A time-division multiplexing synchronous multi-focal plane multiphoton microscopy imaging device and method

Abstract

The present invention discloses a time-division multiplexing synchronous multi-focal plane multi-photon microscopy imaging device and method, which relate to the field of biomedical research. The device comprises a laser light source, a first polarization beam splitter, a first lens pair, a first remote-controlled focusing group, a second lens pair, a third lens pair, a second remote-controlled focusing group, a fourth lens pair, a fourth polarization beam splitter, a third reflector, a fourth reflector, an electro-optical modulator, a signal generator, a fifth polarization beam splitter, a light block, a fifth reflector, a fifth lens pair, a scanning galvanometer, a scanning lens, a sixth reflector, a tube lens, a first dichroic mirror, a third objective lens, an electric three-dimensional translation stage, a second dichroic mirror, a first bandpass filter, a first photomultiplier tube, a second bandpass filter, a second photomultiplier tube, and a computer processing module. The device adopts a remote-controlled focusing method, reduces the spherical aberration caused by adjusting the axial position of the focal plane, and synchronously excites multiple focal planes, thereby realizing simultaneous detection of images with different focal depths.

Description

Technical Field

[0001] The present invention relates to the field of biomedical research, and in particular to a time-division multiplexing synchronous multi-focal plane multi-photon microscopy imaging device and method. Background Art

[0002] Since observing the microscopic world of biological tissues and cells allows us to discover and interpret the biological mechanisms involved in physiological and pathological phenomena, developing fast, efficient, and non-invasive diagnostic tools that can identify the status of biological samples has always been an important part of biomedical research. Since its invention, optical microscopy technology has provided strong support for observing the morphological and functional characteristics of living samples.

[0003] Currently, traditional optical microscopy relies on differences in certain optical properties of the sample to produce image contrast. This lacks chemical selectivity and specificity, and the biological information it provides is very limited, offering only qualitative estimates of cell morphology and growth. To obtain more details related to chemical composition, cell histopathology methods can be used to study biological samples, staining specific cellular structures. However, this approach can affect cell viability and damage tissue structure.

[0004] Therefore, a time-division multiplexing synchronous multi-focal plane multi-photon microscopic imaging device and method are provided to solve the above problems. Summary of the Invention

[0005] The purpose of the present invention is to provide a time-division multiplexing synchronous multi-focal plane multi-photon microscopic imaging device and method, which can achieve imaging results of multiple modes through a single imaging, and can effectively avoid signal crosstalk while achieving automatic image matching. The remote control focusing method is adopted to adjust the spherical aberration caused by the axial position of the focal plane to be small, and multiple focal planes are excited synchronously, thereby realizing the simultaneous detection of images with different focal depths.

[0006] To achieve the above-mentioned objectives, the present invention provides a time-division multiplexed synchronous multi-focal plane multi-photon microscopy imaging device, comprising a light source collection mechanism, a scanning mechanism and an imaging mechanism, wherein the light source collection mechanism is arranged on one side of the scanning mechanism, and the imaging mechanism is connected to the scanning mechanism through a signal line, the light source collection mechanism comprises a laser light source, a first polarization beam splitter, a fourth polarization beam splitter, a first remote control focusing group and a second remote control focusing group, the laser light source is arranged on one side of the first polarization beam splitter, a first lens pair is arranged between the first polarization beam splitter and the first remote control focusing group, a third lens pair is arranged between the first polarization beam splitter and the second remote control focusing group, a fourth lens pair is arranged between the second remote control focusing group and the fourth polarization beam splitter, a second lens pair is arranged between the fourth polarization beam splitter and the first remote control focusing group, and a third reflector is arranged on the other side of the fourth polarization beam splitter.

