Multi-polarization mode two-photon microscopy for second harmonic polarization imaging analysis

Abstract

The application provides a multi-polarization mode two-photon microscope for second harmonic polarization imaging analysis, which comprises a femtosecond laser, an optical isolator, a first plane mirror, a second plane mirror, a polarizer, an electrically controlled rotating frame with a half-wave plate, a 4-bit electrically controlled rotating wheel, a second dichroic mirror (14), a first electrically controlled rotating frame with a polarizer (15), a first electrically controlled translation platform (16), a second filter (17), a first photomultiplier tube (18), a third dichroic mirror (19), a fourth dichroic mirror (20), a third filter (21), a second photomultiplier tube (22), a second electrically controlled rotating frame with a polarizer (23), a second electrically controlled translation platform (24), a fourth filter (25), and a third photomultiplier tube (26). The application can realize excitation light polarization, and can realize several uniform polarization excitations of electric field vectors parallel to an imaging plane, such as 360-degree rotation excitation of linearly polarized light, left-handed and right-handed circular polarization excitation, and elliptical polarization excitation.

Description

Technical Field

[0001] This invention relates to the field of microscopy, and in particular to a multi-polarization mode two-photon microscope for second harmonic polarization imaging analysis. Background Technology

[0002] Polarization-correlated multiphoton microscopy combines the features of multiphoton microscopy and polarization microscopy. It allows for the study of the nonlinear optical properties and polarization states of samples, providing additional information about sample structure and distribution. Polarization-correlated multiphoton microscopy has wide applications in various scientific fields: Biomedical research: Cell imaging: Polarization-correlated multiphoton microscopy can be used to observe and analyze the nonlinear optical properties of living cells, tissues, and biomarkers. It provides information about protein structure, cell membrane polarization properties, and organelles.

[0003] Collagen fiber research: This microscopy technique can be used to study the arrangement and structure of collagen fibers, which helps to understand the mechanical properties of tissues, especially in tissues such as skin, bone and blood vessels.

[0004] Materials Science: Nanomaterial characterization: Polarization-correlated multiphoton microscopy can be used to study the arrangement and orientation of nanostructures and nanoparticles, as well as their nonlinear optical properties. This is extremely useful for the development of nanotechnology and nanomaterials.

[0005] Liquid crystal research: Liquid crystals have specific polarization response characteristics, so polarization-correlated multiphoton microscopy can be used to study the structure and properties of liquid crystals, including applications such as liquid crystal displays and optical modulators.

[0006] Surface science and interface research: Solid-liquid interface: studies the interfacial phenomena between solids and liquids, including charge distribution, molecular adsorption, and dissolution kinetics.

[0007] Solid-gas interface: used to analyze the properties of the solid-gas interface, such as surface reactions, catalysis, and adsorption.

[0008] Optoelectronics and Optical Devices: Nonlinear optical device design: By observing polarization-dependent multiphoton signals, the design and performance of nonlinear optical devices, such as nonlinear optical crystals and optical gratings, can be optimized.

[0009] Environmental Science: Atmospheric research: It can be used to analyze the nonlinear optical properties of particulate matter in the atmosphere, thereby providing information about atmospheric pollutants.

[0010] Water and soil studies: used to study organic matter, minerals, and biological particles in water and soil samples.

[0011] Nanophotonics: In nanophotonics research, polarization-correlated multiphoton microscopy can be used to explore the optical properties of nanostructures, thereby helping to design and develop novel nano-optical devices.

[0012] Optical image processing and computational imaging: This microscopy technique can also be used for image processing and computational imaging to improve resolution and extract more information.

[0013] For example, in the field of medical imaging, there is a clear correlation between the intensity of the SHG signal and the orientation of collagen fibers. The SHG signal intensity is typically strongest when the polarization direction of the excitation laser is parallel to the orientation of the collagen fibers. This is because, in this case, the second nonlinear polarization of the collagen fibers has the greatest overlap with the polarization direction of the excitation light, thus maximizing the SHG signal.

[0014] By analyzing the polarization characteristics of SHG signals, information about the orientation of collagen fibers can be obtained. Specifically, the orientation of collagen fibers can be determined by rotating the polarization direction of the excitation laser and measuring the changes in the SHG signal. This method is widely used in histological and biomedical research to study the arrangement of collagen fibers, the orientation of fiber bundles, and the orderliness of fiber structure.

