An endoscope system based on harmonic imaging and multiphoton fluorescence imaging

Abstract

The present invention discloses an endoscope system based on harmonic imaging and multiphoton fluorescence imaging, comprising a first and a second femtosecond pulse laser, a prism, an X-Y scanning galvanometer, a first long-wavelength dichroic mirror, a coupling objective lens, an optical fiber bundle, an endoscope, and an imaging device; the first and second femtosecond pulse lasers respectively emit near-infrared femtosecond pulse lasers, which are combined by the prism and enter the X-Y scanning galvanometer. After that, they pass through the first long-wavelength dichroic mirror and are coupled into the optical fiber bundle by the coupling objective lens. Finally, they are focused on the tissue to be tested through the endoscope, stimulating the tissue to be tested and its fluorescent substance to produce second harmonics, third harmonics, two-photon fluorescence, and three-photon fluorescence. The harmonics and fluorescence signals return along the original path, are reflected by the first long-wavelength dichroic mirror, and enter the imaging device to obtain second harmonic images, third harmonic images, two-photon fluorescence images, and three-photon fluorescence images of the tissue to be tested. The present invention combines harmonic and multiphoton fluorescence imaging to detect diseased tissue in a cavity.

Description

Technical Field

[0001] The present invention relates to the field of endoscopes, and in particular to an endoscope system based on harmonic imaging and multiphoton fluorescence imaging. Background Art

[0002] With the advent of lasers, nonlinear effects such as harmonics and multiphoton absorption induced by strong light in media have been discovered. Among them, second harmonic generation (SHG), third harmonic generation (THG), two-photon fluorescence (2PEF), and three-photon fluorescence (3PEF) images can be acquired using the same system. Two-photon fluorescence is a third-order nonlinear effect in which a fluorescent molecule simultaneously absorbs two longer-wavelength photons at high photon density, transitioning from the ground state to an excited state and then back to the ground state, resulting in non-radiative energy loss and the release of a short-wavelength photon, emitting a fluorescence signal. Similar to two-photon fluorescence, a fluorescent molecule also releases a short-wavelength photon upon absorbing three photons. This phenomenon is called three-photon fluorescence and is a fifth-order nonlinear effect.

[0003] During second harmonic generation, no photon absorption occurs, and no energy level transitions or energy loss occurs. Instead, under the influence of intense laser light, molecules within the medium undergo second-order nonlinear polarization, radiating coherent light at a doubled frequency. Because second harmonic generation does not involve electron energy level transitions, the sample is not affected by photobleaching. Third harmonic generation, a third-order nonlinear effect generated by intense laser irradiation of the medium's molecules, emits light waves with a frequency three times that of the excitation light.

[0004] The light sources for second harmonic generation, third harmonic generation, and multiphoton fluorescence systems generally utilize femtosecond pulsed lasers in the near-infrared (NIR-I) and near-infrared (NIR-II) regions. These lasers reduce photon scattering and effectively penetrate biological matrices, enabling detection at a certain depth in tissue. The intensity of multiphoton fluorescence, second harmonic generation, and third harmonic generation is closely related to the density of excitation photons. Only molecules at the focal point of the excitation light are excited; molecules outside the focal point do not generate multiphoton fluorescence, second harmonic generation, or third harmonic generation. This results in a high signal-to-noise ratio for the received signal.

[0005] Conventional second harmonic generation (SHG), third harmonic generation (THG), and multiphoton fluorescence microscopy systems are complex and large in size, limiting their use to the detection of surface and ex vivo tissues. Miniaturizing SHG / THG / multiphoton fluorescence imaging systems and applying them to endoscopy allows the technology to observe diseased tissues within body cavities. SHG is typically used to image non-centrosymmetric molecules and ordered structures, such as collagen fibers, microtubules, myosin, skin tissue, and corneal stroma; THG is typically used to image substances or interfaces with a high refractive index (compared to the surrounding water), such as organelles, red or white blood cells, lipid droplets, adipose tissue, axonal myelin, and bone; and multiphoton absorption can stimulate a variety of endogenous fluorophores, such as keratin, melanin, flavin, elastic fibers, and collagen. The structure, blood vessels, protein distribution and morphology of diseased tissues are quite different from those of normal tissues. Therefore, the state of the tissue can be judged by observing the second harmonic / third harmonic / multiphoton fluorescence images. For example, endoscopes are used for intracavitary lesions such as brain vascular imaging, brain glioma diagnosis, early diagnosis of lung cancer, ovarian cancer, cervical cancer, and bladder cancer. Detection using an endoscope avoids the harm of extracting tissue biopsies to patients.

