Super-resolution microscopic imaging method and device based on multiple polarization and illumination modes

Abstract

The present invention discloses a super-resolution microscopic imaging method and device based on multiple polarizations and illumination modes. The present invention realizes three-dimensional polarization control of the focused light spot based on the coherent synthesis of multi-polarization light beams and the focusing of a large numerical aperture objective lens. Compared with the existing polarization imaging technology, one more dimension of polarization information is obtained, and a super-resolution image containing richer information is reconstructed. The present invention adds a change in illumination mode while converting polarization, and the change in illumination mode can overcome the optical diffraction limit. The present invention combines three-dimensional polarization control with illumination mode change to realize super-resolution microscopic imaging.

Description

Technical Field

[0001] The present invention relates to the field of super-resolution microscopic imaging, and in particular to a super-resolution microscopic imaging method and device based on multiple polarization and illumination modes. Background Art

[0002] Intensity, wavelength, and polarization are the three basic properties of light. Conventional imaging can only obtain the intensity and wavelength information of an object. The reflected light and scattered light of the interaction between light and an object contain polarization information determined by the object's own characteristics. By detecting polarization information, physical information such as birefringence, stress, and surface roughness that cannot be detected by conventional imaging can be obtained. Polarization imaging technology has a wide range of applications in research fields such as optical detection, microscopic imaging, and Raman spectroscopy. Conventional polarization imaging can only detect polarization information in the x and y two dimensions. Summary of the invention

[0003] The purpose of the present invention is to address the deficiencies of the prior art and provide a super-resolution microscopic imaging method and device based on multiple polarization and illumination modes. The present invention performs polarization modulation on the focused light spot of a large numerical aperture objective lens, realizes the acquisition of three-dimensional polarization information of x, y, and z, and modulates the illumination pattern at the same time, and obtains a super-resolution microscopic image by post-processing the images collected under multiple polarization and illumination modes.

[0004] The present invention is achieved through the following technical solutions:

[0005] On the one hand, the present invention provides a super-resolution microscopic imaging method based on multiple polarization and illumination modes, the method comprising:

[0006] The light beam emitted by the laser is collimated and converted into linear polarized light by a polarizer, and then incident on the first polarization modulator to modulate the polarization direction.

[0007] The modulated light beam is split into linear polarized light in the x-direction and the y-direction by the polarization beam splitter. The linear polarized light in the y-direction is converted into radial polarized light by the vortex half-wave plate and then passes through the annular aperture. The linear polarized light in the x-direction is incident on the second polarization modulator to modulate the polarization direction, and the modulated light beam is converted into a vortex beam by the vortex phase plate.

[0008] The two beams of light are combined by a beam splitter and enter the confocal microscope. The light beam is focused on the sample by an objective lens with a large numerical aperture, and the numerical aperture NA is usually greater than 0.8. The reflected or scattered photons pass through the analyzer and are focused by the lens on a small hole conjugate with the sample and received by the detector. The scanning mechanism is controlled to scan the light spot on the sample surface to obtain a microscopic image.

[0009] The inputs of the first polarization modulator and the second polarization modulator are adjusted to obtain microscopic images under different polarization and illumination modes, and a super-resolution microscopic image is obtained by a post-processing algorithm.

[0010] Furthermore, the principle of polarization modulation is as follows: the intensity ratio of the two beams of light separated by the polarization beam splitter depends on the input of the first polarization modulator. Among them, after the linear polarized light in the y direction passes through the vortex half-wave plate and the annular aperture, the light beam passes through the large numerical aperture objective lens to form a focused light spot whose polarization is mainly in the z direction. After the linear polarized light in the x direction passes through the second polarization modulator, it has two polarization components in the x and y directions. The intensity ratio of the two polarization components depends on the input of the second polarization modulator. The polarization of the focused light spot formed by the polarized light in the two directions passing through the large numerical aperture objective lens is mainly in the x and y directions respectively. Therefore, the inputs of the first polarization modulator and the second polarization modulator are adjusted to obtain focused light spots with different polarization directions.

[0011] Furthermore, the working process of the polarization modulator is as follows: a periodic voltage signal is input to the first polarization modulator and the second polarization modulator, and within one cycle, the polarization state of the light beam is gradually converted from x polarization to y polarization, and then from y polarization to x polarization, wherein the repetition frequency of the second polarization modulator is much higher than the repetition frequency of the first polarization modulator. At the same time, the detector collects the light intensity, and each time the voltage of the first polarization modulator passes through a cycle, an intensity under the multi-polarization and illumination mode at a corresponding spatial position is obtained, and the scanning mechanism focuses the light beam at the next spatial position to collect the signal at the next spatial position. When the signal collection of all spatial positions is completed, an image of the multi-polarization and illumination mode is obtained.