[0015] Existing technical solutions have limitations. Firstly, they offer a limited number of polarization modes, making it impossible to freely switch between multiple polarization states of the excitation source. Secondly, they cannot simultaneously perform polarization detection and analysis on both forward and backward SHG signals. Summary of the Invention

[0016] In view of this, the purpose of the present invention is to provide a multi-polarization mode two-photon microscope for second harmonic polarization imaging analysis, which realizes excitation light polarization and uniform polarization excitation with electric field vectors parallel to the imaging plane, including linearly polarized light 360-degree rotation excitation, left and right circular polarization excitation, and elliptical polarization excitation.

[0017] To achieve the above objectives, the present invention adopts the following technical solution: a multi-polarization mode two-photon microscope for second harmonic polarization imaging analysis, comprising a femtosecond laser (1), an optical isolator (2), a first plane mirror (3), a second plane mirror (4), a polarizer (5), an electrically controlled rotating frame with a half-wave plate (6), a 4-position electric rotating wheel (7), a first filter (8), a scanning galvanometer unit (9), a first dichroic mirror (10), an objective lens (11), a stage (12), a condenser lens (13), a second dichroic mirror (14), a first electrically controlled rotating frame with an analyzer (15), a first electrically controlled translation platform (16), a second filter (17), a first photomultiplier tube (18), a third dichroic mirror (19), a fourth dichroic mirror (20), a third filter (21), a second photomultiplier tube (22), a second electrically controlled rotating frame with an analyzer (23), a second electrically controlled translation platform (24), a fourth filter (25), and a third photomultiplier tube (26). A femtosecond laser (1) is used as an excitation source to excite SHG and two-photon fluorescence TPEF signals; The femtosecond laser beam emitted by the femtosecond laser (1) first passes through the optical isolator (2), the first plane mirror (3), and the second plane mirror (4); The first plane mirror (3) and the second plane mirror (4) are combined to adjust the light output from the laser into the imaging optical path; The polarizer (5) is used to purify the linearly polarized light output by the laser to obtain pure linearly polarized light in the horizontal direction. The electrically controlled rotating frame (6) with a half-wave plate has the initial position of the main shaft of the half-wave plate in the horizontal direction. The rotation direction of the main shaft of the half-wave plate can be continuously controlled by the stepper motor of the electrically controlled rotating frame, thereby realizing the control of the polarization direction of the linearly polarized light passing through the half-wave plate. When it is not necessary to change the polarization state of the transmitted light, the main shaft of the half-wave plate automatically resets to the initial horizontal direction. The four holes of the 4-position electric rotary wheel (7) are respectively: blank hole (7-1), S-wave plate (7-2), quarter-wave plate with fast axis at a positive 45 degrees to the horizontal direction (7-3), and quarter-wave plate with fast axis at a negative 45 degrees to the horizontal direction (7-4). By controlling the motor, the required hole can be rotated into the optical path. By cooperating with the electrically controlled rotating frame (6) with half-wave plate and the 4-position electric rotary wheel (7), the polarization state of the excited laser can be controlled. The first filter (8) has high transmittance for light waves in the excitation band and filters out stray light; The scanning galvanometer unit (9) converts the laser beam into a high-speed vibrating beam, enabling the laser to scan the sample at a specific frequency and trajectory; The first dichroic mirror (10) is used to project light waves through the excitation band and reflect light waves in the SHG and two-photon fluorescence bands; The objective lens (11) is used to focus the excitation light onto the sample and collect the reversed SHG and two-photon fluorescence signals to provide a magnified view of the sample; The stage (12) is used to place the specimen to be observed and to allow the specimen to move left and right and front and back. A condenser lens (13) is used to collect forward SHG signals for imaging; The second dichroic mirror (14) is used to reflect the SHG band for subsequent imaging and transmit the excitation light band to prevent the excitation light from damaging the photomultiplier tube. The first electrically controlled rotating frame (15) with a polarizer can rotate the polarizer by a stepper motor to collect SHG light wave signals with different polarization directions; The first electrically controlled translation platform (16) is used to control whether the first electrically controlled rotating frame (15) with an analyzer is placed in the imaging optical path; when it is necessary to analyze the polarization state of the SHG signal, the first electrically controlled rotating frame (15) with an analyzer is translated into the imaging optical path; when it is not necessary to analyze the polarization state of the subharmonic signal, the first electrically controlled rotating frame (15) with an analyzer is translated out of the imaging optical path. The second filter (17) has high transmittance for the SHG signal band and filters out stray light from other bands; The first photomultiplier tube (18) is used to receive photons of the forward SHG signal, convert the photons into electrons, multiply these electrons through secondary emission, and send the signal to the computer. After processing, the image is displayed on the computer monitor. The third dichroic mirror (19) reflects light waves in the SHG band and transmits light waves in the two-photon fluorescence band. The fourth dichroic mirror (20) reflects light waves in the two-photon fluorescence band and transmits light waves in the stray band; The third filter (21) is used for high light transmission in the two-photon fluorescence band and to filter out stray light in the stray band. The second photomultiplier tube (22) is used to receive photons of two-photon fluorescence signals, convert photons into electrons, multiply these electrons through secondary emission, and transmit the signal to the computer. After processing, the image is displayed on the computer monitor. The second electrically controlled rotating frame (23) with a polarizer can rotate the polarizer by a stepper motor to collect SHG light wave signals with different polarization directions; The second electrically controlled translation platform (24) is used to control whether the second electrically controlled rotating frame (23) with an analyzer is placed in the imaging optical path; when it is necessary to analyze the polarization state of the SHG signal, the second electrically controlled rotating frame (23) with an analyzer is translated into the imaging optical path; when it is not necessary to analyze the polarization state of the subharmonic signal, the second electrically controlled rotating frame (23) with an analyzer is translated out of the imaging optical path. The fourth filter (25) has high transmittance for the SHG signal band and filters out stray light from other bands; The third photomultiplier tube (26) is used to receive photons of the backward SHG signal, convert the photons into electrons, multiply these electrons through secondary emission, and send the signal to the computer. After processing, the image is displayed on the computer monitor.

