Optical fiber endoscopic device and method for simultaneous two-photon imaging of nadh and fad

Abstract

The application discloses a kind of optical fiber endoscope device and method for NADH and FAD synchronous two-photon imaging, it is related to biological imaging technical field.The device includes laser collimation beam expander module, light beam switching module, pulse chirp module, endoscopic detection module and fluorescence acquisition and processing module.Wherein, laser collimation beam expander module carries out collimation beam expansion to the spatial light of laser exit;Light beam switching module alternately cuts into endoscopic system with double-color excitation light;Pulse chirp module compensates the broadening of optical pulse after transmission through optical fiber;Endoscopic detection module transmits excitation light to sample and realizes light beam and fluorescence signal scanning;Fluorescence acquisition and processing module separates, collects different waveband fluorescence signal and reconstructs image.The device effectively solves the problems in the prior art, such as unstable excitation, four-wave mixing and inability to dynamically control the time interval of double-color excitation light pulse, etc., realizes synchronous two-photon endoscopic imaging of NADH and FAD, and improves imaging quality and system adaptability.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of bio-imaging, and particularly relates to a fiber endoscope device and method for NADH and FAD synchronous two-photon imaging. BACKGROUND

[0002] Two-photon fluorescence (TPF) microscopy occupies an important position in the field of biomedical imaging due to its sub-micron three-dimensional spatial resolution, high signal-to-noise ratio, weak out-of-focus photobleaching, and excellent penetration ability. As a label-free technology, it can achieve imaging using endogenous fluorophores in biological tissues without introducing exogenous biomarkers, greatly reducing the complexity and potential risks of imaging. Two-photon fluorescence endoscopic imaging, as an extension of TPF microscopy, perfectly inherits the characteristics of label-free, strong penetration, optical sectioning ability, and small out-of-focus light damage. With flexible endoscopic imaging, the technology can penetrate into gastrointestinal tract, respiratory tract and other luminal organs to achieve high-resolution in-situ imaging and real-time monitoring of moving neural activity, providing support for neural function research and disease mechanism exploration. It plays an irreplaceable role in key application scenarios such as in-vivo deep structure imaging, long-term in-vivo follow-up, minimally invasive biopsy, and intraoperative navigation.

[0003] In cells, reduced nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FAD) are two key coenzymes widely distributed in mitochondria and cytoplasm. NADH is the reduced form of NAD + , carrying electrons for the respiratory chain; FAD acts as a precursor of FADH2 in redox reactions, participating in various dehydrogenase catalytic processes. NADH / NAD + and FAD / FADH2 are crucial for maintaining cellular energy balance, regulating redox state, and supporting gene expression. They are cyclically converted in metabolic pathways such as the tricarboxylic acid cycle, the electron transport chain, and fatty acid oxidation, directly reflecting the metabolic level and health status of cells. NADH and FAD both have endogenous autofluorescence, and their fluorescence intensity and spectral characteristics change significantly with their binding state, local microenvironment (such as pH, protein binding), and redox potential. This allows them to be detected with high sensitivity in multi-photon or fluorescence lifetime imaging without exogenous dyes, avoiding the problems of toxicity, non-specific binding, and functional interference caused by exogenous labeling. With the help of two-photon fluorescence (TPF) imaging technology, the dynamic fluorescence signal of NADH / FAD can be non-invasively monitored in real time in vivo or living tissues, and then the key physiological and pathological processes such as cell differentiation, proliferation, and apoptosis, and tumor metabolic reprogramming can be evaluated. This imaging strategy provides strong technical support for early disease diagnosis, efficacy evaluation, and research on metabolic disorder-related diseases such as cancer and neurodegenerative diseases.