[0012] Furthermore, the principle of illumination mode transformation is as follows: the intensity ratio of the two beams of light separated by the polarization beam splitter depends on the input of the first polarization modulator. Among them, after the linear polarized light in the y direction passes through the vortex half-wave plate and the annular aperture, the focusing spot formed by the light beam passing through the large numerical aperture objective lens is a solid spot. The linear polarized light in the x direction has two polarization components in the x and y directions after passing through the second polarization modulator. The polarized light in the x direction passes through the vortex phase plate, and the focusing spot formed by the light beam passing through the large numerical aperture objective lens is an elliptical hollow spot, and the major axis of the ellipse is along the x direction; the polarized light in the y direction passes through the vortex phase plate, and the focusing spot formed by the light beam passing through the large numerical aperture objective lens is an elliptical hollow spot, and the major axis of the ellipse is along the y direction. Therefore, adjusting the input of the first polarization modulator and the second polarization modulator can realize the transformation of the illumination mode.

[0013] On the other hand, the present invention provides a super-resolution microscopic imaging device based on multiple polarization and illumination modes, which includes a laser, a polarizer, two polarization modulators, a polarization beam splitter, a vortex half-wave plate, an annular aperture, a vortex phase plate, a beam splitter and a scanning imaging module; the scanning imaging module includes a scanning mechanism, a tube lens, a large numerical aperture objective lens, a lens, a pinhole and a detector.

[0014] The light beam emitted by the laser is collimated and converted into linear polarized light by the polarizer, and then incident on the first polarization modulator to modulate the polarization direction.

[0015] The modulated light beam is split into linear polarized light in the x-direction and the y-direction by the polarization beam splitter. The linear polarized light in the y-direction passes through the vortex half-wave plate and the annular aperture in sequence. The linear polarized light in the x-direction passes through the second polarization modulator and the vortex phase plate in sequence. The two beams are combined by the beam splitter and enter the scanning imaging module.

[0016] The scanning mechanism enables the focused light spot formed by the tube lens and the large numerical aperture objective lens to scan the sample surface. The reflected light and scattered light on the sample surface are focused on the small hole conjugate with the sample through the lens, and are received by the detector to obtain a microscopic image.

[0017] The inputs of the first polarization modulator and the second polarization modulator are adjusted to obtain microscopic images under different polarization and illumination modes.

[0018] Furthermore, a D-shaped mirror is used to deflect the light path before the light beam is incident on the first polarization modulator, thereby reducing the incident angle and improving the performance of the polarization modulator.

[0019] Furthermore, the orientation of the vortex half-wave plate is adjusted so that the linear polarization in the y direction is converted into radial polarization.

[0020] Furthermore, the scanning mechanism is a scanning galvanometer, and the midpoint of the line connecting the centers of the two galvanometers is conjugate with the entrance pupil of the objective lens.

[0021] Furthermore, the polarization modulator can be implemented by an electro-optic modulator (EOM) or a half-wave plate driven by a motor. The modulation direction of the electro-optic modulator is at a 45-degree angle to the polarization direction of the incident light, so that the polarization of the outgoing light is converted between the polarization of the incident light and the polarization of the perpendicular incident light; the half-wave plate is driven by a motor to rotate, so that the polarization direction of the incident light rotates accordingly.

[0022] Furthermore, the reflected light and scattered light of the sample are focused on the small hole, and the small hole is used to eliminate the out-of-focus stray light, which is achieved by using a pinhole or a multi-mode optical fiber.

[0023] Compared with the prior art, the present invention has the following beneficial technical effects: The present invention realizes three-dimensional polarization control of the focused light spot based on the coherent synthesis of multi-polarized light beams and the focusing of a large numerical aperture objective lens. Compared with the existing polarization imaging technology, one more dimension of polarization information is obtained, and a super-resolution image containing richer information is reconstructed. The present invention adds a change in illumination mode while converting polarization, and the change in illumination mode can overcome the optical diffraction limit. The present invention combines three-dimensional polarization control with illumination mode change to realize super-resolution microscopic imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A schematic diagram of a super-resolution microscopic imaging device based on multiple polarization and illumination modes provided in an embodiment of the present invention;

[0025] Figure 2 This is the input voltage timing diagram of the two electro-optic modulators when the device is working. DETAILED DESCRIPTION

[0026] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific embodiments of the present invention are described in detail below with reference to the accompanying drawings.