[0007] Preferably, the scanning mechanism includes an electro-optical modulator, a fifth polarization beam splitter, a scanning galvanometer, a sixth reflector and a third objective lens. A fourth reflector is provided on one side of the electro-optical modulator, and the fourth reflector is horizontally symmetrically arranged with the third reflector. The electro-optical modulator is connected to the signal generator through the signal line. The fifth polarization beam splitter is provided on the other side of the electro-optical modulator. A light block is provided on the side of the fifth polarization beam splitter away from the electro-optical modulator. A fifth reflector is provided directly below the fifth polarization beam splitter. A fifth lens pair is provided between the fifth reflector and the scanning galvanometer. A scanning lens is provided between the scanning galvanometer and the sixth reflector. A tube lens, a first dichroic mirror and the third objective lens are provided above the sixth reflector from bottom to top in sequence, and an electric three-dimensional translation stage is provided on the top of the third objective lens.

[0008] Preferably, the imaging mechanism includes a computer processing module, a first photomultiplier tube, and a second photomultiplier tube. The first photomultiplier tube and the second photomultiplier tube are both connected to the computer processing module through the signal line. The computer processing module is connected to the signal generator and the scanning galvanometer through the signal line. A first bandpass filter and a second dichroic mirror are arranged in sequence below the first photomultiplier tube, and a second bandpass filter is arranged between the second dichroic mirror and the second photomultiplier tube.

[0009] Preferably, the first polarization beam splitter, the third lens pair, the second remote control focusing group and the fourth lens pair are centrally symmetrically arranged with respect to the first lens pair, the first remote control focusing group, the second lens pair and the fourth polarization beam splitter.

[0010] Preferably, the fifth reflector and the sixth reflector are arranged symmetrically in a vertical direction, the sixth reflector and the first dichroic mirror are arranged parallel to each other, and the first dichroic mirror and the second dichroic mirror are arranged symmetrically in a vertical direction.

[0011] Preferably, the first remote control focusing group includes a second polarization beam splitter, a first quarter wave plate, a first objective lens and a first reflector, and the second polarization beam splitter, the first quarter wave plate, the first objective lens and the first reflector are arranged in sequence from left to right; the second remote control focusing group includes a third polarization beam splitter, a second quarter wave plate, a second objective lens and a second reflector, and the second reflector, the second objective lens, the second quarter wave plate and the third polarization beam splitter are arranged in sequence from left to right.

[0012] Preferably, the first lens pair includes a first lens and a second lens symmetrically arranged, the second lens pair includes a third lens and a fourth lens symmetrically arranged, the third lens pair includes a fifth lens and a sixth lens symmetrically arranged, the fourth lens pair includes a seventh lens and an eighth lens symmetrically arranged, and the fifth lens pair includes a ninth lens and a tenth lens symmetrically arranged.

[0013] A time-division multiplexing synchronous multi-focal plane multiphoton microscopy method comprises the following steps:

[0014] S1: The first polarization beam splitter splits the laser light emitted by the laser light source into two beams with orthogonal polarization directions;

[0015] S2: After the first light beam and the second light beam are respectively controlled by the first remote control focusing group and the second remote control focusing group, they are combined into mutually orthogonal light beams with different focal depths at the fourth polarization beam splitter;

[0016] S3: passes through the third reflector, the fourth reflector, the electro-optic modulator, the fifth polarization beam splitter and the light block;

[0017] S4: The light after passing through the fifth polarization beam splitter is transmitted to the detection sample on the motorized three-dimensional translation stage through the fifth reflector, the fifth lens, the sixth lens, the scanning galvanometer, the scanning lens, the sixth reflector, the tube lens, the first dichroic mirror, and the third objective lens;

[0018] S5: The sample detection light passes through the third objective lens, the first dichroic mirror, and the second dichroic mirror and is split into two beams of light with different wavelengths. One beam passes through the first filter and the first photomultiplier tube and is then transmitted to the computer processing module. The other beam passes through the second filter and the second photomultiplier tube and is then transmitted to the computer processing module.

[0019] S6: The computer processing module generates the pixel clock of the microscope as the external trigger signal of the signal generator, and its period is 4μs. After receiving the pixel clock signal, the signal generator generates a square wave signal with a period of 8μs and a duty cycle of 50% as the trigger signal of the electro-optical modulator. By fine-tuning the time delay of the electrical signal, the sample information of a certain focal plane is collected within the residence time of each pixel, thereby realizing pixel-level focal plane switching.