[0004] Currently, researchers continue to innovate around the excitation light regulation and signal acquisition technology, and have made significant progress in the field of NADH and FAD synchronous fluorescence imaging, and gradually promoted the application of related technologies in the field of optical fiber endoscopy. One of the existing technologies is a monochromatic two-photon fluorescence lifetime endoscope, which successfully obtains the two-photon fluorescence lifetime reconstruction image of NADH at an excitation wavelength of 760nm. The basic principle of this scheme is universal, and through sequential excitation, the fluorescence signal acquisition and image reconstruction of NADH and FAD can be realized in turn. Another existing technology uses a dual-output femtosecond laser to output two excitation lights of different wavelengths, and uses wavelength mixing technology to effectively realize the time-space overlap of the two excitation light pulses, thereby generating an additional excitation path, and finally the equivalent three excitation lights act on the sample together. Based on this technology, the benchtop microscope constructed by it realizes the two-photon synchronous excitation of NADH and FAD in human skin and nematodes, and completes the reconstruction of fluorescence lifetime images. Another existing technology introduces an electro-optical modulator into a two-photon fluorescence microscopy system, which can quickly switch the excitation light of 750nm and 860nm by virtue of its fast switching light path characteristics, and constructs a spatially overlapping time-multiplexed excitation light pulse sequence, successfully obtaining the synchronous autofluorescence images of NADH and FAD in freshly excised mouse colon tissue. A dual-color two-photon microscopic endoscope has also been developed in the existing technology, which uses 830nm and 1050nm dual-color excitation light, generates a third excitation light of 927nm through an optical parametric oscillator, and realizes in vivo two-photon synchronous endoscopic imaging for four specific excitation wavelength fluorescent proteins in the mouse brain. In summary, the research on two-photon fluorescence imaging technology of NADH and FAD has gradually developed from single-color excitation light to dual-color excitation light, from benchtop microscope to optical fiber endoscope application, and through the use of electro-optical modulators, parametric oscillators and other devices to realize synchronous excitation and image reconstruction of dual-color excitation light.

[0005] However, the prior art has some disadvantages. On the one hand, the existing desktop synchronous imaging scheme is difficult to adapt to the demand of optical fiber endoscopy, and there are problems of unstable excitation and four-wave mixing. The current synchronous two-photon imaging technology of NADH and FAD is mostly based on the construction of a desktop two-photon microscope system, although certain achievements have been made in imaging resolution and fluorescence channel separation, but the application in the optical fiber endoscopy system still faces significant technical obstacles. In the desktop system, the time multiplexing of double-color light pulses is realized by using a polarization beam splitter, which requires multiple beam splitting, beam combining and polarization state adjustment, and the optical path is complex and the optical loss is large. The polarization state of the excitation light after polarization modulation is easy to change during fiber transmission, which leads to the decrease of system stability and significantly increases the difficulty of structure design and debugging. At the same time, the wavelength mixing technology needs to realize the accurate space-time overlap of double-color excitation light at the sample. In the application of optical fiber endoscopy, due to the bending, vibration and sample movement (such as breathing, peristalsis) of the optical fiber, the excitation light is easy to have space-time mismatch, thereby causing the asynchronization of imaging signals and the error of image reconstruction. In addition, this kind of scheme easily induces four-wave mixing effect in low dispersion optical fiber, produces strong background noise and wastes excitation power, which leads to the decrease of image resolution and contrast, and seriously affects the imaging quality. The single-color light excitation scheme often causes fluorescence signal crosstalk due to the wavelength overlap of NADH and FAD excitation, and it is difficult to realize effective separation of fluorescence channels.

[0006] On the other hand, the double-color excitation light pulse sequence cannot realize dynamic time regulation, and it is difficult to meet the needs of imaging different types of samples. The existing double-color excitation light construction method usually relies on adjusting the optical path difference in the propagation path of the two excitation lights, thereby indirectly controlling the time sequence of the arrival of the two excitation light pulses at the sample surface, to realize two-photon excitation. But this time control scheme based on optical path difference has obvious limitations. This method needs to build an additional optical path and relies on multiple precise optical devices, which occupies a large space and has a complex system structure, which is not conducive to the miniaturization and integration of the optical fiber endoscopy equipment. The adjustment of the time difference needs to move the optical path elements physically, and the adjustment precision is limited, which is difficult to quickly respond to the actual needs. More importantly, due to the difference in the concentration distribution of NADH and FAD in different samples, the time interval of the excitation light must be manually adjusted before imaging to match the sample characteristics, which is a tedious and time-consuming process. For medical workers without an optical background, it increases the threshold for using the equipment, which is not conducive to the popularization and routine application of synchronous two-photon endoscopy technology in clinical scenarios. SUMMARY

[0007] The purpose of the present application is to provide an optical fiber endoscopy device and method for synchronous two-photon imaging of NADH and FAD, which solves the problems of unstable excitation, four-wave mixing and inability to dynamically regulate the time interval of double-color excitation light pulses in the prior art by using a double-input single-output structure optical switch to switch two excitation lights at high speed, and the synergistic effect of the optical switch, Y-type optical fiber and photodetector.

