Two-photon imaging system and method based on vector light field regulation and control

Abstract

The invention discloses a two-photon imaging system and method based on vector light field regulation and control, and belongs to the technical field of microscopic imaging. The two-photon imaging system comprises a laser beam expanding module, a vector light field modulation module, a light beam scanning module and an imaging module which are arranged in sequence; the laser beam expanding module comprises a laser, a first half-wave plate, a first polarization beam splitter, a first lens and a second lens which are arranged in sequence; a laser beam generated by the laser passes through the first half-wave plate and the first polarization beam splitter for laser power adjustment, and then passes through the first lens and the second lens for beam diameter adjustment. According to the two-photon imaging system based on vector light field regulation and control, the focal field is regulated and controlled by regulating and controlling the amplitude, the phase and the polarization of the vector light field, and the imaging resolution or the volume imaging speed of the system can be improved.

Description

Technical Field

[0001] The present invention relates to the field of microscopic imaging technology, and in particular to a two-photon imaging system and method based on vector light field regulation. Background Art

[0002] Two-photon microscopy is a nonlinear optical imaging technology that uses the nonlinear effect of matter under the action of strong lasers to obtain high-resolution, high-contrast images by detecting the two-photon signal generated by the sample. Unlike single-photon excitation fluorescence microscopy, two-photon excitation fluorescence requires the simultaneous absorption of two photons to excite fluorescent molecules. The technology that uses this nonlinear process for microscopic imaging is called two-photon excitation fluorescence microscopy. This microscopy technology has unique advantages. It can deeply reveal the microscopic structure and functional information inside the sample without damaging the biological sample, and has important applications in the biological field.

[0003] The excitation beam of a traditional two-photon microscope is generally linearly polarized, and its focus is diffraction-limited, which limits the improvement of lateral resolution. In the process of three-dimensional volume imaging, scanning in the Z direction is required, which greatly reduces the speed of volume imaging. Summary of the invention

[0004] The purpose of the present invention is to provide a two-photon imaging system and method based on vector light field control to solve the problems mentioned in the background technology.

[0005] To achieve the above-mentioned purpose, the present invention provides a two-photon imaging system based on vector light field control, comprising a laser beam expansion module, a vector light field modulation module, a beam scanning module and an imaging module which are arranged in sequence; The laser beam expansion module includes a laser, a first half-wave plate, a first polarization beam splitter, a first lens, and a second lens which are arranged in sequence; the laser beam generated by the laser passes through the first half-wave plate and the first polarization beam splitter to adjust the laser power, and then passes through the first lens and the second lens to adjust the beam diameter.

[0006] Preferably, the vector light field modulation module includes a second half-wave plate, a second polarization beam splitter, a first reflector, a third half-wave plate, a spatial light modulator, a second reflector, a third reflector, a first translation stage, a fourth reflector, a fifth reflector, a second translation stage, a fourth half-wave plate, a sixth reflector, a third polarization beam splitter and a quarter-wave plate; the third reflector is arranged on the first translation stage, and the fifth reflector is arranged on the second translation stage; The laser beam passes through the second half-wave plate to adjust the polarization direction, and then passes through the second polarization beam splitter to be divided into two beams of light, one of which passes through the first reflector and the third half-wave plate in sequence, passes through the spatial light modulator for phase modulation, and then passes through the fourth reflector and the fifth reflector for beam adjustment, and the other beam passes through the second reflector and the third reflector, passes through the spatial light modulator for phase modulation, and then passes through the fourth half-wave plate for beam conversion and is reflected by the sixth reflector. Finally, both beams of light pass through the third polarization beam splitter for beam combination, and then are converted into left-handed circularly polarized beams and right-handed circularly polarized beams by the quarter-wave plate.

[0007] Preferably, the beam scanning module includes a third lens, a fourth lens, an XY scanning galvanometer, a scanning lens and a sleeve lens; the laser beam is transmitted to the XY scanning galvanometer through the third lens and the fourth lens, and the laser beam is scanned point by point in the XY plane through the XY scanning galvanometer, and finally the laser beam passes through the scanning lens and the sleeve lens in sequence.