[0027] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0028] The present invention provides a super-resolution microscopic imaging method based on multiple polarization and illumination modes, comprising:

[0029] The light beam emitted by the laser is collimated and converted into linear polarized light by a polarizer, and then incident on the first polarization modulator to modulate the polarization direction.

[0030] The modulated light beam is split into linear polarized light in the x-direction and the y-direction by the polarization beam splitter. The linear polarized light in the y-direction is converted into radial polarized light by the vortex half-wave plate and then passes through the annular aperture. The linear polarized light in the x-direction is incident on the second polarization modulator to modulate the polarization direction, and the modulated light beam is converted into a vortex beam by the vortex phase plate.

[0031] The two beams of light are combined by a beam splitter and enter the confocal microscope. The light beam is focused on the sample by an objective lens with a large numerical aperture. The numerical aperture NA is usually greater than 0.8. The reflected or scattered photons pass through the analyzer and are focused by the lens on a small hole conjugate with the sample and received by the detector. The scanning mechanism is controlled to scan the light spot on the sample surface to obtain a microscopic image.

[0032] The inputs of the first polarization modulator and the second polarization modulator are adjusted to obtain microscopic images under different polarization and illumination modes, and a super-resolution microscopic image is obtained by a post-processing algorithm.

[0033] The principle of polarization modulation is as follows: the intensity ratio of the two beams of light separated by the polarization beam splitter depends on the input of the first polarization modulator. Among them, after the linear polarized light in the y direction passes through the vortex half-wave plate and the annular aperture, the light beam passes through the large numerical aperture objective lens to form a focused light spot whose polarization is mainly in the z direction. After the linear polarized light in the x direction passes through the second polarization modulator, it has two polarization components in the x and y directions. The intensity ratio of the two polarization components depends on the input of the second polarization modulator. The polarized light in two directions passes through the large numerical aperture objective lens to form a focused light spot whose polarization is mainly in the x and y directions respectively. Therefore, the inputs of the first polarization modulator and the second polarization modulator are adjusted to obtain focused light spots with different polarization directions.

[0034] The working process of the polarization modulator is as follows: a periodic voltage signal is input to the first polarization modulator and the second polarization modulator. In one cycle, the polarization state of the light beam is gradually converted from x polarization to y polarization, and then from y polarization to x polarization. The repetition frequency of the second polarization modulator is much higher than that of the first polarization modulator. At the same time, the detector collects light intensity. Every time the voltage of the first polarization modulator passes through a cycle, an intensity under the multi-polarization and illumination mode at a corresponding spatial position is obtained. The scanning mechanism focuses the light beam at the next spatial position and collects the signal at the next spatial position. When the signal collection of all spatial positions is completed, an image of the multi-polarization and illumination mode is obtained.

[0035] The principle of illumination mode transformation is as follows: the intensity ratio of the two beams of light separated by the polarization beam splitter depends on the input of the first polarization modulator. Among them, after the linear polarized light in the y direction passes through the vortex half-wave plate and the annular aperture, the focusing spot formed by the light beam passing through the large numerical aperture objective lens is a solid spot. The linear polarized light in the x direction has two polarization components in the x and y directions after passing through the second polarization modulator. The polarized light in the x direction passes through the vortex phase plate, and the focusing spot formed by the light beam passing through the large numerical aperture objective lens is an elliptical hollow spot, and the major axis of the ellipse is along the x direction; the polarized light in the y direction passes through the vortex phase plate, and the focusing spot formed by the light beam passing through the large numerical aperture objective lens is an elliptical hollow spot, and the major axis of the ellipse is along the y direction. Therefore, adjusting the input of the first polarization modulator and the second polarization modulator can realize the transformation of the illumination mode.

[0036] The following is a specific implementation example of the device of the present invention, but is not limited thereto. The super-resolution microscopic imaging device based on multiple polarization and illumination modes in this example is as follows: Figure 1As shown, it includes a laser generating and collimating device 1, a half-wave plate 2, a first polarizer 3, a first electro-optic modulator 4, a polarization beam splitter prism 5, a vortex half-wave plate 6, an annular aperture 7, a second electro-optic modulator 8, a first reflector 9, a vortex phase plate 10, a second reflector 11, a first beam splitter prism 12, a second beam splitter prism 13, a scanning galvanometer 14, a scanning lens 15, a third reflector 16, a tube lens 17, an objective lens 18, a stage 19, a second polarizer 20, a lens 21, a pinhole 22, and an avalanche photodiode 23.