[0008] To achieve the above object, the application provides a fiber endoscope device for NADH and FAD synchronous two-photon imaging, which comprises a laser collimation and expansion module, a beam switching module, a pulse chirp module, an endoscope detection module and a fluorescence collection and processing module.

[0009] The laser collimation and expansion module is used for collimating and expanding spatial light emitted by a laser, and comprises a laser, a lens pair one and a lens pair two.

[0010] The beam switching module is used for switching double-color excitation light into the endoscope system alternately, and comprises a fiber coupler one, a fiber coupler two, a Y-shaped fiber one, a Y-shaped fiber two, a photodetector one, a photodetector two, an optical switch, a fiber one and a fiber coupler three.

[0011] The pulse chirp module is used for compensating the broadening of the excitation light pulse after passing through the fiber, and comprises a beam splitter, a prism pair and a mirror.

[0012] The endoscope detection module is used for transmitting excitation light to a sample, and realizes beam scanning and fluorescence signal scanning on the sample, and comprises a dichroic mirror one, a fiber coupler four, a fiber two, a resonant driver, a packaging shell, a micro objective, a sample and a controller.

[0013] The fluorescence collection and processing module is used for separating and collecting fluorescence signals of different wavebands and reconstructing images, and comprises a dichroic mirror two, a band-pass filter one, a coupling lens one, a photomultiplier tube one, a band-pass filter two, a coupling lens two, a photomultiplier tube two, a data acquisition card and a computer.

[0014] In the laser collimation and expansion module, the laser provides two continuous femtosecond excitation lights, and the lens pair one and the lens pair two collimate and expand the two spatial excitation lights respectively.

[0015] In the beam switching module, the fiber coupler one couples the excitation light processed by the lens pair one into the Y-shaped fiber one, the fiber coupler two couples the excitation light processed by the lens pair two into the Y-shaped fiber two, two branches of the Y-shaped fiber one split light at a ratio of 1:9, the branch with a light splitting ratio of 90% is connected to the optical switch, and the branch with a light splitting ratio of 10% is connected to the photodetector one, two branches of the Y-shaped fiber two split light at a ratio of 1:9, the branch with a light splitting ratio of 90% is connected to the optical switch, and the branch with a light splitting ratio of 10% is connected to the photodetector two, the output channel of the optical switch is connected to the fiber one, and the fiber one transmits the excitation light converted into spatial light by the fiber coupler three.

[0016] In the pulse chirp module, the beam splitter is used for splitting light and light path turning of the spatial light, and the prism pair and the mirror are used for compressing the excitation light pulse and compensating the broadening of the excitation light pulse after passing through the fiber.

[0017] In the endoscopic detection module, a dichroic mirror reflects the excitation light and transmits fluorescence; a fiber coupler couples the spatial excitation light into a second fiber; the second fiber transmits the excitation light to the sample; a resonant driver drives the end of the second fiber to perform scanning motion; a housing is used to insulate and waterproof the resonant driver and the micro-objective; the micro-objective focuses the excitation light emitted from the second fiber onto the sample and couples the fluorescence of the sample to the second fiber; and a controller controls the operation of the resonant driver.

[0018] In the fluorescence acquisition and processing module, dichroic mirror 2 transmits and reflects NADH and FAD fluorescence respectively. After passing through bandpass filter 1 and coupling lens 1, the NADH fluorescence is converted into an analog electrical signal by photomultiplier tube 1. After passing through bandpass filter 2 and coupling lens 2, the FAD fluorescence is converted into an analog electrical signal by photomultiplier tube 2. The data acquisition card acquires and digitizes the analog electrical signals output by photomultiplier tube 1 and photomultiplier tube 2. The computer processes the obtained digital signals and reconstructs the image.

[0019] Preferably, in the beam switching module, photodetector one and photodetector two convert the detected optical signal into an analog electrical signal, which is then acquired and digitized by the data acquisition card and transmitted to the computer for processing.

[0020] Preferably, in the beam switching module, the optical switch has a dual-input single-output structure, and the output channel can only be connected to one input channel at a time. The computer switches the optical switch via a data acquisition card.

[0021] Preferably, in the endoscopic detection module, the computer generates a scanning drive signal through a data acquisition card, and the drive controller receives and controls the resonant driver to complete the beam scanning.