[0020] Therefore, the present invention adopts the above-mentioned time-division multiplexing synchronous multi-focal plane multiphoton microscopy imaging device and method, which has the following beneficial effects:

[0021] (1) The present invention adopts multi-focal plane synchronous excitation, eliminating the possibility of image mismatch caused by sample displacement or concentration change.

[0022] (2) The present invention adopts a remote control focusing method, and the spherical aberration caused by adjusting the axial position of the focal plane can be ignored.

[0023] (3) The present invention can eliminate signal crosstalk between focal planes without any prior knowledge or high-frequency phase-locked circuit.

[0024] (4) The pixel-level focal plane switching of the present invention realizes automatic demultiplexing, does not require complex demultiplexing circuits or algorithms, and does not require any internal modification of commercial microscopes, and thus has a wide range of application scenarios.

[0025] (5) The present invention can realize multi-mode multi-photon microscopy imaging.

[0026] The technical solution of the present invention is further described in detail below through the accompanying drawings and embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a schematic structural diagram of a time-division multiplexing synchronous multi-focal plane multiphoton microscopic imaging device of the present invention;

[0028] Figure 2 A schematic diagram of a reflective remote control focusing portion of a time-division multiplexing synchronous multi-focal plane multiphoton microscopy imaging device according to the present invention;

[0029] Figure 3 Schematic diagram of the light emitted from the fourth polarization beam splitter of a time-division multiplexed synchronous multi-focal plane multiphoton microscopic imaging device of the present invention;

[0030] Figure 4 Schematic diagram of the light emitted from the fifth polarization beam splitter of a time-division multiplexed synchronous multi-focal plane multiphoton microscopy imaging device of the present invention;

[0031] Figure 5 A schematic diagram of a pixel clock discovery in a time-division multiplexing synchronous multi-focal plane multiphoton microscopy imaging device of the present invention;

[0032] Figure 6 This is a schematic diagram of the signal generator output of a time-division multiplexing synchronous multi-focal plane multiphoton microscopy imaging device of the present invention;

[0033] Figure 7 This is a schematic diagram of focal plane scanning of a time-division multiplexing synchronous multi-focal plane multiphoton microscopy imaging device of the present invention;

[0034] Reference numerals

[0035] 1. Laser light source; 2. First polarization beam splitter; 3. First lens pair; 31. First lens; 32. Second lens; 4. First remote control focus group; 41. Second polarization beam splitter; 42. First quarter wave plate; 43. First objective lens; 44. First reflector; 5. Second lens pair; 51. Third lens; 52. Fourth lens; 6. Third lens pair; 61. Fifth lens; 62. Sixth lens; 7. Second remote control focus group; 71. Third polarization beam splitter; 72. Second quarter wave plate; 73. Second objective lens; 74. Second reflector; 8. Fourth lens pair; 81. Seventh lens; 82. Eighth lens; 9. Fourth polarization beam splitter Beam splitter; 10. third reflector; 11. fourth reflector; 12. electro-optic modulator; 13. signal generator; 14. fifth polarization beam splitter; 15. light block; 16. fifth reflector; 17. fifth lens pair; 171. ninth lens; 172. tenth lens; 18. scanning galvanometer; 19. scanning lens; 20. sixth reflector; 21. tube lens; 22. first dichroic mirror; 23. third objective lens; 24. electric three-dimensional translation stage; 25. second dichroic mirror; 26. first band-pass filter; 27. first photomultiplier tube; 28. second band-pass filter; 29. ​​second photomultiplier tube; 30. computer processing module. DETAILED DESCRIPTION

[0036] The technical solution of the present invention is further described below with reference to the accompanying drawings and embodiments.

[0037] Unless otherwise defined, the technical or scientific terms used in the present invention shall have the usual meanings understood by persons of ordinary skill in the field to which the present invention belongs. The words "first", "second" and similar terms used in the present invention do not indicate any order, quantity or importance, but are only used to distinguish different components. Words such as "include" or "comprise" mean that the elements or objects preceding the word include the elements or objects listed after the word and their equivalents, without excluding other elements or objects. Words such as "connect" or "connected" are not limited to physical or mechanical connections, but may include electrical connections, whether direct or indirect. "Up", "down", "left", "right" and the like are only used to indicate relative positional relationships. When the absolute position of the object being described changes, the relative positional relationship may also change accordingly.