[0037] When the device is working, the laser generating and collimating device 1 becomes linear polarized light in the y direction after passing through the half-wave plate 2 and the first polarizer 3, and is incident on the first electro-optic modulator 4. The modulation direction of the first electro-optic modulator 4 is 45 degrees to the y direction.

[0038] The modulated light beam is split into linear polarized light in the x direction and the y direction by the polarization beam splitter prism 5. The linear polarized light in the y direction is converted into radial polarized light by the vortex half-wave plate 6, and then passes through the annular aperture 7; the linear polarized light in the x direction passes through the second electro-optic modulator 8, the first reflector 9, the vortex phase plate 10, and the second reflector 11. The modulation direction of the second electro-optic modulator 8 is 45 degrees to the x direction, and the modulated light beam is converted into a vortex light beam by the vortex phase plate 10.

[0039] The two light beams are combined by the first beam splitter prism 12, and enter the scanning galvanometer 14 through the second beam splitter prism 13. The midpoint of the line connecting the centers of the two galvanometers is conjugate with the entrance pupil of the objective lens. The light beams pass through the scanning lens 15, the third reflector 16, and the tube lens 17 in sequence, and are focused on the sample surface on the stage 19 by the objective lens 18 with a large numerical aperture. The reflected light and scattered light of the sample pass through the objective lens 18, the tube lens 17, the third reflector 16, the scanning lens 15, and the scanning galvanometer 14 in sequence, and are reflected by the second beam splitter prism 13, and are focused on the small hole 22 conjugate with the object through the second polarizer 20 and the lens 21. The small hole is connected to the avalanche photodiode 23 to collect the light signal. The scanning galvanometer 14 scans the light spot on the sample surface to obtain a microscopic image.

[0040] The working process of the electro-optic modulator is as follows: -V is input to the first electro-optic modulator EOM1 and the second electro-optic modulator EOM2 π ~V π The periodic voltage signal, V π is the full-wave voltage of the electro-optic modulator EOM, where the repetition frequency of EOM2 is much higher than that of EOM1, such as Figure 2 As shown. At the same time, the detector collects light intensity. Every time the voltage of EOM1 passes through a cycle, an intensity under the multi-polarization and illumination mode at a corresponding spatial position is obtained. The scanning galvanometer 14 focuses the light beam at the next spatial position to collect the signal at the next spatial position. When the signal collection of all spatial positions is completed, an image of the multi-polarization and illumination mode is obtained.

[0041] The above is only a preferred embodiment of the present invention. Although the present invention has been disclosed as a preferred embodiment, it is not intended to limit the present invention. Any technician familiar with the art can make many possible changes and modifications to the technical solution of the present invention by using the above disclosed methods and technical contents without departing from the scope of the technical solution of the present invention, or modify it into an equivalent embodiment of equivalent changes. Therefore, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still falls within the scope of protection of the technical solution of the present invention.

Claims

1. A super-resolution microscopic imaging method based on multiple polarization and illumination modes, characterized in that: include: The light beam emitted by the laser is collimated and converted into linear polarized light by a polarizer, and then incident on the first polarization modulator to modulate the polarization direction; The modulated light beam is split into linear polarized light in the x-direction and the y-direction by the polarization beam splitter; the linear polarized light in the y-direction is converted into radial polarized light by the vortex half-wave plate and then passes through the annular aperture; the linear polarized light in the x-direction is incident on the second polarization modulator to modulate the polarization direction, and the modulated light beam is converted into a vortex light beam by the vortex phase plate; The two beams of light are combined by a beam splitter and enter the confocal microscope. The light beams are focused on the sample by a large numerical aperture objective lens. The reflected or scattered photons pass through the analyzer and are focused by the lens on a small hole conjugate with the sample and received by the detector. The scanning mechanism is controlled to scan the light spot on the sample surface to obtain a microscopic image. The inputs of the first polarization modulator and the second polarization modulator are adjusted to obtain microscopic images under different polarization and illumination modes, and a super-resolution microscopic image is obtained by a post-processing algorithm.

2. The super-resolution microscopic imaging method based on multiple polarization and illumination modes according to claim 1, characterized in that: The principle of polarization modulation is as follows: the intensity ratio of the two beams of light separated by the polarization beam splitter depends on the input of the first polarization modulator; among them, after the linear polarized light in the y direction passes through the vortex half-wave plate and the annular aperture, the light beam passes through the large numerical aperture objective lens to form a focused light spot whose polarization is mainly in the z direction; the linear polarized light in the x direction has two polarization components in the x and y directions after passing through the second polarization modulator, and the intensity ratio of the two polarization components depends on the input of the second polarization modulator, and the polarization of the focused light spot formed by the polarized light in the two directions passing through the large numerical aperture objective lens is mainly in the x and y directions respectively; therefore, by adjusting the inputs of the first polarization modulator and the second polarization modulator, focused light spots with different polarization directions are obtained.