[0008] Preferably, the imaging module includes a dichroic mirror, an objective lens, a sample and a photomultiplier tube; a laser beam is transmitted through the dichroic mirror as excitation light, and then the excitation light is focused onto the sample through the objective lens to generate signal light, the signal light is reflected to the photomultiplier tube through the dichroic mirror, and then the signal light is collected by the photomultiplier tube.

[0009] Preferably, the second reflector and the fourth reflector are both right-angle prism reflectors, and the third reflector and the fifth reflector are both roof prism reflectors.

[0010] Preferably, the angle between the fast axis of the second half-wave plate and the horizontal direction is 22.5°, and the angle between the fast axis of the third half-wave plate and the vertical direction is 45°.

[0011] Preferably, the angle between the fourth half-wave plate and the horizontal direction is 45°, and the angle between the quarter-wave plate and the horizontal direction is 45°.

[0012] Preferably, the spatial light modulator is located at the back focal length of the second lens and at the front focal length of the third lens.

[0013] Preferably, the XY scanning galvanometer is located at the back focal length of the fourth lens and at the front focal length of the scanning lens.

[0014] Preferably, the second half-wave plate is used to adjust the polarization direction of the light beam and can be replaced by two polarizers. The second half-wave plate and the second polarization beam splitter are used to adjust the ratio of the two beams of light and can be replaced by a combination of a beam splitter and an attenuation plate. The spatial light modulator is used to add a phase to the light beam, which can be replaced by a digital micromirror device or a specially processed phase plate. The second reflector and the third reflector are used to reflect the light beam and adjust the optical path, and can be replaced by ordinary reflectors. The XY scanning galvanometer is used to perform point-by-point scanning in the XY plane by adjusting the reflection angle, and can be replaced by a spatial light modulator or a digital micromirror device. The third polarization beam splitter is used to combine the two beams of light and can be replaced by a Wollaston prism or a semi-transparent and semi-reflective mirror. The vector light field modulation module is used to generate a radially polarized vector light field and can be replaced by a vortex wave plate.

[0015] The present invention provides a two-photon imaging method based on vector light field control, comprising the following steps: The laser generates a femtosecond laser beam, the laser power is adjusted by a first half-wave plate and a first polarization beam splitter, and then the beam is collimated and expanded by a first lens and a second lens, and the beam diameter is adjusted to match the spatial light modulator; The laser beam is polarized by the second half-wave plate and then split into X-polarized light X1 and Y-polarized light Y1 by the second polarization beam splitter. Y1 is reflected by the first reflector and then converted into an X-polarized beam X2 by the third half-wave plate; X1 passes through the second reflector and the third reflector, and through the second reflector, the third reflector and the first translation stage so that the optical paths of X1 and X2 reaching the spatial light modulator are equal; X1 and X2 are converted into X1' and X2' respectively through the spatial light modulator, X1' is converted into a Y polarized light beam Y2' through the fourth half-wave plate, and Y2' is reflected by the sixth reflector; X2' is adjusted through the fourth reflector and the fifth reflector to make the optical path of X2' and Y2' equal and the pulses completely overlap; X2' and Y2' are combined into one beam through the third polarization beam splitter; X2' and Y2' are converted into a left-handed circularly polarized beam and a right-handed circularly polarized beam by a quarter wave plate, and a target VOF is generated by the left-handed circularly polarized beam and the right-handed circularly polarized beam; The VOF is transmitted to the XY scanning galvanometer through the third lens and the fourth lens. The XY scanning galvanometer scans the XY plane point by point by adjusting the reflection angle, and then passes through the scanning lens and the sleeve lens in sequence; VOF is used as excitation light to transmit through a dichroic mirror, and then the excitation light is focused onto the sample through an objective lens to generate signal light. The signal light is reflected by the dichroic mirror to a photomultiplier tube, which collects the signal light.