[0022] The present invention also provides a fiber optic endoscopy method for simultaneous two-photon imaging of NADH and FAD, using the aforementioned fiber optic endoscopy device for simultaneous two-photon imaging of NADH and FAD, and the steps are as follows:

[0023] Step S1: The laser emits two continuous femtosecond excitation beams, which are collimated and expanded by lens pair one and lens pair two, respectively, and then coupled into Y-type fiber one and Y-type fiber two by fiber coupler one and fiber coupler two, respectively.

[0024] In step S2, Y-fiber 1 and Y-fiber 2 divide the excitation light into two parts. 10% of the excitation light is transmitted to photodetector 1 and photodetector 2 respectively, and 90% of the excitation light is transmitted to the two input channels of the optical switch respectively. Photodetector 1 and photodetector 2 convert the detected optical signal into an analog electrical signal and transmit it to the data acquisition card.

[0025] Step S3: The computer captures the time sequence of the two excitation light pulses arriving at the optical switch in real time based on the analog electrical signal received by the data acquisition card, and controls the optical switch to dynamically and accurately switch the excitation light pulses. The switched excitation light is transmitted through fiber optic cable one to fiber optic coupler three and converted into spatial light.

[0026] Step S4: Spatial light enters the pulse chirping module through the beam splitter. The prism pair and the reflector compensate for the broadening of the excitation light pulse after it passes through the optical fiber. The compensated excitation light is reflected by the dichroic mirror to the optical fiber coupler and coupled into the optical fiber.

[0027] Step S5: The second fiber transmits the excitation light to the end, and under the drive of the resonant driver, it performs scanning motion. The excitation light is focused onto the sample by the micro-objective, and the sample is excited to produce NADH and FAD fluorescence.

[0028] Step S6: The fluorescence is collected by the micro-objective and enters the second optical fiber. It is transmitted to the fourth optical fiber coupler and converted into spatial light. It is then transmitted through the first dichroic mirror to the second dichroic mirror, where the second dichroic mirror separates the NADH and FAD fluorescence.

[0029] Step S7: After passing through bandpass filter one and coupling lens one, NADH fluorescence is converted into an analog electrical signal by photomultiplier tube one. After passing through bandpass filter two and coupling lens two, FAD fluorescence is converted into an analog electrical signal by photomultiplier tube two. The data acquisition card acquires and digitizes these two analog electrical signals, and the computer processes the obtained digital signals and reconstructs the image.

[0030] Preferably, the optical switch, photomultiplier tube one, photomultiplier tube two, and controller are all connected to the data acquisition card and share a sampling clock to ensure synchronous triggering of the two-photon excitation and acquisition process.

[0031] Preferably, in step S5, the resonant driver performs point-by-point scanning at a fixed frequency, and the time-multiplexed dual-color excitation light achieves synchronous excitation at each pixel.

[0032] Therefore, this invention proposes a fiber optic endoscope and method for simultaneous two-photon imaging of NADH and FAD, with the following advantages:

[0033] (1) The present invention uses a dual-input single-output optical switch to switch the two excitation beams at high speed, forming a time-multiplexed excitation beam pulse sequence, avoiding the time overlap of the two beams at the sample, effectively suppressing the four-wave mixing effect and fluorescence signal crosstalk, reducing background noise and excitation power loss, and significantly improving the signal-to-noise ratio and resolution of imaging; at the same time, the optical switch has no effect on the polarization state of the excitation beam, reducing the interference of polarization state changes in fiber transmission on the system, ensuring excitation stability, simplifying the system structure and debugging process, and making it more suitable for fiber optic endoscopy scenarios.

[0034] (2) This invention constructs a dual-color excitation pulse timing control mechanism that does not rely on optical path difference adjustment through the synergistic effect of an optical switch, a Y-type optical fiber, and a photodetector. It can dynamically adjust and respond in real time to the time interval of the dual-color excitation pulse. This mechanism has a compact structure and high integration, avoiding the structural complexity and inefficient adjustment problems caused by physically moving optical components. It can adapt to the fluorescence signals of NADH and FAD in different concentration ranges, realize balanced detection of multi-channel signals, ensure imaging quality and quantitative accuracy, reduce the threshold for operators, and is more conducive to rapid deployment and routine application in clinical scenarios.