[0018] In a preferred embodiment; linearly polarized light: at this time, the blank aperture (7-1) is rotated in the optical path, and the polarization direction of the linearly polarized light can be rotated by rotating the electrically controlled rotating frame (6) with a half-wave plate by a stepper motor; when the main axis of the half-wave plate is initially in the horizontal direction, the polarization state of the excited polarized light is linearly polarized light in the horizontal direction. When the main axis of the half-wave plate rotates by an angle θ, the polarization direction of the excited light rotates by 2θ; by controlling the rotation angle of the electrically controlled rotating frame (6) with a half-wave plate by a stepper motor, the polarization direction of the excitation light source can be controlled, and the excitation of linearly polarized light with arbitrary polarization direction can be realized.

[0019] In a preferred embodiment; radially polarized light: at this time, the S-wave plate (7-2) is rotated in the optical path, and the main axis of the half-wave plate of the electrically controlled rotating frame (6) with the half-wave plate is located in the initial horizontal direction.

[0020] In a preferred embodiment; circularly polarized light: at this time, the main axis of the half-wave plate of the electrically controlled rotating frame (6) with the half-wave plate is located in the initial horizontal direction; when the fast axis is at a positive 45 degrees with the horizontal direction of the quarter-wave plate (7-3) is rotated in the optical path, left-hand circularly polarized light excitation is achieved; when the fast axis is at a negative 45 degrees with the horizontal direction of the quarter-wave plate (7-3) is rotated in the optical path, right-hand circularly polarized light excitation is achieved.

[0021] In a preferred embodiment; Elliptically polarized light: At this time, a quarter-wave plate (7-3) with a fast axis at a positive 45 degrees to the horizontal direction is rotated in the optical path, and elliptically polarized light with different ellipticity is excited by controlling the rotation angle of the electrically controlled rotating frame (6) with a half-wave plate.