[0006] In summary, ordinary single-photon fluorescence endoscopes usually use lasers with shorter wavelengths to excite tissue fluorescence. However, light in this wavelength band is easily reflected and scattered by tissues. In addition, since the excitation light and fluorescence wavelengths are close, the excitation light cannot be completely filtered out, resulting in interference in the fluorescence image and a reduced signal-to-noise ratio. Harmonic and multiphoton fluorescence imaging use femtosecond pulsed lasers in the first and second near-infrared regions (800-1700nm) as light sources, which are less affected by tissue reflection and scattering. Since second harmonic / third harmonic / multiphoton fluorescence can only be excited in the area near the focus of the illumination light path, and the wavelength difference between the excitation light and second harmonic / third harmonic / multiphoton fluorescence is large, the excitation light is easily filtered out. Therefore, images collected using a confocal system are not interfered by stray light and have a high signal-to-noise ratio. However, such systems are mostly implemented using confocal microscope systems, which are complex and bulky. They are usually used for detection of surface and ex vivo tissues, and their scope of use is limited. Summary of the Invention

[0007] The purpose of the present invention is to provide an endoscope system based on harmonic imaging and multiphoton fluorescence imaging, which miniaturizes the second harmonic / third harmonic / multiphoton fluorescence imaging system and applies it to the endoscope, so that second harmonic, third harmonic, two-photon fluorescence, and three-photon fluorescence imaging can be performed simultaneously; in addition, the illumination and imaging share the same optical path, and the scanning device of the harmonic and multiphoton fluorescence imaging system is designed to be outside the endoscope, so that the endoscope system can observe lesions in smaller body cavities.

[0008] The technical solutions adopted in the present invention are as follows:

[0009] In a first aspect, the present invention provides an endoscope system based on harmonic imaging and multiphoton fluorescence imaging, the endoscope system comprising a first femtosecond pulse laser, a second femtosecond pulse laser, a laser optical path, an endoscope, and an imaging device;

[0010] The laser optical path includes a prism, an XY scanning galvanometer, a first long-wavelength dichroic mirror, a coupling objective lens, and an optical fiber bundle, which are arranged in sequence. The end of the optical fiber bundle is connected to the endoscope. The first femtosecond pulse laser and the second femtosecond pulse laser respectively emit near-infrared femtosecond pulse lasers. The two near-infrared femtosecond pulse lasers merge through the prism and enter the XY scanning galvanometer. After that, they pass through the first long-wavelength dichroic mirror and are coupled into the optical fiber bundle by the coupling objective lens. Finally, they are focused on the tissue to be measured through the endoscope.

[0011] Two near-infrared femtosecond pulse lasers scan the measured tissue under the action of the XY scanning galvanometer, exciting the measured tissue and its fluorescent substances to produce second harmonics, third harmonics, two-photon fluorescence and three-photon fluorescence. The second harmonics, third harmonics, two-photon fluorescence and three-photon fluorescence return along the original path, are reflected by the first long-wave pass dichroic mirror and enter the imaging device, obtaining the second harmonic image, third harmonic image, two-photon fluorescence image and three-photon fluorescence image of the measured tissue.

[0012] In the above scheme, the wavelength range of the near-infrared femtosecond pulse laser emitted by the first femtosecond pulse laser is 850-1250nm, which is used to excite the tissue under test and its fluorescent substances to produce second harmonics and two-photon fluorescence; the wavelength range of the near-infrared femtosecond pulse laser emitted by the second femtosecond pulse laser is 1300-1350nm, which is used to excite the tissue under test and its fluorescent substances to produce third harmonics and three-photon fluorescence.