[0038] In this embodiment, the present invention provides a time-division multiplexing synchronous multi-focal plane multiphoton microscopy imaging device, such as Figure 1-Figure 7As shown, it includes a light source collection mechanism, a scanning mechanism and an imaging mechanism. The light source collection mechanism is arranged on one side of the scanning mechanism, and the imaging mechanism is connected to the scanning mechanism through a signal line. The light source collection mechanism includes a laser light source 1, a first polarization beam splitter 2, a fourth polarization beam splitter 9, a first remote control focusing group 4 and a second remote control focusing group 7. The laser light source 1 is arranged on one side of the first polarization beam splitter 2, a first lens pair 3 is arranged between the first polarization beam splitter 2 and the first remote control focusing group 4, a third lens pair 6 is arranged between the first polarization beam splitter 2 and the second remote control focusing group 7, a fourth lens pair 8 is arranged between the second remote control focusing group 7 and the fourth polarization beam splitter 9, a second lens pair 5 is arranged between the fourth polarization beam splitter 9 and the first remote control focusing group 4, and a third reflector 10 is arranged on the other side of the fourth polarization beam splitter 9.

[0039] The scanning mechanism includes an electro-optical modulator 12, a fifth polarization beam splitter 14, a scanning galvanometer 18, a sixth reflector 20 and a third objective lens 23. A fourth reflector 11 is provided on one side of the electro-optical modulator 12, and the fourth reflector 11 is horizontally symmetrically arranged with the third reflector 10. The electro-optical modulator 12 is connected to the signal generator 13 through a signal line. A fifth polarization beam splitter 14 is provided on the other side of the electro-optical modulator 12. A light block 15 is provided on the side of the fifth polarization beam splitter 14 away from the electro-optical modulator 12. A fifth reflector 16 is provided directly below the fifth polarization beam splitter 14. A fifth lens pair 17 is provided between the fifth reflector 16 and the scanning galvanometer 18. A scanning lens 19 is provided between the scanning galvanometer 18 and the sixth reflector 20. Above the sixth reflector 20, a tube lens 21, a first dichroic mirror 22 and a third objective lens 23 are provided in sequence from bottom to top. An electric three-dimensional translation stage 24 is provided on the top of the third objective lens 23.

[0040] The electro-optic modulator 12 periodically deflects the light from the fourth polarization beam splitter 9, achieving optical information with two alternating focal planes in the same polarization direction. The first dichroic mirror 22 reflects the optical signal from the motorized three-dimensional translation stage 24 to the second dichroic mirror 25, separating the optical signals of different wavelengths. The first and second bandpass filters 26 and 28 respectively ensure that the optical signals of the corresponding wavelengths are not interfered with by stray light.

[0041] The imaging mechanism includes a computer processing module 30, a first photomultiplier tube 27, and a second photomultiplier tube 29. The first photomultiplier tube 27 and the second photomultiplier tube 29 are both connected to the computer processing module 30 via signal lines, and the computer processing module 30 is connected to the signal generator 13 and the scanning galvanometer 18 via signal lines. A first bandpass filter 26 and a second dichroic mirror 25 are arranged in sequence below the first photomultiplier tube 27, and a second bandpass filter 28 is arranged between the second dichroic mirror 25 and the second photomultiplier tube 29.

[0042] Computer processing module 30 receives signals from first and second photomultiplier tubes 27 and 29, respectively. The two photomultiplier tubes collect spectral information in different wavelength bands, enriching the scanned image information. The laser light source is a Ti:sapphire mode-locked pulsed laser with a central wavelength tuned to 870 nm, a repetition rate of approximately 80 MHz, and a pulse width at half maximum of approximately 100 fs. The laser light emitted by laser source 1 passes through first polarization beam splitter 2, converting it into two beams with mutually orthogonal polarization directions. First and second remote control focusing groups 4 and 7 control the focal spots of the two orthogonal polarized beams, thereby achieving simultaneous imaging of multiple focal planes.