[0022] Compared with the prior art, the present invention has the following beneficial effects: (1) By controlling the polarization state of the excitation source, in addition to realizing the polarization of the excitation light, it is possible to achieve uniform polarization excitation with electric field vectors parallel to the imaging plane, such as linearly polarized light rotating 360 degrees, left and right circular polarization excitation, and elliptical polarization excitation. It is also possible to freely switch to vector non-uniform vector polarization light excitation with electric field vectors perpendicular to the imaging plane, and perform SHG imaging of the sample with different polarization state excitation light, thereby obtaining more comprehensive structural information of the sample.

[0023] (2) It can collect forward and backward SHG signals simultaneously and achieve selective collection of polarization states of forward and backward SHG signals, so as to realize the analysis of polarization state of forward and backward SHG signals of sample under different excitation polarization states. Attached Figure Description

[0024] Figure 1 This is a schematic diagram of a system according to a preferred embodiment of the present invention; Figure 2 This is a structural diagram of a 4-bit electric polarization control system according to a preferred embodiment of the present invention. Detailed Implementation

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0026] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0027] It should be noted that the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the exemplary implementations according to this application; as used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise; furthermore, it should be understood that when the terms “comprising” and / or “including” are used in this specification, they indicate the presence of features, steps, operations, devices, components and / or combinations thereof.

[0028] Multi-polarization mode two-photon microscope for second harmonic polarization imaging analysis, reference Figure 1-2 The components include a femtosecond laser (1), an optical isolator (2), a first plane mirror (3), a second plane mirror (4), a polarizer (5), an electrically controlled rotating frame with a half-wave plate (6), a 4-position electric rotating wheel (7), a first filter (8), a scanning galvanometer unit (9), a first dichroic mirror (10), an objective lens (11), a stage (12), a condenser lens (13), a second dichroic mirror (14), a first electrically controlled rotating frame with an analyzer (15), a first electrically controlled translation platform (16), a second filter (17), a first photomultiplier tube (18), a third dichroic mirror (19), a fourth dichroic mirror (20), a third filter (21), a second photomultiplier tube (22), a second electrically controlled rotating frame with an analyzer (23), a second electrically controlled translation platform (24), a fourth filter (25), and a third photomultiplier tube (26). A femtosecond laser (1) is used as an excitation source to excite SHG (SHG) and two-photon fluorescence (TPEF) signals.

[0029] The femtosecond laser beam emitted by the femtosecond laser first passes through the optical isolator (2), the first plane mirror (3), and the second plane mirror (4). The first plane mirror (3) and the second plane mirror (4) are combined to adjust the light output from the laser into the imaging optical path.

[0030] The polarizer (5) is used to purify the linearly polarized light output by the laser to obtain pure linearly polarized light in the horizontal direction.

[0031] An electrically controlled rotating frame (6) with a half-wave plate has the initial position of the half-wave plate's spindle in the horizontal direction. The rotation direction of the half-wave plate's spindle can be continuously controlled by the stepper motor of the electrically controlled rotating frame, thereby controlling the polarization direction of the linearly polarized light passing through the half-wave plate. When it is not necessary to change the polarization state of the transmitted light, the spindle of the half-wave plate automatically returns to its initial horizontal position.

[0032] The four holes of the 4-position electric rotary wheel (7) are respectively: a blank hole (7-1), an S-wave plate (7-2), a quarter-wave plate with its fast axis at a positive 45-degree angle to the horizontal (7-3), and a quarter-wave plate with its fast axis at a negative 45-degree angle to the horizontal (7-4). By controlling the motor, the required hole can be rotated into the optical path. Through the cooperation of the electrically controlled rotating frame (6) with half-wave plates and the 4-position electric rotary wheel (7), the polarization state of the excited laser can be controlled.

[0033] The first filter (8) has high transmittance for light waves in the excitation band and filters out stray light.

[0034] The scanning galvanometer unit (9) converts the laser beam into a high-speed vibrating beam, enabling the laser to scan the sample at a specific frequency and trajectory.

[0035] The first dichroic mirror (10) is used to project light waves through the excitation band and reflect light waves in the SHG and two-photon fluorescence bands.

[0036] The objective lens (11) is used to focus the excitation light onto the sample and collect the reversed SHG and two-photon fluorescence signals to provide a magnified view of the sample.

[0037] The stage (12) is used to place the specimen to be observed and to allow the specimen to move in the left-right and front-back directions.