[0035] (3) Compared with existing desktop synchronous imaging solutions, this invention is more in line with the application requirements of fiber optic endoscopy systems. It can achieve high-resolution in situ imaging of cavitary and respiratory tracts and other cavitary organs, providing more reliable technical support for key application scenarios such as in vivo deep structure imaging, long-term in vivo follow-up, minimally invasive biopsy and intraoperative navigation, and helping the development of fields such as neurological function research, disease mechanism exploration and early disease diagnosis.

[0036] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0037] Figure 1 This is an overall structural diagram of a fiber optic endoscope for simultaneous two-photon imaging of NADH and FAD according to the present invention.

[0038] Figure Labels

[0039] 1. Laser; 2. Lens pair 1; 3. Lens pair 2; 4. Fiber optic coupler 1; 5. Fiber optic coupler 2; 6. Y-fiber 1; 7. Y-fiber 2; 8. Photodetector 1; 9. Photodetector 2; 10. Optical switch; 11. Fiber 1; 12. Fiber optic coupler 3; 13. Beam splitter; 14. Prism pair; 15. Mirror; 16. Dichroic mirror 1; 17. Fiber optic coupler 4; 18. Fiber 2; 19. Resonant driver; 20. Encapsulation shell; 21. Micro-objective; 22. Sample; 23. Dichroic mirror 2; 24. Bandpass filter 1; 25. Coupled lens 1; 26. Photomultiplier tube 1; 27. Bandpass filter 2; 28. Coupled lens 2; 29. ​​Photomultiplier tube 2; 30. Controller; 31. Data acquisition card; 32. Computer. Detailed Implementation

[0040] To make the technical solutions, advantages, and objectives of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the described embodiments of the present invention without creative effort are within the protection scope of the present invention.

[0041] Unless otherwise defined, the technical or scientific terms used in this invention shall have the ordinary meaning as understood by one of ordinary skill in the art to which this invention pertains.

[0042] Example 1

[0043] like Figure 1 The diagram shown is an overall structural diagram of a fiber optic endoscope for simultaneous two-photon imaging of NADH and FAD according to the present invention, including a laser collimation and beam expansion module, a beam switching module, a pulse chirp module, an endoscope detection module, and a fluorescence acquisition and processing module.

[0044] The laser collimation and beam expanding module is used to collimate and expand the spatial light emitted from the laser, including: laser 1, lens pair 1 2, and lens pair 2 3;

[0045] The beam switching module is used to alternately switch the dual-color excitation light into the endoscope system, including: fiber coupler 1 4, fiber coupler 2 5, Y-type fiber 1 6, Y-type fiber 2 7, photodetector 1 8, photodetector 2 9, optical switch 10, fiber 1 11 and fiber coupler 3 12.

[0046] The pulse chirp module is used to compensate for the broadening of the optical pulse after passing through the optical fiber, and includes: beam splitter 13, prism pair 14 and reflector 15;

[0047] The endoscopic detection module is used to transmit excitation light to the sample and realize beam scanning and fluorescence signal scanning on the sample. It includes: a dichroic mirror 16, an optical fiber coupler 4 17, an optical fiber 2 18, a resonant driver 19, a package shell 20, a micro-objective 21, a sample 22, and a controller 30.

[0048] The fluorescence acquisition and processing module is used to separate and collect fluorescence signals of different wavelengths and reconstruct images. It includes: a dichroic mirror 23, a bandpass filter 24, a coupling lens 25, a photomultiplier tube 26, a bandpass filter 27, a coupling lens 28, a photomultiplier tube 29, a data acquisition card 31, and a computer 32.

[0049] In the laser collimation and beam expansion module, laser 1 provides two continuous femtosecond excitation beams, and lens pair 1 2 and lens pair 2 3 collimate and expand the two spatial excitation beams respectively.

[0050] In the beam switching module, fiber coupler 4 couples the excitation light processed by lens pair 2 into Y-type fiber 6, and fiber coupler 5 couples the excitation light processed by lens pair 3 into Y-type fiber 7.

[0051] The two branches of the Y-type optical fiber 6 are split in a 1:9 ratio. The branch with a 90% split ratio is connected to the optical switch 10, and the branch with a 10% split ratio is connected to the photodetector 8. The photodetector 8 converts the detected optical signal into an analog electrical signal, which is then acquired and digitized by the data acquisition card 31 and transmitted to the computer 32 for processing.