[0043] The first polarization beam splitter 2, the third lens pair 6, the second remote focus group 7, and the fourth lens pair 8 are arranged symmetrically with the first lens pair 3, the first remote focus group 4, the second lens pair 5, and the fourth polarization beam splitter 9. The second lens pair 5 and the fifth lens pair 17 form two 4f optical systems, imaging the pupil of the first objective lens 43 onto the midplane of the scanning galvanometer 18. The fourth lens pair 8 and the fifth lens pair 17 form two 4f optical systems, imaging the pupil of the second objective lens 73 onto the midplane of the scanning galvanometer 18. The tube lens 21 collimates the light beam for output, and the scanning lens 19 and the tube lens 21 also form a 4f system, imaging the midplane of the scanning galvanometer 18 onto the pupil of the third objective lens 23. The scanning galvanometer 18 receives the polarized light separated by the fifth polarization beam splitter 14 and transmits it to the third objective lens 23.

[0044] The scanning mirror 18 is a reflector mounted on a galvanometer. A servo driver inputs electrical signals to the galvanometer to control the rotation of the scanning mirror 18, thereby changing the direction of the light beam to scan the focal spot across the focal plane. The scanning mirror 18 achieves progressive scanning of the focal plane through the synchronization of its two mirrors. The fast-axis mirror and the slow-axis mirror work together, receiving a sawtooth-like input signal to perform line scanning. After each line is scanned, the fast-axis mirror outputs return and turn signals to proceed to the next line. When these coordinated operations complete the preset number of scan lines, a frame of image acquisition is complete.

[0045] The fifth reflector 16 and the sixth reflector 20 are arranged symmetrically in the vertical direction. The sixth reflector 20 and the first dichroic mirror 22 are arranged parallel to each other. The first dichroic mirror 22 and the second dichroic mirror 25 are arranged symmetrically in the vertical direction.

[0046] The first remote control focusing group 4 includes a second polarization beam splitter 41, a first quarter wave plate 42, a first objective lens 43 and a first reflector 44, and the second polarization beam splitter 41, the first quarter wave plate 42, the first objective lens 43 and the first reflector 44 are arranged in sequence from left to right; the second remote control focusing group 7 includes a third polarization beam splitter 71, a second quarter wave plate 72, a second objective lens 73 and a second reflector 74, and the second reflector 74, the second objective lens 73, the second quarter wave plate 72 and the third polarization beam splitter 71 are arranged in sequence from left to right.

[0047] By controlling the distance between the first objective lens 43 and the first reflector 44, the degree of convergence and divergence of the light beam can be changed; since the light passes through the first quarter-wave plate 42 twice, the polarization direction of the light changes, so that the light can continue to propagate through the second polarization beam splitter 41; by controlling the distance between the second objective lens 73 and the second reflector 74, the degree of convergence and divergence of the light beam can be changed; and since the light passes through the second quarter-wave plate 72 twice, the polarization direction of the light changes, so that the reflected light can be reflected by the third polarization beam splitter 71.

[0048] The first lens pair 3 includes a symmetrically arranged first lens 31 and a second lens 32. The second lens pair 5 includes a symmetrically arranged third lens 51 and a fourth lens 52. The third lens pair 6 includes a symmetrically arranged fifth lens 61 and a symmetrically arranged sixth lens 62. The fourth lens pair 8 includes a symmetrically arranged seventh lens 81 and an eighth lens 82. The fifth lens pair 17 includes a symmetrically arranged ninth lens 171 and a tenth lens 172. The first lens pair 3 and the third lens pair 6 expand the beam diameter to accommodate the pupil aperture of the first objective lens 43 and the second objective lens 73. The fourth polarization beam splitter 9 combines the first and second beams.

[0049] The fifth polarization beam splitter 14 reflects perpendicularly polarized light, and through the coordination of a series of components, optical signals with different focal depths alternate. During imaging, the computer processing module 30 separates the two signal beams carrying microscopic information, enabling simultaneous imaging at different focal depths. After time-division multiplexing the two beams of light with different focal depths, the system applies the composite beam to the sample and completely separates the two optical signals through demultiplexing at the signal acquisition end, allowing simultaneous acquisition of two imaging results in a single imaging experiment.