[0038] A condenser lens (13) is used to collect forward SHG signals for imaging.

[0039] The second dichroic mirror (14) is used to reflect the SHG band for subsequent imaging and transmit the excitation light band to prevent the excitation light from damaging the photomultiplier tube.

[0040] The first electrically controlled rotating frame (15) with an analyzer can collect SHG light wave signals with different polarization directions by rotating the analyzer through a stepper motor.

[0041] The first electrically controlled translation platform (16) is used to control whether the first electrically controlled rotating frame (15) with an analyzer is placed in the imaging optical path. When it is necessary to analyze the polarization state of the SHG signal, the first electrically controlled rotating frame (15) with an analyzer is translated into the imaging optical path. When it is not necessary to analyze the polarization state of the subharmonic signal, the first electrically controlled rotating frame (15) with an analyzer is translated out of the imaging optical path.

[0042] The second filter (17) has high transmittance for the SHG signal band and filters out stray light from other bands.

[0043] The first photomultiplier tube (18) is used to receive photons of the forward SHG signal, convert the photons into electrons, multiply these electrons through secondary emission, and send the signal to the computer. After processing, the image is displayed on the computer monitor.

[0044] The third dichroic mirror (19) reflects light waves in the SHG band and transmits light waves in the two-photon fluorescence band.

[0045] The fourth dichroic mirror (20) reflects light waves in the two-photon fluorescence band and transmits light waves in the stray band.

[0046] The filter (21) is used for high light transmission in the two-photon fluorescence band and to filter out light in the stray band.

[0047] The photomultiplier tube (22) is used to receive photons of two-photon fluorescence signals, convert photons into electrons, multiply these electrons through secondary emission, and transmit the signal to the computer. After processing, the image is displayed on the computer monitor.

[0048] The electrically controlled rotating frame (23) with an analyzer can rotate the analyzer by a stepper motor to collect SHG light wave signals with different polarization directions.

[0049] The electrically controlled translation platform (24) is used to control whether the second electrically controlled rotating frame (23) with an analyzer is placed in the imaging optical path. When it is necessary to analyze the polarization state of the SHG signal, the second electrically controlled rotating frame (23) with an analyzer is translated into the imaging optical path. When it is not necessary to analyze the polarization state of the subharmonic signal, the second electrically controlled rotating frame (23) with an analyzer is translated out of the imaging optical path.

[0050] The filter (25) has high transmittance for the SHG signal band and filters out stray light from other bands.

[0051] The photomultiplier tube (26) is used to receive photons of the backward SHG signal, convert the photons into electrons, multiply these electrons through secondary emission, and send the signal to the computer. After processing, the image is displayed on the computer monitor.

[0052] The polarization state of the excited laser has the following modes: (a) Linearly polarized light: At this time, the blank aperture (7-1) is rotated in the optical path. By rotating the electrically controlled rotating frame (6) with a half-wave plate by a stepper motor, the polarization direction of the linearly polarized light can be rotated. When the main axis of the half-wave plate is initially in the horizontal direction, the polarization state of the excited polarized light is linearly polarized light in the horizontal direction. When the main axis of the half-wave plate rotates by an angle θ, the polarization direction of the excited light rotates by 2θ. Therefore, by controlling the rotation angle of the electrically controlled rotating frame (6) with a half-wave plate by a stepper motor, the polarization direction of the excitation source can be controlled, and the excitation of linearly polarized light with arbitrary polarization direction can be realized.

[0053] (b) Radial polarized light: At this time, the S-wave plate (7-2) is rotated in the optical path, and the main axis of the half-wave plate of the electrically controlled rotating frame (6) with the half-wave plate is located in the initial horizontal direction.

[0054] (c) Circularly polarized light: At this time, the main axis of the half-wave plate of the electrically controlled rotating frame (6) with the half-wave plate is located in the initial horizontal direction. When the quarter-wave plate (7-3) with the fast axis at a positive 45 degrees to the horizontal direction is rotated in the optical path, left-hand circularly polarized light excitation is achieved. When the quarter-wave plate (7-3) with the fast axis at a negative 45 degrees to the horizontal direction is rotated in the optical path, right-hand circularly polarized light excitation is achieved.