[0016] Therefore, the present invention adopts the above-mentioned two-photon imaging system and method based on vector light field control, which has the following beneficial effects: (1) The present invention adds a vector light field (VOF) modulation module on the basis of the traditional two-photon microscope. The VOF is generated by beam splitting with a polarization beam splitter, adjusting the phase with a spatial light modulator (SLM), converting the polarization state with a quarter-wave plate and combining the beams with a polarization beam splitter. This can control the shape of the excitation light beam at the focus.

[0017] (2) The present invention uses a spatial light modulator to indirectly generate a rotary polarized vortex vector light field (APV-VOF). The APV-VOF can generate a smaller focus point, which directly improves the imaging resolution by improving the scanning accuracy of the two-photon microscope.

[0018] (3) The present invention uses SLM to indirectly generate a radially polarized vector light field (RP-VOF), performs binary multi-ring regulation on the RP-VOF to generate longitudinal light needles, and reduces the Z-direction scanning time of the two-photon microscope through the longitudinal light needles, thereby improving the three-dimensional volume imaging speed.

[0019] (4) The present invention utilizes a phase-type spatial light modulator to simultaneously modulate two light beams and control the amplitude, phase and polarization of the light field through coherent superposition, thereby generating a vector light field with arbitrary polarization and phase.

[0020] (5) The present invention can perform high-resolution imaging of living tissues and cells, and is particularly suitable for long-term in vivo research. It causes little light damage to living cells and tissues. In the field of neuroscience, it is used for imaging brain nerves and cerebral blood vessels. It can perform three-dimensional structural imaging of deep hippocampal neurons and high-speed dynamic real-time imaging of cerebral blood vessels.

[0021] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 is a schematic structural diagram of an imaging system according to an embodiment of the present invention; Figure 2 A schematic diagram of loading phase of a spatial light modulator according to an embodiment of the present invention; Reference numerals 1. Laser; 2. First half-wave plate; 3. First polarization beam splitter; 4. First lens; 5. Second lens; 6. Second half-wave plate; 7. Second polarization beam splitter; 8. First reflector; 9. Third half-wave plate; 10. Spatial light modulator; 11. Second reflector; 12. Third reflector; 13. First translation stage; 14. Fourth reflector; 15. Fifth reflector; 16. Second translation stage; 17. Fourth half-wave plate; 18. Sixth reflector; 19. Third polarization beam splitter; 20. Quarter-wave plate; 21. Third lens; 22. Fourth lens; 23. XY scanning galvanometer; 24. Scanning lens; 25. Sleeve lens; 26. Dichroic mirror; 27. Objective lens; 28. Sample; 29. ​​Photomultiplier tube. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. The components of the embodiments of the present invention generally described and shown in the drawings here can be arranged and designed in various different configurations. Therefore, the following detailed description of the embodiments of the present invention provided in the drawings is not intended to limit the scope of the claimed invention, but merely represents selected embodiments of the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0024] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.

[0025] Professional term explanation VOF (Vector Optical Field) is a vector light field, which means that different positions on the same wavefront have different polarization states at the same time.

[0026] Example like Figure 1 As shown, the present invention provides a two-photon imaging system based on vector light field regulation, including a laser beam expansion module, a vector light field (VOF) modulation module, a beam scanning module and an imaging module which are arranged in sequence.

[0027] The laser beam expansion module includes a laser 1, a first half-wave plate 2, a first polarization beam splitter 3, a first lens 4 and a second lens 5 which are arranged in sequence; the laser beam generated by the laser 1 first passes through the first half-wave plate 2 and the first polarization beam splitter 3 for laser power adjustment, and then passes through the first lens 4 and the second lens 5 for beam diameter adjustment.

[0028] The vector light field (VOF) modulation module is used to generate VOF with different polarizations and phases for modulating the excitation focus, and includes: a second half-wave plate 6, a second polarization beam splitter 7, a first reflector 8, a third half-wave plate 9, a spatial light modulator 10, a second reflector 11, a third reflector 12, a first translation stage 13, a fourth reflector 14, a fifth reflector 15, a second translation stage 16, a fourth half-wave plate 17, a sixth reflector 18, a third polarization beam splitter 19 and a quarter-wave plate 20. The third reflector 12 is arranged on the first translation stage 13, and the fifth reflector 15 is arranged on the second translation stage 16. The second reflector 11 and the fourth reflector 14 are both right-angle prism reflectors, and the third reflector 12 and the fifth reflector 15 are both roof prism reflectors. The third reflector 12 is arranged on the first translation stage 13, and the fifth reflector 15 is arranged on the second translation stage 16.