[0050] A time-division multiplexing synchronous multi-focal plane multiphoton microscopy method comprises the following steps:

[0051] S1: The first polarization beam splitter splits the laser light emitted by the laser light source into two beams with orthogonal polarization directions;

[0052] S2: After the first light beam and the second light beam are respectively controlled by the first remote control focusing group and the second remote control focusing group, they are combined into mutually orthogonal light beams with different focal depths at the fourth polarization beam splitter;

[0053] S3: passes through the third reflector, the fourth reflector, the electro-optic modulator, the fifth polarization beam splitter and the light block;

[0054] S4: The light after passing through the fifth polarization beam splitter is transmitted to the detection sample on the motorized three-dimensional translation stage through the fifth reflector, the fifth lens, the sixth lens, the scanning galvanometer, the scanning lens, the sixth reflector, the tube lens, the first dichroic mirror, and the third objective lens;

[0055] S5: The sample detection light passes through the third objective lens, the first dichroic mirror, and the second dichroic mirror and is split into two beams of light with different wavelengths. One beam passes through the first filter and the first photomultiplier tube and is then transmitted to the computer processing module. The other beam passes through the second filter and the second photomultiplier tube and is then transmitted to the computer processing module.

[0056] S6: The computer processing module generates the pixel clock of the microscope as the external trigger signal of the signal generator, and its period is 4μs. After receiving the pixel clock signal, the signal generator generates a square wave signal with a period of 8μs and a duty cycle of 50% as the trigger signal of the electro-optical modulator. By fine-tuning the time delay of the electrical signal, the sample information of a certain focal plane is collected within the residence time of each pixel, thereby realizing pixel-level focal plane switching.

[0057] Specific implementation steps:

[0058] (1) The first polarization beam splitter 2 splits the laser light source 1 of the Ti:sapphire mode-locked pulse laser into two beams of horizontal polarization and vertical polarization. A first lens 31 and a second lens 32 are placed in the direction of travel of the vertically polarized beam to expand the beam, which then passes through a second polarization beam splitter 41, a first quarter-wave plate 42, a first objective lens 43, and a first reflector 44. A fifth lens 61 and a sixth lens 62 are placed in the direction of travel of the horizontally polarized beam to expand the beam, which then passes through a third polarization beam splitter 71, a second quarter-wave plate 72, a second objective lens 73, and a second reflector 74. The positions of the first reflector 44 and the second reflector 74 along the optical axis are adjustable. A fourth polarization beam splitter 9 combines the horizontally polarized and vertically polarized beams.

[0059] (2) When the voltage of the electro-optical modulator 12 is 0, the directions of the horizontally polarized light and the vertically polarized light remain unchanged; when the voltage of the electro-optical modulator 12 is λ / 2, the directions of the horizontally polarized light and the vertically polarized light are interchanged. When the input voltage of the electro-optical modulator 12 switches between 0 and λ / 2V, the polarization directions of the light beams are always perpendicular to each other. The modulation signal of the electro-optical modulator 12 comes from the signal generator 13, and the voltage applied to the electro-optical modulator 12 switches between 0 and λ / 2V at a high frequency (the frequency of the square wave is 250kHz and the duty cycle is 50%). At this time, the two mutually perpendicular polarized light beams switch between each other at a high frequency. The fifth polarization beam splitter 14 is located after the electro-optical modulator 12 and is used to remove the horizontally polarized component in the laser.

[0060] (3) The laser scanning microscope is an IX71 research-grade inverted microscope. The second lens pair 5 and the fifth lens pair 17 form two 4f optical systems, imaging the pupil of the first objective lens 43 onto the midplane of the scanning galvanometer 18. The fourth lens pair 8 and the fifth lens pair 17 form two 4f optical systems, imaging the pupil of the second objective lens 73 onto the midplane of the scanning galvanometer 18. The tube lens 21 collimates the light beam for output. The scanning lens 19 and the tube lens 21 also form a 4f system, imaging the midplane of the scanning galvanometer 18 onto the pupil of the third objective lens 23. The third objective lens 23 focuses the laser onto the sample, which is placed on the motorized three-dimensional translation stage 24.