[0055] (d) Elliptically polarized light: At this time, a quarter-wave plate (7-3) with a fast axis at a positive 45 degrees to the horizontal direction is rotated in the optical path. Elliptically polarized light with different ellipticity is excited by controlling the rotation angle of the electrically controlled rotating frame (6) with a half-wave plate.

[0056] Specific examples: The laser source uses a Chameleon Ultra automode-locked Ti:sapphire femtosecond laser from Coherent, with the excitation wavelength set at 810 nm to achieve the optimal SHG signal of collagen.

[0057] The polarizer (5) can be a polarizing beam splitter with a working wavelength range of 620-1000 nm.

[0058] An electrically controlled rotating frame (6) with a half-wave plate, wherein the half-wave plate is selected to operate at a wavelength of 325-1100 nm.

[0059] The central working wavelength of the S-wave plate (7-2) is 810 nm. The working wavelengths of the quarter-wave plate (7-3) with its fast axis at a positive 45 degrees to the horizontal direction and the quarter-wave plate (7-4) with its fast axis at a negative 45 degrees to the horizontal direction are 325-1100 nm.

[0060] The first filter (8) is a bandpass filter with a working center wavelength of 810 nm and a bandpass range of 10 nm.

[0061] The second filter (17) is a bandpass filter with a working center wavelength of 405 nm and a bandpass range of 20 nm.

[0062] The fourth filter (25) is a bandpass filter with a working center wavelength of 405 nm and a bandpass range of 20 nm.

[0063] The third filter (21) is a bandpass filter with a working center wavelength of 580 nm and a bandpass range of 110 nm.

[0064] The first dichroic mirror (10) is a long-pass dichroic mirror with a starting wavelength of 730 nm.

[0065] The second dichroic mirror (14) is a long-pass dichroic mirror with a starting wavelength of 425 nm.

[0066] The third dichroic mirror (19) is a long-pass dichroic mirror with a starting wavelength of 425 nm.

[0067] The fourth dichroic mirror (20) is a long-pass dichroic mirror with a starting wavelength of 730 nm.

[0068] The polarizers in the first electrically controlled rotating frame (15) with polarizer and the second electrically controlled rotating frame (23) with polarizer are ultra-wideband linear grid polarizers with a working wavelength range of 250nm - 4µm.

[0069] The first photomultiplier tube (18), the second photomultiplier tube (22), and the third photomultiplier tube (26) are GaAsP photomultiplier tubes with a detection spectral range of 300 - 720 nm.

[0070] When imaging tissue samples, two-photon fluorescence signals with wavelengths ranging from 480 to 695 nm and SHG signals with wavelengths ranging from 395 to 415 nm can be obtained.

[0071] By combining and adjusting the electrically controlled rotating frame (6) with a half-wave plate and the 4-position electric rotating wheel (7), radially polarized light, left-handed circularly polarized light, right-handed polarized light, elliptically polarized light with arbitrary length-to-short axis ratio, and linearly polarized light with arbitrary polarization direction can be used to excite the sample. Then, the forward SHG signal collected by the first photomultiplier tube (18) can be acquired by combining the control of the first electrically controlled rotating frame (15) with an analyzer and the first electrically controlled translation platform (16) with polarization direction. The backward SHG signal collected by the third photomultiplier tube (26) can be acquired by combining the control of the second electrically controlled rotating frame (23) with an analyzer and the second electrically controlled translation platform (24) with polarization direction. The two-photon fluorescence signal collected by the second photomultiplier tube (22) can also be acquired.

[0072] This invention achieves automatic control of the polarization state of the excitation source of a multiphoton microscope by using an electrically controlled rotating frame (6) with a half-wave plate and a 4-position electric rotating wheel (7), and by placing blank holes (7-1), an S-wave plate (7-2), a quarter-wave plate with a fast axis at a positive 45 degrees to the horizontal direction (7-3), and a quarter-wave plate with a fast axis at a negative 45 degrees to the horizontal direction (7-4) in the 4-position electric rotating wheel (7). It can realize the excitation of radially polarized light, left-handed circularly polarized light, right-handed polarized light, elliptically polarized light with arbitrary length-to-short axis ratio, and linearly polarized light with arbitrary polarization direction.