[0029] The laser beam passes through the second half-wave plate 6 to adjust the polarization direction, and then passes through the second polarization beam splitter 7 to be divided into two beams of light. One beam of light passes through the first reflector 8 and the third half-wave plate 9 in sequence, and then passes through the spatial light modulator 10 for phase modulation, and then passes through the fourth reflector 14 and the fifth reflector 15 for beam adjustment. The other beam of light passes through the second reflector 11 and the third reflector 12, and then passes through the spatial light modulator 10 for phase modulation. The loaded phase diagram is shown in FIG. Figure 2 As shown, the two beams are then converted through the fourth half-wave plate 17 and reflected by the sixth reflector 18. Finally, the two beams are combined through the third polarization beam splitter 19 and then converted into left-handed circularly polarized beams and right-handed circularly polarized beams through the quarter-wave plate 20.

[0030] The beam scanning module is used to drive the beam to scan the entire sample 28, including: a third lens 21, a fourth lens 22, an XY scanning galvanometer 23, a scanning lens 24 and a sleeve lens 25; the laser beam is transmitted to the XY scanning galvanometer 23 through the third lens 21 and the fourth lens 22, and the laser beam is scanned point by point in the XY plane through the XY scanning galvanometer 23, and finally the laser beam passes through the scanning lens 24 and the sleeve lens 25 in sequence.

[0031] The imaging module is used to collect sample signals, and includes: a dichroic mirror 26, an objective lens 27, a sample 28, and a photomultiplier tube 29. The laser beam is transmitted through the dichroic mirror 26 as the excitation light, and then the excitation light is focused on the sample 28 through the objective lens 27 to generate signal light, and the signal light is reflected by the dichroic mirror 26 to the photomultiplier tube 29, and then the signal light is collected by the photomultiplier tube 29.

[0032] The fast axis of the second half-wave plate 6 is at an angle of 22.5° to the horizontal direction, and the fast axis of the third half-wave plate 9 is at an angle of 45° to the vertical direction. The fourth half-wave plate 17 is at an angle of 45° to the horizontal direction. The quarter-wave plate 20 is at an angle of 45° to the horizontal direction. The spatial light modulator 10 is located at the front focal length of the second lens 5 and the front focal length of the third lens 21. The XY scanning galvanometer 23 is located at the back focal length of the fourth lens 22 and the front focal length of the scanning lens 24.

[0033] The functions of each device in each module are as follows: Laser beam expansion module: Laser 1 is used to provide a femtosecond laser illumination beam; the first half-wave plate 2 and the first polarization beam splitter 3 are used to adjust the laser power; the first lens 4 and the second lens 5 are used to collimate and expand the illumination beam, and the beam diameter can be adjusted to match the spatial light modulator 10.

[0034] VOF modulation module: the light beam passes through the second half-wave plate 6 to adjust the polarization direction; the second polarization beam splitter 7 divides the light beam into X-polarized light X1 and Y-polarized light Y1; Y1 is reflected by the first reflector 8 and is parallel to X1; Y1 is converted into X-polarized light beam X2 by the third half-wave plate 9; the second reflector 11, the third reflector 12 and the first displacement stage 13 are used to adjust the optical path so that the optical path of X1 and X2 to the spatial light modulator 10 is equal; the spatial light modulator 10 is pre-loaded with the designed vortex phase diagram to modulate the phases of the two light beams and convert the light beams X1' is changed to X1' and X2'; X1' passes through the fourth half-wave plate 17 and is converted into a Y-polarized light beam Y2'; X2' and Y2' are synthesized into a beam of light through the third polarization beam splitter 19; the fourth reflector 14, the fifth reflector 15 and the second translation stage 16 are used to make the optical path of X2' and Y2' equal and the pulses completely overlap when synthesized by the third polarization beam splitter 19; X2' and Y2' are converted into a left-handed circularly polarized light beam L and a right-handed circularly polarized light beam R through the quarter-wave plate 20, L and R are the basis vectors of the synthesized VOF, and finally the target VOF is generated.