[0061] (4) The backward multiphoton signal generated by the sample is collected by a first dichroic mirror 22. The second dichroic mirror 25, in combination with a first bandpass filter 26 and a second bandpass filter 28, separates the signals of different wavelengths. The signals are amplified by the corresponding first and second photomultiplier tubes 27 and 29. While collecting the signals, the imaging software drives the scanning galvanometer 18, restores the image from the different acquisition channels, removes noise, and finally displays it on the computer screen.

[0062] (5) The computer processing module 30 issues the following Figure 5 The pixel clock shown is used as an external trigger signal of the signal generator 13, and its period is 2μs. Figure 6 The square wave signal shown has a period of 4 μs and a duty cycle of 50%. The modulation signal of the electro-optical modulator 12 comes from the signal generator 13. By fine-tuning the delay of the modulated electrical signal, it is possible to ensure that only the optical signal excited by the laser beam in a single focal plane is collected during the dwell time of each pixel.

[0063] Imaging achieves imaging with two different focal depths in a single frame. The imaging beam distribution on the same frame is as follows: Figure 7As shown, two adjacent columns of pixels are composed of fluorescence signals excited by the first and second beams, respectively, representing different focal depths. By extracting the bright and dark fringes separately, we can obtain an image of the sample at the corresponding focal depth. Based on this, in the first frame, the first beam is aligned at -5μm and the second beam at 0μm; in the second frame, the first beam is aligned at -4μm and the second beam at 1μm; in the third frame, the first beam is aligned at -3μm and the second beam at 2μm; in the fourth frame, the first beam is aligned at -2μm and the second beam at 3μm; in the fifth frame, the first beam is aligned at -1μm and the second beam at 4μm. In the sixth frame, the first beam is again aligned at -5μm and the second beam at 0μm, and this cycle is repeated, thus achieving three-dimensional multiphoton microscopy imaging with an imaging depth of -5μm to 4μm.

[0064] Therefore, the present invention adopts the above-mentioned time-division multiplexing synchronous multi-focal plane multi-photon microscopy imaging device and method, which can achieve imaging results of multiple modes through one imaging, and can effectively avoid signal crosstalk while achieving automatic image matching; adopting a remote control focusing method, the spherical aberration caused by adjusting the axial position of the focal plane is small, and multiple focal planes are synchronously excited, thereby realizing simultaneous detection of images with different focal depths.

[0065] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the same. 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 solutions of the present invention with equivalents, and these modifications or equivalent replacements cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A time-division multiplexing synchronous multi-focal plane multiphoton microscopy imaging device, characterized by: The invention comprises a light source collection mechanism, a scanning mechanism and an imaging mechanism, wherein the light source collection mechanism is arranged on one side of the scanning mechanism, and the imaging mechanism is connected to the scanning mechanism via a signal line. The light source collection mechanism comprises a laser light source, a first polarization beam splitter, a fourth polarization beam splitter, a first remote control focus group and a second remote control focus group. The laser light source is arranged on one side of the first polarization beam splitter, a first lens pair is arranged between the first polarization beam splitter and the first remote control focus group, a third lens pair is arranged between the first polarization beam splitter and the second remote control focus group, a fourth lens pair is arranged between the second remote control focus group and the fourth polarization beam splitter, a second lens pair is arranged between the fourth polarization beam splitter and the first remote control focus group, and a third reflector is arranged on the other side of the fourth polarization beam splitter. The scanning mechanism includes an electro-optical modulator, a fifth polarization beam splitter, a scanning galvanometer, a sixth reflector and a third objective lens. A fourth reflector is provided on one side of the electro-optical modulator, and the fourth reflector is horizontally symmetrically arranged with the third reflector. The electro-optical modulator is connected to a signal generator through the signal line. The fifth polarization beam splitter is provided on the other side of the electro-optical modulator. A light block is provided on the side of the fifth polarization beam splitter away from the electro-optical modulator. A fifth reflector is provided directly below the fifth polarization beam splitter. A fifth lens pair is provided between the fifth reflector and the scanning galvanometer. A scanning lens is provided between the scanning galvanometer and the sixth reflector. A tube lens, a first dichroic mirror and the third objective lens are provided above the sixth reflector from bottom to top. An electric three-dimensional translation stage is provided on the top of the third objective lens. The first polarization beam splitter, the third lens pair, the second remote-controlled focus group, and the fourth lens pair are centrally symmetrically arranged with respect to the first lens pair, the first remote-controlled focus group, the second lens pair, and the fourth polarization beam splitter; The first remote control focusing group includes a second polarization beam splitter, a first quarter wave plate, a first objective lens, and a first reflector, and the second polarization beam splitter, the first quarter wave plate, the first objective lens, and the first reflector are arranged in sequence from left to right; the second remote control focusing group includes a third polarization beam splitter, a second quarter wave plate, a second objective lens, and a second reflector, and the second reflector, the second objective lens, the second quarter wave plate, and the third polarization beam splitter are arranged in sequence from left to right; The imaging mechanism includes a computer processing module, a first photomultiplier tube, and a second photomultiplier tube. The first photomultiplier tube and the second photomultiplier tube are both connected to the computer processing module via the signal line. The computer processing module is connected to the signal generator and the scanning galvanometer via the signal line. A first bandpass filter and a second dichroic mirror are sequentially arranged below the first photomultiplier tube, and a second bandpass filter is arranged between the second dichroic mirror and the second photomultiplier tube.