[0073] By controlling the polarization state of the excitation source, in addition to achieving excitation light polarization, it is possible to achieve several uniform polarization excitations with electric field vectors parallel to the imaging plane, such as linearly polarized light rotating 360 degrees, left- and right-hand circular polarization excitation, and elliptical polarization excitation. It is also possible to freely switch to vector non-uniform vector polarization excitation with electric field vectors perpendicular to the imaging plane, and to perform SHG imaging of the sample with excitation light of different polarization states, thereby obtaining more comprehensive structural information of the sample.

[0074] It can simultaneously collect forward and backward SHG signals and achieve selective collection of the polarization states of forward and backward SHG signals, enabling analysis of the polarization state of forward and backward SHG signals of samples under different excitation polarization states.

Claims

1. A multi-polarization mode two-photon microscope for second harmonic polarization imaging analysis, characterized in that, Includes a femtosecond laser (1), an optical isolator (2), a first plane mirror (3), a second plane mirror (4), a polarizer (5), an electrically controlled rotating frame with a half-wave plate (6), a 4-position electric rotating wheel (7), a first filter (8), a scanning galvanometer unit (9), a first dichroic mirror (10), an objective lens (11), a stage (12), a condenser lens (13), a second dichroic mirror (14), a first electrically controlled rotating frame with an analyzer (15), a first electrically controlled translation platform (16), a second filter (17), a first photomultiplier tube (18), a third dichroic mirror (19), a fourth dichroic mirror (20), a third filter (21), a second photomultiplier tube (22), a second electrically controlled rotating frame with an analyzer (23), a second electrically controlled translation platform (24), a fourth filter (25), and a third photomultiplier tube (26). A femtosecond laser (1) is used as an excitation source to excite SHG and two-photon fluorescence TPEF signals; The femtosecond laser beam emitted by the femtosecond laser (1) first passes through the optical isolator (2), the first plane mirror (3), and the second plane mirror (4); The first plane mirror (3) and the second plane mirror (4) are combined to adjust the light output from the laser into the imaging optical path; The polarizer (5) is used to purify the linearly polarized light output by the laser to obtain pure linearly polarized light in the horizontal direction. The electrically controlled rotating frame (6) with a half-wave plate has the initial position of the main shaft of the half-wave plate in the horizontal direction. The rotation direction of the main shaft of the half-wave plate can be continuously controlled by the stepper motor of the electrically controlled rotating frame, thereby realizing the control of the polarization direction of the linearly polarized light passing through the half-wave plate. When it is not necessary to change the polarization state of the transmitted light, the main shaft of the half-wave plate automatically resets to the initial horizontal direction. The four holes of the 4-position electric rotary wheel (7) are respectively: blank hole (7-1), S-wave plate (7-2), quarter-wave plate with fast axis at a positive 45 degrees to the horizontal direction (7-3), and quarter-wave plate with fast axis at a negative 45 degrees to the horizontal direction (7-4). By controlling the motor, the required hole can be rotated into the optical path. By cooperating with the electrically controlled rotating frame (6) with half-wave plate and the 4-position electric rotary wheel (7), the polarization state of the excited laser can be controlled. The first filter (8) has high transmittance for light waves in the excitation band and filters out stray light; The scanning galvanometer unit (9) converts the laser beam into a high-speed vibrating beam, enabling the laser to scan the sample at a specific frequency and trajectory; The first dichroic mirror (10) is used to project light waves through the excitation band and reflect light waves in the SHG and two-photon fluorescence bands; The objective lens (11) is used to focus the excitation light onto the sample and collect the reversed SHG and two-photon fluorescence signals to provide a magnified view of the sample; The stage (12) is used to place the specimen to be observed and to allow the specimen to move in the left-right and front-back directions; A condenser lens (13) is used to collect forward SHG signals for imaging; The second dichroic mirror (14) is used to reflect the SHG band for subsequent imaging and transmit the excitation light band to prevent the excitation light from damaging the photomultiplier tube. The first electrically controlled rotating frame (15) with an analyzer can rotate the analyzer by a stepper motor to collect SHG light wave signals with different polarization directions; The first electrically controlled translation platform (16) is used to control whether the first electrically controlled rotating frame (15) with an analyzer is placed in the imaging optical path; when it is necessary to analyze the polarization state of the SHG signal, the first electrically controlled rotating frame (15) with an analyzer is translated into the imaging optical path; when it is not necessary to analyze the polarization state of the subharmonic signal, the first electrically controlled rotating frame (15) with an analyzer is translated out of the imaging optical path. The second filter (17) has high transmittance for the SHG signal band and filters out stray light from other bands; The first photomultiplier tube (18) is used to receive photons of the forward SHG signal, convert the photons into electrons, multiply these electrons through secondary emission, and send the signal to the computer. After processing, the image is displayed on the computer monitor. The third dichroic mirror (19) reflects light waves in the SHG band and transmits light waves in the two-photon fluorescence band. The fourth dichroic mirror (20) reflects light waves in the two-photon fluorescence band and transmits light waves in the stray band; The third filter (21) is used for high light transmission in the two-photon fluorescence band and to filter out stray light in the stray band. The second photomultiplier tube (22) is used to receive photons of two-photon fluorescence signals, convert photons into electrons, multiply these electrons through secondary emission, and transmit the signal to the computer. After processing, the image is displayed on the computer monitor. The second electrically controlled rotating frame (23) with a polarizer can rotate the polarizer by a stepper motor to collect SHG light wave signals with different polarization directions; The second electrically controlled translation platform (24) is used to control whether the second electrically controlled rotating frame (23) with an analyzer is placed in the imaging optical path; when it is necessary to analyze the polarization state of the SHG signal, the second electrically controlled rotating frame (23) with an analyzer is translated into the imaging optical path; when it is not necessary to analyze the polarization state of the subharmonic signal, the second electrically controlled rotating frame (23) with an analyzer is translated out of the imaging optical path. The fourth filter (25) has high transmittance for the SHG signal band and filters out stray light from other bands; The third photomultiplier tube (26) is used to receive photons of the backward SHG signal, convert the photons into electrons, multiply these electrons through secondary emission, and send the signal to the computer. After processing, the image is displayed on the computer monitor.