[0035] Light beam scanning module: The third lens 21 and the fourth lens 22 transmit the light beam modulated by the spatial light modulator 10 to the XY scanning galvanometer 23; the XY scanning galvanometer 23 performs point-by-point scanning of the XY plane by adjusting the reflection angle; then the phase plane is transmitted to the entrance pupil of the objective lens 27 through the scanning lens 24 and the sleeve lens 25, and the light beam size is adjusted to the entrance pupil distance of the objective lens 27.

[0036] Imaging module: VOF is used as excitation light and is focused onto sample 28 through objective lens 27 to generate signal light; after the sample is excited, the excitation light and signal light are collected together by objective lens 27, and after passing through dichroic mirror 26, the excitation light passes through dichroic mirror 26, and the signal light is reflected by dichroic mirror 26, so as to separate the excitation light and the signal light; the signal light is reflected by dichroic mirror 26 to photomultiplier tube 29 and collected by photomultiplier tube 29. By using vector light field to excite sample 28, a two-photon fluorescence imaging system with higher spatial resolution and faster volume imaging speed is realized, which can stably obtain fast / high-resolution images of biological samples without labeling, and is beneficial to practical applications.

[0037] In addition to generating VOF, the imaging system can also generate other forms of excitation light sources, such as non-diffraction light, structured light, etc., by regulating the spatial light modulator 10, thereby forming an imaging system that is more convenient for switching the excitation light source.

[0038] This imaging system is the same as the traditional two-photon microscope, and both use ultrashort pulse laser to excite the sample. The traditional two-photon microscope is a linearly polarized beam, and this system uses the spatial light modulator 10 to control the polarization and phase of the excitation beam; the incident light of the second half-wave plate 6 is linearly polarized in the X direction, and the outgoing light of the second half-wave plate 6 is 45° linearly polarized. After being split by the second polarization beam splitter 7, the two beams have the same intensity and are orthogonal polarization states. ; The quarter wave plate 20 orthogonal basis vector Convert to When the spatial light modulator 10 is loaded with the radial polarization vector light field RP-VOF phase, the expressions of the output light beams of the spatial light modulator 10 are respectively ; ; Where E1 and E2 represent the electric field strength of the two outgoing light beams, respectively, and e is a natural constant. represents the imaginary unit, φ It represents the handedness angle in polar coordinate system at any position on the wave surface, and its range is , Indicates the polarization direction x The orthogonal basis vectors of .

[0039] At this time, the incident beam of the objective lens 27 is expressed as ; Where E represents the electric field intensity of the incident light beam of the objective lens, Indicates the polarization direction x and y A pair of orthogonal basis vectors is generated, and RP-VOF is generated. When the following two formulas are satisfied, the chiral polarized vortex vector light field APV-VOF is generated: ; .

[0040] The second half-wave plate 6 is used to adjust the polarization direction of the light beam, and it can be replaced by two polarizers; the second half-wave plate 6 and the second polarization beam splitter 7 are used to adjust the ratio of the two beams of light, and can be replaced by a combination of a beam splitter and an attenuation plate; the spatial light modulator 10 is used to add a phase to the light beam, and it can be replaced by a digital micromirror device (DMD) or a specially processed phase plate; the second reflector 11 and the third reflector 12 are used to reflect the light beam and adjust the optical path, and can be replaced by ordinary reflectors, etc.; the XY scanning galvanometer 23 is used to perform point-by-point scanning in the XY plane by adjusting the reflection angle, and can be replaced by a spatial light modulator or a DMD or other device; the third polarization beam splitter 19 is used to combine the two beams of light, and can be replaced by a Wollaston prism or a semi-transparent and semi-reflective mirror or other device; the VOF modulation module is used to generate a radially polarized vector light field (RP-VOF), and the whole can be replaced by a vortex wave plate.