2. The time-division multiplexing synchronous multi-focal plane multiphoton microscopy imaging device according to claim 1, characterized in that: The fifth reflector and the sixth reflector are arranged symmetrically in a vertical direction, the sixth reflector and the first dichroic mirror are arranged in parallel, and the first dichroic mirror and the second dichroic mirror are arranged symmetrically in a vertical direction.

3. The time-division multiplexing synchronous multi-focal plane multiphoton microscopy imaging device according to claim 2, characterized in that: The first lens pair includes a first lens and a second lens symmetrically arranged, the second lens pair includes a third lens and a fourth lens symmetrically arranged, the third lens pair includes a fifth lens and a sixth lens symmetrically arranged, the fourth lens pair includes a seventh lens and an eighth lens symmetrically arranged, and the fifth lens pair includes a ninth lens and a tenth lens symmetrically arranged.

4. A time-division multiplexing synchronous multi-focal plane multiphoton microscopy method, wherein the time-division multiplexing synchronous multi-focal plane multiphoton microscopy method uses the time-division multiplexing synchronous multi-focal plane multiphoton microscopy device according to claims 1-3, characterized in that: The following steps are involved: S1: The first polarization beam splitter splits the laser light emitted by the laser light source into two beams with orthogonal polarization directions; S2: After the first light beam and the second light beam are respectively controlled by the first remote control focusing group and the second remote control focusing group, they are combined into mutually orthogonal light beams with different focal depths at the fourth polarization beam splitter; S3: passes through the third reflector, the fourth reflector, the electro-optic modulator, the fifth polarization beam splitter and the light block; S4: The light after passing through the fifth polarization beam splitter is transmitted to the detection sample on the motorized three-dimensional translation stage through the fifth reflector, the third lens pair, the scanning galvanometer, the scanning lens, the sixth reflector, the tube lens, the first dichroic mirror, and the third objective lens; S5: The sample detection light passes through the third objective lens, the first dichroic mirror, and the second dichroic mirror and is split into two beams of light with different wavelengths. One beam passes through the first bandpass filter and the first photomultiplier tube and is then transmitted to the computer processing module. The other beam passes through the second bandpass filter and the second photomultiplier tube and is then transmitted to the computer processing module. S6: The computer processing module generates the pixel clock of the microscope as the external trigger signal of the signal generator, and its period is 4μs. After receiving the pixel clock signal, the signal generator generates a square wave signal with a period of 8μs and a duty cycle of 50% as the trigger signal of the electro-optical modulator. By fine-tuning the time delay of the electrical signal, the sample information of a certain focal plane is collected within the residence time of each pixel, thereby realizing pixel-level focal plane switching.

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