3. The super-resolution microscopic imaging method based on multiple polarization and illumination modes according to claim 1, characterized in that: The working process of the polarization modulator is as follows: a periodic voltage signal is input to the first polarization modulator and the second polarization modulator. Within one cycle, the polarization state of the light beam is gradually converted from x polarization to y polarization, and then from y polarization to x polarization. The repetition frequency of the second polarization modulator is much higher than the repetition frequency of the first polarization modulator. At the same time, the detector collects light intensity. Whenever the voltage of the first polarization modulator passes through a cycle, an intensity under the multi-polarization and illumination mode at a corresponding spatial position is obtained. The scanning mechanism focuses the light beam at the next spatial position and collects signals at the next spatial position. When the signal collection of all spatial positions is completed, an image of the multi-polarization and illumination mode is obtained.

4. The super-resolution microscopic imaging method based on multiple polarization and illumination modes according to claim 1, characterized in that: The principle of illumination mode conversion is as follows: the intensity ratio of the two beams of light separated by the polarization beam splitter depends on the input of the first polarization modulator; wherein, after the linear polarized light in the y direction passes through the vortex half-wave plate and the annular aperture, the focusing spot formed by the light beam passing through the large numerical aperture objective lens is a solid spot; the linear polarized light in the x direction has two polarization components in the x and y directions after passing through the second polarization modulator, the polarized light in the x direction passes through the vortex phase plate, and the focusing spot formed by the light beam passing through the large numerical aperture objective lens is an elliptical hollow spot, and the major axis of the ellipse is along the x direction; the polarized light in the y direction passes through the vortex phase plate, and the focusing spot formed by the light beam passing through the large numerical aperture objective lens is an elliptical hollow spot, and the major axis of the ellipse is along the y direction; therefore, adjusting the inputs of the first polarization modulator and the second polarization modulator can realize the conversion of the illumination mode.

5. The super-resolution microscopic imaging method based on multiple polarization and illumination modes according to claim 1, characterized in that: The orientation of the vortex half-wave plate is adjusted to convert the linear polarization in the y direction into radial polarization.

6. A super-resolution microscopic imaging device based on multiple polarization and illumination modes, characterized in that: The device comprises a laser, a polarizer, two polarization modulators, a polarization beam splitter, a vortex half-wave plate, an annular aperture, a vortex phase plate, a beam splitter and a scanning imaging module; the scanning imaging module comprises a scanning mechanism, a tube lens, a large numerical aperture objective lens, a lens, a pinhole and a detector; The light beam emitted by the laser is collimated and converted into linear polarized light by the polarizer, and incident on the first polarization modulator to modulate the polarization direction; The modulated light beam is split into linear polarized light in the x-direction and the y-direction by the polarization beam splitter; the linear polarized light in the y-direction passes through the vortex half-wave plate and the annular aperture in sequence; the linear polarized light in the x-direction passes through the second polarization modulator and the vortex phase plate in sequence; the two beams are combined by the beam splitter and enter the scanning imaging module; The scanning mechanism causes the focused light spot formed by the tube lens and the large numerical aperture objective lens to scan on the sample surface; the reflected light and scattered light on the sample surface are focused on the small hole conjugate with the sample through the lens, and are received by the detector to obtain a microscopic image; The inputs of the first polarization modulator and the second polarization modulator are adjusted to obtain microscopic images under different polarization and illumination modes.

7. The super-resolution microscopic imaging device based on multiple polarization and illumination modes according to claim 6, characterized in that: A D-shaped mirror is used to deflect the light path before the light beam enters the first polarization modulator.

8. The super-resolution microscopic imaging device based on multiple polarization and illumination modes according to claim 6, characterized in that: The scanning mechanism is a scanning galvanometer, and the midpoint of the line connecting the centers of the two galvanometers is conjugate with the entrance pupil of the objective lens.

9. The super-resolution microscopic imaging device based on multiple polarization and illumination modes according to claim 6, characterized in that: The polarization modulator is realized by an electro-optic modulator or a half-wave plate driven by a motor.

10. The super-resolution microscopic imaging device based on multiple polarization and illumination modes according to claim 6, characterized in that: The reflected light and scattered light of the sample are focused on the small hole, which is used to eliminate the out-of-focus stray light and is achieved by using a pinhole or multimode optical fiber.

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