[0041] The present invention also provides a two-photon imaging method based on vector light field control, comprising the following steps: The laser 1 generates a femtosecond laser beam, the laser power is adjusted by the first half-wave plate 2 and the first polarization beam splitter 3, and then the beam is collimated and expanded by the first lens 4 and the second lens 5, and the beam diameter is adjusted to match the spatial light modulator 10; The laser beam is polarized by the second half-wave plate 6 and then split into X-polarized light X1 and Y-polarized light Y1 by the second polarization beam splitter 7; Y1 is reflected by the first reflector 8 and then converted into an X-polarized light beam X2 by the third half-wave plate 9; X1 passes through the second reflecting mirror 11 and the third reflecting mirror 12, and the second reflecting mirror 11, the third reflecting mirror 12 and the first translation stage 13 so that the optical paths of X1 and X2 reaching the spatial light modulator 10 are equal; X1 and X2 are converted into X1' and X2' respectively through the spatial light modulator 10, X1' is converted into a Y polarized light beam Y2' through the fourth half-wave plate 17, and Y2' is reflected by the sixth reflector 18; X2' passes through the fourth reflector 14 and the fifth reflector 15 to adjust the light beam, so that the optical path of X2' and Y2' are equal and the pulses completely overlap; X2' and Y2' pass through the third polarization beam splitter 19 to combine into one beam; X2' and Y2' are converted into a left-handed circularly polarized beam and a right-handed circularly polarized beam by a quarter wave plate 20, and a target VOF is generated by the left-handed circularly polarized beam and the right-handed circularly polarized beam; The VOF is transmitted to the XY scanning galvanometer 23 through the third lens 21 and the fourth lens 22. The XY scanning galvanometer 23 scans the XY plane point by point by adjusting the reflection angle, and then passes through the scanning lens 24 and the sleeve lens 25 in sequence; The VOF is transmitted through the dichroic mirror 26 as the excitation light, and then the excitation light is focused on the sample 28 through the objective lens 27 to generate signal light. The signal light is reflected by the dichroic mirror 26 to the photomultiplier tube 29, and the photomultiplier tube 29 collects the signal light.

[0042] Therefore, the present invention adopts the above-mentioned two-photon imaging system and method based on vector light field control, which has higher spatial resolution and faster volume imaging speed than traditional two-photon fluorescence imaging systems. It can stably obtain fast / high-resolution images of biological samples without labeling, and the resolution can be improved by 20%, and the volume imaging speed can be increased by 3-5 times.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that they can still modify or replace the technical solution of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solution to deviate from the spirit and scope of the technical solution of the present invention.

Claims

1. A two-photon imaging system based on vector light field control, characterized in that: It comprises a laser beam expansion module, a vector light field modulation module, a beam scanning module and an imaging module which are arranged in sequence; the laser beam expansion module comprises a laser, a first half-wave plate, a first polarization beam splitter, a first lens and a second lens which are arranged in sequence; the laser beam generated by the laser passes through the first half-wave plate and the first polarization beam splitter to adjust the laser power, and then passes through the first lens and the second lens to adjust the beam diameter.

2. A two-photon imaging system based on vector light field control according to claim 1, characterized in that: The vector light field modulation module includes a second half-wave plate, a second polarization beam splitter, a first reflector, a third half-wave plate, a spatial light modulator, a second reflector, a third reflector, a first translation stage, a fourth reflector, a fifth reflector, a second translation stage, a fourth half-wave plate, a sixth reflector, a third polarization beam splitter and a quarter-wave plate; the third reflector is arranged on the first translation stage, and the fifth reflector is arranged on the second translation stage; The laser beam passes through the second half-wave plate to adjust the polarization direction, and then passes through the second polarization beam splitter to be divided into two beams of light, one of which passes through the first reflector and the third half-wave plate in sequence, passes through the spatial light modulator for phase modulation, and then passes through the fourth reflector and the fifth reflector for beam adjustment, and the other beam of light passes through the second reflector and the third reflector, passes through the spatial light modulator for phase modulation, and then passes through the fourth half-wave plate for beam conversion and is reflected by the sixth reflector. Finally, both beams of light pass through the third polarization beam splitter for beam combination, and then are converted into a left-handed circularly polarized beam and a right-handed circularly polarized beam by the quarter-wave plate.

