter out stray light, a parallel beam is obtained by the second lens 4. It is converted into a linearly polarized parallel beam by the polarizer 5. The linearly polarized parallel beam is reflected by the semi-transparent semi-reflective mirror 8 and enters the objective lens 7. The objective lens 7 focuses the linearly polarized parallel beam on 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 measured according to the designed step size and direction.

[0035] The scattered light collection module includes an objective lens 7, a semi-transparent semi-reflective mirror 8, and a third lens 9. The scattered light of the sample to be measured passes through the objective lens 7 and the semi-transparent semi-reflective mirror 8 and is then focused on the full Stokes metasurface 10 by the third lens 9. It should be noted that the objective lens 7 and the semi-transparent semi-reflective mirror 8 here can be the same as those in the illumination module of the illumination module.

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

[0037] The light intensity detection module includes a fourth lens 11 and a area array detector 12 arranged in sequence along the optical path. The four focal spot intensities of the polarization projection of the full Stokes parameter metasurface 10 are collected by the area array detector 12 after passing through the fourth lens 11.

[0038] Such as Figure 2As shown, since there are many optical elements in the optical path of the entire confocal polarization microscopy imaging device, it will affect the polarization of the light beam. In this embodiment, the eigenvalue correction method is used to correct the polarization of the optical path. When correcting, it is considered 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 sequentially placed at the position of the objective lens 7: polarizer ( ), polarizer ( ), quarter-wave plate ( ). After collecting the data, the correction is performed according to the eigenvalue correction program.

[0039] Figure 3 Figure 10 is a scanning electron microscope image of the full Stokes parameter metasurface. The full Stokes parameter metasurface 10 uses techniques such as electron beam lithography to process an array of elliptical cylindrical particles on a transparent glass substrate using silicon. 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 aforementioned device, the present invention also provides a confocal polarization microscopy imaging method based on a functional small hole. The light emitted by the laser 1 passes through the first lens 2 and is focused at the aperture 3. After the stray light is filtered by the aperture 3, a parallel light beam is obtained by the second lens 4, converted into linearly polarized light by the polarizer 5, and reflected by the semi-transmissive semi-reflective mirror 8 into the objective lens 7. The objective lens 7 focuses the incident parallel light on the sample to be measured;

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

[0042] The full Stokes parameter metasurface 10 projects the incident uniform polarized light onto four focal points. The polarization directions of the four focal points are respectively: (horizontal direction), (diagonal, at an angle of with the horizontal direction), (vertical direction), (right-handed circular polarization direction); the four focal points are spatially separated, and the intensity is the projection of the incident light under the corresponding polarization basis vectors;

[0043] The fourth lens 11 in the light intensity detection module images the focal spot obtained by the 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 measured. 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 in each image collected by the area array detector, and the intensity information of the focal points is extracted;

[0044] The entire device is provided with parallel polarized light required for illumination by the illumination module. The sample scanning module places different regions (the number of scanning points in the x and y directions are N and M respectively) on the sample to be measured at the laser focal point. The scattered light of the sample to be measured is polarization focused by the full Stokes parameter metasurface 10, passes through the fourth lens 11 and is imaged on the area array detector 12 to obtain N×M images. The intensity information in these N×M images is extracted and a scanned image of the sample to be measured is drawn using a computer. The electron microscope image of the two-dimensional micro-structure sample is as shown in Figure 4 (a) in, and the size of one unit structure is , and the entire scanning field of view is . The images collected by a traditional confocal microscopy system using the same objective lens and a matched-sized pinhole (about the size of an Airy disk) are as shown in Figure 4 (b) in. No other information can be found in each unit structure. In the example of the present invention, the information entropy of the image is calculated to characterize the amount of information contained in the picture. The results show that the full Stokes parameter images obtained by the confocal polarization microscopy device contain more information, such as the Stokes parameter S0 of the two-dimensional micro-structure sample in Figure 4 (c), the Stokes parameter S1 of the two-dimensional micro-structure sample in Figure 4 (d), the Stokes parameter S2 of the two-dimensional micro-structure sample in Figure 4 (e), and the Stokes parameter S3 of the two-dimensional micro-structure sample in Figure 4 (f). For the two-dimensional micro-structure sample, the information entropy obtained by the confocal polarization microscopy device is approximately twice that of the traditional confocal microscopy.