[0052] The two branches of the Y-type optical fiber 7 split the light in a 1:9 ratio. The branch with a 90% split ratio is connected to the optical switch 10, and the branch with a 10% split ratio is connected to the photodetector 9. The photodetector 9 converts the detected optical signal into an analog electrical signal, which is then acquired and digitized by the data acquisition card 31 and transmitted to the computer 32 for processing.

[0053] The optical switch 10 has a dual-input single-output structure. At any given time, the output channel can only be connected to one input channel. The computer 32 switches the switch on and off via the data acquisition card 31.

[0054] The output channel of the optical switch 10 is connected to the optical fiber 11. The excitation light transmitted by the optical fiber 11 is converted into spatial light by the optical fiber coupler 12.

[0055] In the pulse chirp module, the beam splitter 13 is used to split the spatial light and fold the optical path, while the prism pair 14 and the reflector 15 are used to compress the excitation light pulse and compensate for the broadening of the excitation light pulse after passing through the optical fiber.

[0056] In the endoscopic detection module, a dichroic mirror 16 reflects the excitation light and transmits fluorescence. Fiber optic coupler 17 couples the spatial excitation light into fiber optic cable 18, which then transmits the excitation light to the sample 22. Computer 32 generates a scanning drive signal via data acquisition card 31. Drive controller 30 receives the drive signal and controls resonant driver 19 to drive the end of fiber optic cable 18 to perform scanning motion. Encapsulation shell 20 is used to insulate and waterproof the resonant driver 19 and micro-objective 21. Micro-objective 21 focuses the excitation light emitted from fiber optic cable 18 onto the sample 22, exciting the sample 22 to produce NADH and FAD fluorescence. The fluorescence is coupled to fiber optic cable 18 via micro-objective 21.

[0057] In the fluorescence acquisition and processing module, dichroic mirror 23 transmits and reflects NADH and FAD fluorescence respectively. After passing through bandpass filter 24 and coupling lens 25, the NADH fluorescence is converted into an analog electrical signal by photomultiplier tube 26. After passing through bandpass filter 27 and coupling lens 28, the FAD fluorescence is converted into an analog electrical signal by photomultiplier tube 29. Data acquisition card 31 acquires and digitizes the analog electrical signals output by photomultiplier tube 26 and photomultiplier tube 29. Computer 32 processes the obtained digital signals and reconstructs the image.

[0058] Example 2

[0059] This invention also provides a fiber optic endoscopy method for simultaneous two-photon imaging of NADH and FAD, using the aforementioned fiber optic endoscopy device for simultaneous two-photon imaging of NADH and FAD, with the following steps:

[0060] S1. Laser 1 emits two continuous femtosecond excitation beams, which are collimated and expanded by lens pair 1 2 and lens pair 2 3 respectively, and then coupled into Y-type fiber 1 6 and Y-type fiber 2 7 by fiber coupler 1 4 and fiber coupler 2 5 respectively.

[0061] S2, Y-type fiber 16 and Y-type fiber 27 divide the excitation light into two parts. 10% of the excitation light is transmitted to photodetector 18 and photodetector 29 respectively, and 90% of the excitation light is transmitted to the two input channels of optical switch 10 respectively. Photodetector 18 and photodetector 29 convert the detected optical signal into an analog electrical signal and transmit it to data acquisition card 31.

[0062] S3. The computer 32 captures the time sequence of the two excitation light pulses arriving at the optical switch 10 in real time according to the analog electrical signal received by the data acquisition card 31, and controls the optical switch 10 to dynamically and accurately switch the excitation light pulses. The switched excitation light is transmitted through the optical fiber 11 to the optical fiber coupler 12 and converted into spatial light.

[0063] S4. Spatial light enters the pulse chirping module through beam splitter 13. Prism pair 14 and reflector 15 compensate for the broadening of the excitation light pulse after passing through the optical fiber. The compensated excitation light is reflected by dichroic mirror 16 to fiber coupler 17 and coupled into optical fiber 18.

[0064] S5: Fiber 2 18 transmits the excitation light to the end, and performs scanning motion under the drive of resonant driver 19. The excitation light is focused onto sample 22 by micro objective 21, and the sample 22 is excited to produce NADH and FAD fluorescence. The encapsulation shell 20 is used to insulate and waterproof the resonant driver 19 and micro objective 21.

[0065] The resonant driver 19 performs point-by-point scanning at a fixed frequency to ensure that the time-multiplexed dual-color excitation light is synchronously excited at each pixel, thereby achieving synchronous acquisition of fluorescence images of NADH and FAD.