2. The multi-polarization mode two-photon microscope for second harmonic polarization imaging analysis according to claim 1, characterized in that; Linearly polarized light: At this time, the blank aperture (7-1) is rotated in the optical path. By rotating the electrically controlled rotating frame (6) with a half-wave plate by a stepper motor, the polarization direction of the linearly polarized light can be rotated. When the main axis of the half-wave plate is in the initial horizontal direction, the polarization state of the excited polarized light is linearly polarized light in the horizontal direction. When the main shaft of the half-wave plate rotates by an angle θ, the polarization direction of the excitation light rotates by 2θ. By controlling the rotation angle of the electrically controlled rotating frame (6) with the half-wave plate through a stepper motor, the polarization direction of the excitation light source can be adjusted, thereby realizing the excitation of linearly polarized light with arbitrary polarization direction.

3. The multi-polarization mode two-photon microscope for second harmonic polarization imaging analysis according to claim 1, Its characteristics are: Radial polarized light: At this time, the S-wave plate (7-2) is rotated in the optical path, and the main axis of the half-wave plate of the electrically controlled rotating frame (6) with the half-wave plate is located in the initial horizontal direction.

4. The multi-polarization mode two-photon microscope for second harmonic polarization imaging analysis according to claim 1, Its characteristics are: Circularly polarized light: At this time, the main axis of the half-wave plate of the electrically controlled rotating frame (6) with the half-wave plate is located in the initial horizontal direction; when the fast axis is at a positive 45 degrees with the horizontal direction, the quarter-wave plate (7-3) is rotated in the optical path to realize left-hand circularly polarized light excitation; when the fast axis is at a negative 45 degrees with the horizontal direction, the quarter-wave plate (7-3) is rotated in the optical path to realize right-hand circularly polarized light excitation.

5. The multi-polarization mode two-photon microscope for second harmonic polarization imaging analysis according to claim 1, Its characteristics are: Elliptically polarized light: At this time, a quarter-wave plate (7-3) with a fast axis at a positive 45 degrees to the horizontal direction is rotated in the optical path. Elliptically polarized light with different ellipticity is excited by controlling the rotation angle of the electrically controlled rotating frame (6) with a half-wave plate.

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