3. The two-photon imaging system based on vector light field control according to claim 1, characterized in that: The beam scanning module includes a third lens, a fourth lens, an XY scanning galvanometer, a scanning lens and a sleeve lens; the laser beam is transmitted to the XY scanning galvanometer through the third lens and the fourth lens, and the laser beam is scanned point by point in the XY plane through the XY scanning galvanometer, and finally the laser beam passes through the scanning lens and the sleeve lens in sequence.

4. The two-photon imaging system based on vector light field control according to claim 1, characterized in that: The imaging module includes a dichroic mirror, an objective lens, a sample and a photomultiplier tube; a laser beam is transmitted through the dichroic mirror as excitation light, and then the excitation light is focused onto the sample through the objective lens to generate signal light, the signal light is reflected to the photomultiplier tube through the dichroic mirror, and then the signal light is collected by the photomultiplier tube.

5. The two-photon imaging system based on vector light field control according to claim 2, characterized in that: The second reflector and the fourth reflector are both right-angle prism reflectors, and the third reflector and the fifth reflector are both roof prism reflectors.

6. A two-photon imaging system based on vector light field control according to claim 2, characterized in that: The included angle between the fast axis of the second half-wave plate and the horizontal direction is 22.5°, and the included angle between the fast axis of the third half-wave plate and the vertical direction is 45°.

7. The two-photon imaging system based on vector light field control according to claim 2, characterized in that: The angle between the fourth half-wave plate and the horizontal direction is 45°, and the angle between the quarter-wave plate and the horizontal direction is 45°.

8. The two-photon imaging system based on vector light field control according to claim 2, characterized in that: The spatial light modulator is located at a back focal length of the second lens and at a front focal length of the third lens.

9. The two-photon imaging system based on vector light field control according to claim 3, characterized in that: The XY scanning galvanometer is located at the back focal length of the fourth lens and the front focal length of the scanning lens.

10. A two-photon imaging method based on vector light field control, applied to the two-photon imaging system based on vector light field control as claimed in claims 1-9, characterized in that: The following steps are involved: The laser generates a femtosecond laser beam, the laser power is adjusted by a first half-wave plate and a first polarization beam splitter, and then the beam is collimated and expanded by a first lens and a second lens, and the beam diameter is adjusted to match the spatial light modulator; The laser beam is polarized by the second half-wave plate and then split into X-polarized light X1 and Y-polarized light Y1 by the second polarization beam splitter. Y1 is reflected by the first reflector and then converted into an X-polarized beam X2 by the third half-wave plate; X1 passes through the second reflector and the third reflector, and through the second reflector, the third reflector and the first translation stage so that the optical paths of X1 and X2 reaching the spatial light modulator are equal; X1 and X2 are converted into X1' and X2' respectively through the spatial light modulator, X1' is converted into a Y polarized light beam Y2' through the fourth half-wave plate, and Y2' is reflected by the sixth reflector; X2' is adjusted through the fourth reflector and the fifth reflector to make the optical path of X2' and Y2' equal and the pulses completely overlap; X2' and Y2' are combined into one beam through the third polarization beam splitter; X2' and Y2' are converted into a left-handed circularly polarized beam and a right-handed circularly polarized beam by a quarter wave plate, and a target VOF is generated by the left-handed circularly polarized beam and the right-handed circularly polarized beam; The VOF is transmitted to the XY scanning galvanometer through the third lens and the fourth lens. The XY scanning galvanometer scans the XY plane point by point by adjusting the reflection angle, and then passes through the scanning lens and the sleeve lens in sequence; VOF is used as excitation light to transmit through a dichroic mirror, and then the excitation light is focused onto the sample through an objective lens to generate signal light. The signal light is reflected by the dichroic mirror to a photomultiplier tube, which collects the signal light.

Images