[0066] S6. Fluorescence is collected by micro-objective 21 and enters fiber optic 18. It is transmitted to fiber optic coupler 17 and converted into spatial light. It is then transmitted through dichroic mirror 16 to dichroic mirror 23. Dichroic mirror 23 separates NADH and FAD fluorescence.

[0067] S7 and NADH fluorescence pass through bandpass filter 24 and coupling lens 25, and are converted into analog electrical signals by photomultiplier tube 26. FAD fluorescence passes through bandpass filter 27 and coupling lens 28, and is converted into analog electrical signals by photomultiplier tube 29. Data acquisition card 31 acquires and digitizes these two analog electrical signals, and computer 32 processes the obtained digital signals and reconstructs the image.

[0068] Optical switch 10, photomultiplier tube 1 26, photomultiplier tube 2 29 and controller 30 are all connected to data acquisition card 31 and share a sampling clock to ensure synchronous triggering of the two-photon excitation and acquisition process.

[0069] It is worth noting that all contents not described in detail in this invention are existing technologies and are well known to those skilled in the art.

[0070] Therefore, this invention provides a fiber optic endoscope device and method for simultaneous two-photon imaging of NADH and FAD. It achieves high-speed switching of dual-color excitation light through a dual-input single-output optical switch and combines the synergistic effect of the optical switch, Y-type fiber and photodetector. This effectively solves the problems of unstable excitation, four-wave mixing and inability to dynamically adjust the pulse time interval of dual-color excitation light in the prior art, improves imaging quality and system adaptability, and expands application scenarios.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. A fiber optic endoscope for simultaneous two-photon imaging of NADH and FAD, characterized in that, It includes a laser collimation and beam expansion module, a beam switching module, a pulse chirp module, an endoscopic detection module, and a fluorescence acquisition and processing module; The laser collimation and beam expanding module is used to collimate and expand the spatial light emitted from the laser, and includes: laser, lens pair one, and lens pair two; The beam switching module is used to alternately switch the dual-color excitation light into the endoscope system, and includes: fiber coupler one, fiber coupler two, Y-fiber one, Y-fiber two, photodetector one, photodetector two, optical switch, fiber one, and fiber coupler three; The pulse chirp module is used to compensate for the broadening of optical pulses after passing through optical fibers, and includes: a beam splitter, a pair of prisms, and a reflector; The endoscopic detection module is used to transmit excitation light to the sample and realize beam scanning and fluorescence signal scanning on the sample. It includes: a dichroic mirror, a fiber coupler, a fiber, a resonant driver, a package shell, a micro-objective, a sample, and a controller. The fluorescence acquisition and processing module is used to separate and collect fluorescence signals of different wavelengths and reconstruct images. It includes: two dichroic mirrors, one bandpass filter, one coupling lens, one photomultiplier tube, two bandpass filters, two coupling lenses, two photomultiplier tubes, a data acquisition card, and a computer. In the laser collimation and beam expansion module, the laser provides two continuous femtosecond excitation beams, and lens pair one and lens pair two collimate and expand the two spatial excitation beams respectively. In the beam switching module, fiber coupler one couples the excitation light processed by lens pair one into Y-type fiber one, and fiber coupler two couples the excitation light processed by lens pair two into Y-type fiber two. The two branches of Y-type fiber one split the light in a 1:9 ratio. The branch with a 90% split ratio is connected to an optical switch, and the branch with a 10% split ratio is connected to photodetector one. The two branches of Y-type fiber two split the light in a 1:9 ratio. The branch with a 90% split ratio is connected to an optical switch, and the branch with a 10% split ratio is connected to photodetector two. The output channel of the optical switch is connected to fiber one. The excitation light transmitted by fiber one is converted into spatial light by fiber coupler three. In the pulse chirp module, the beam splitter is used to split the spatial light and fold the optical path, and the prism pair and the reflector are used to compress the excitation light pulse and compensate for the broadening of the excitation light pulse after passing through the optical fiber. In the endoscopic detection module, a dichroic mirror reflects the excitation light and transmits fluorescence; a fiber coupler couples the spatial excitation light into a second fiber; the second fiber transmits the excitation light to the sample; a resonant driver drives the end of the second fiber to perform scanning motion; a housing is used to insulate and waterproof the resonant driver and the micro-objective; the micro-objective focuses the excitation light emitted from the second fiber onto the sample and couples the fluorescence of the sample to the second fiber; and a controller controls the operation of the resonant driver. In the fluorescence acquisition and processing module, dichroic mirror 2 transmits and reflects NADH and FAD fluorescence respectively. After passing through bandpass filter 1 and coupling lens 1, the NADH fluorescence is converted into an analog electrical signal by photomultiplier tube 1. After passing through bandpass filter 2 and coupling lens 2, the FAD fluorescence is converted into an analog electrical signal by photomultiplier tube 2. The data acquisition card acquires and digitizes the analog electrical signals output by photomultiplier tube 1 and photomultiplier tube 2. The computer processes the obtained digital signals and reconstructs the image.