[0045] The confocal polarization microscopy device and method based on a functional small hole of the present invention utilize the full Stokes parameter projection characteristics, design a full Stokes parameter metasurface with a suitable size to replace the traditional confocal pinhole, and realize high-resolution polarization imaging of the sample. This method has the characteristics of high resolution, polarization sensitivity, compact structure, and good compatibility, and can realize high-resolution multi-dimensional microscopic imaging of the sample.

[0046] The specific embodiments described above further elaborate on the object, technical solution and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A confocal polarization microscopy imaging device based on functional small holes, 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. Among them, the illumination module is used to provide a laser with a fixed polarization state; the sample scanning module is used to place different regions of the sample to be measured at the laser focus according to the designed S-shaped movement route; the scattered light collection module is used to collect the scattered light of the sample to be measured; the functional pinhole module is used to focus the scattered light of the sample to be measured and project it polarized onto four foci with different polarization directions separated in space; among them, the functional pinhole module includes a full Stokes parameter metasurface, and the full Stokes parameter metasurface, as the pinhole of the confocal polarization microscopy imaging device, projects the scattered light of the sample to be measured incident on the metasurface onto four foci with different polarization directions separated in space; the light intensity detection module is used to collect the focal spots polarized and focused by the functional pinhole module, and calculate the intensity to draw a scan image.

2. The confocal polarization microscopy imaging device based on functional small holes according to claim 1, wherein, The illumination module includes a laser, a first lens, a diaphragm, a second lens, a polarizer, a beam splitter, and an objective lens. Among them, the beam emitted by the laser is focused at the diaphragm by the first lens, and after filtering out stray light by the diaphragm, a parallel beam is obtained by the second lens, which is converted into a linearly polarized parallel beam by the polarizer, and then reflected by the beam splitter into the objective lens, and the objective lens focuses the linearly polarized parallel beam onto the sample to be measured.

3. The confocal polarization microscopy imaging device based on functional small holes according to claim 2, wherein, The sample scanning module includes a piezoelectric displacement platform, and the piezoelectric displacement platform moves the sample to be measured according to the designed step size and direction.

4. The confocal polarization microscopy imaging device based on functional small holes according to claim 3, characterized in that, The scattered light collection module includes an objective lens, a beam splitter, and a third lens. The scattered light of the sample to be measured is focused on the full Stokes parameter metasurface by the third lens after passing through the objective lens and the beam splitter.

5. The confocal polarization microscopy imaging device based on functional small holes according to claim 4, wherein, The light intensity detection module includes a fourth lens and a area array detector. The four focal spots with different polarization directions projected by the full Stokes parameter metasurface are collected by the area array detector after passing through the fourth lens.

6. The confocal polarization microscopy imaging device based on functional small holes according to claim 1, characterized in that, The full Stokes parameter metasurface is an array of elliptical cylindrical particles processed from silicon on a light-transmitting glass substrate, and the material of the particle array is metal or a high refractive index medium.

7. A confocal polarization microscopy imaging device based on functional small holes according to claim 1, characterized in that, The polarization directions of the four foci with different polarization directions are the horizontal direction, the direction at a 45° angle to the horizontal direction, the vertical direction, and the right-handed circular polarization direction respectively.

8. A confocal polarization microscopy imaging method based on functional small holes, characterized in that, It includes the following steps: The illumination module provides a laser with a fixed polarization state and generates a laser focus; The sample scanning module places different regions of the sample to be measured at the laser focus according to the designed S-shaped movement route, and excites the sample to be measured to scatter; The scattered light collection module collects the scattered light of the sample to be measured; The functional pinhole module focuses the scattered light of the sample to be measured and projects it polarized onto four foci with different polarization directions separated in space; among them, the functional pinhole module includes a full Stokes parameter metasurface; the full Stokes parameter metasurface, as the pinhole of the confocal polarization microscopy imaging device, projects the scattered light of the sample to be measured incident on the metasurface onto four foci with different polarization directions separated in space; The light intensity detection module collects the focal spots polarized and focused by the functional pinhole module, and calculates the intensity to draw a scan image.

9. A confocal polarization microscopy imaging method based on functional small holes according to claim 8, characterized in that, The sample scanning module moves the sample to be measured so that the sample to be measured is illuminated at different positions in the x and y directions. The number of scanning points in the x and y directions are N and M respectively. The scattered light of the sample to be measured is focused by the full Stokes parameter metasurface polarization projection of the functional small hole module and imaged by the area array detector of the light intensity detection module to obtain N×M images, and the intensities are calculated respectively, so as to obtain a confocal scanning image of the sample to be measured with resolutions of N and M in the x and y directions respectively.

Images