2. The fiber optic endoscope for simultaneous two-photon imaging of NADH and FAD according to claim 1, characterized in that, In the beam switching module, photodetector one and photodetector two convert the detected optical signals into analog electrical signals, which are then acquired and digitized by the data acquisition card and transmitted to the computer for processing.

3. The fiber optic endoscope for simultaneous two-photon imaging of NADH and FAD according to claim 1, characterized in that, In the beam switching module, the optical switch has a dual-input single-output structure. At any given time, the output channel can only be connected to one input channel. The computer switches the switch on and off via a data acquisition card.

4. The fiber optic endoscope for simultaneous two-photon imaging of NADH and FAD according to claim 1, characterized in that, In the endoscopic detection module, the computer generates scanning drive signals through the data acquisition card, and the drive controller receives and controls the resonant driver to complete the beam scanning.

5. A fiber optic endoscopy method for simultaneous two-photon imaging of NADH and FAD, characterized in that, Using the apparatus according to any one of claims 1-4, the steps are as follows: Step S1: The laser emits two continuous femtosecond excitation beams, which are collimated and expanded by lens pair one and lens pair two, respectively, and then coupled into Y-type fiber one and Y-type fiber two by fiber coupler one and fiber coupler two, respectively. In step S2, Y-fiber 1 and Y-fiber 2 divide the excitation light into two parts. 10% of the excitation light is transmitted to photodetector 1 and photodetector 2 respectively, and 90% of the excitation light is transmitted to the two input channels of the optical switch respectively. Photodetector 1 and photodetector 2 convert the detected optical signal into an analog electrical signal and transmit it to the data acquisition card. Step S3: The computer captures the time sequence of the two excitation light pulses arriving at the optical switch in real time based on the analog electrical signal received by the data acquisition card, and controls the optical switch to dynamically and accurately switch the excitation light pulses. The switched excitation light is transmitted through fiber optic cable one to fiber optic coupler three and converted into spatial light. Step S4: Spatial light enters the pulse chirping module through the beam splitter. The prism pair and the reflector compensate for the broadening of the excitation light pulse after it passes through the optical fiber. The compensated excitation light is reflected by the dichroic mirror to the optical fiber coupler and coupled into the optical fiber. Step S5: The second fiber transmits the excitation light to the end, and under the drive of the resonant driver, it performs scanning motion. The excitation light is focused onto the sample by the micro-objective, and the sample is excited to produce NADH and FAD fluorescence. Step S6: The fluorescence is collected by the micro-objective and enters the second optical fiber. It is transmitted to the fourth optical fiber coupler and converted into spatial light. It is then transmitted through the first dichroic mirror to the second dichroic mirror, where the second dichroic mirror separates the NADH and FAD fluorescence. Step S7: After passing through bandpass filter one and coupling lens one, NADH fluorescence is converted into an analog electrical signal by photomultiplier tube one. After passing through bandpass filter two and coupling lens two, FAD fluorescence is converted into an analog electrical signal by photomultiplier tube two. The data acquisition card acquires and digitizes these two analog electrical signals, and the computer processes the obtained digital signals and reconstructs the image.

6. The fiber optic endoscopy method for simultaneous two-photon imaging of NADH and FAD according to claim 5, characterized in that, The optical switch, photomultiplier tube 1, photomultiplier tube 2, and controller are all connected to the data acquisition card and share a sampling clock to ensure synchronous triggering of the two-photon excitation and acquisition process.

7. A fiber optic endoscopy method for simultaneous two-photon imaging of NADH and FAD according to claim 5, characterized in that, In step S5, the resonant driver performs point-by-point scanning at a fixed frequency, and the time-multiplexed dual-color excitation light achieves synchronous excitation at each pixel.

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