[0013] In the above solution, the wavelength of the near-infrared femtosecond pulse laser emitted by the first femtosecond pulse laser is 920 nm, and the wavelength of the near-infrared femtosecond pulse laser emitted by the second femtosecond pulse laser is 1300 nm.

[0014] In the above solution, the imaging device includes a second long-wave pass dichroic mirror, a third long-wave pass dichroic mirror, a fourth long-wave pass dichroic mirror, a third harmonic imaging sub-device, a second harmonic imaging sub-device, a two-photon fluorescence imaging sub-device, and a three-photon fluorescence imaging sub-device;

[0015] A second long-wavelength pass dichroic mirror, a third long-wavelength pass dichroic mirror, and a fourth long-wavelength pass dichroic mirror are sequentially arranged; the second harmonic, the third harmonic, the two-photon fluorescence, and the three-photon fluorescence entering the imaging device sequentially pass through the second long-wavelength pass dichroic mirror, the third long-wavelength pass dichroic mirror, and the fourth long-wavelength pass dichroic mirror;

[0016] Among them, the third harmonic is reflected by the second long-wavelength pass dichroic mirror and enters the third harmonic imaging sub-device to obtain a third harmonic image; then the second harmonic is reflected by the third long-wavelength pass dichroic mirror and enters the second harmonic imaging sub-device to obtain a second harmonic image; finally, the two-photon fluorescence is reflected by the fourth long-wavelength pass dichroic mirror and enters the two-photon fluorescence imaging sub-device to obtain a two-photon fluorescence image, while the three-photon fluorescence passes through the fourth long-wavelength pass dichroic mirror and enters the three-photon fluorescence imaging sub-device to obtain a three-photon fluorescence image.

[0017] In the above solution, each imaging sub-device includes a narrow-band filter, a converging lens and a photomultiplier tube.

[0018] In the above scheme, the endoscope system also includes a control system, which is connected to the XY scanning galvanometer and the photomultiplier tubes of each imaging sub-device, and is used to control the movement of the XY scanning galvanometer and receive signals from each photomultiplier tube, and combine the scanning trajectory of the XY scanning galvanometer with the signals of each photomultiplier tube to reconstruct each image to obtain second harmonic images, third harmonic images, two-photon fluorescence images and three-photon fluorescence images.

[0019] In the above solution, the control system is also connected to the first femtosecond pulse laser and the second femtosecond pulse laser, and is used to control the opening and closing of the first femtosecond pulse laser and the second femtosecond pulse laser.

[0020] In the above scheme, the endoscope includes an achromatic objective lens and a portion of the optical fiber bundle; the portion of the optical fiber bundle is connected to the end of the optical fiber bundle, and two near-infrared femtosecond pulse lasers are focused on the measured tissue through the achromatic objective lens in the endoscope.

[0021] In the above solution, the endoscope is equipped with a handle, and the outer diameter of the working insertion portion of the endoscope is less than 6 mm.

[0022] In a second aspect, the present invention provides an imaging method of an endoscope system based on harmonic imaging and multiphoton fluorescence imaging according to any one of the above technical solutions, comprising the following steps:

[0023] The first femtosecond pulse laser and the second femtosecond pulse laser each emit near-infrared femtosecond pulse lasers. The two near-infrared femtosecond pulse lasers are combined through a prism and then enter the XY scanning galvanometer. They then pass through a first long-wavelength dichroic mirror and are coupled into a fiber bundle by a coupling objective lens. Finally, they are focused on the tissue being tested through an endoscope.

[0024] Two near-infrared femtosecond pulse lasers excite the tissue under test and its fluorescent substances to generate second harmonics, third harmonics, two-photon fluorescence, and three-photon fluorescence. The second harmonics, third harmonics, two-photon fluorescence, and three-photon fluorescence return along the original path and are reflected by the first long-wavelength dichroic mirror into the imaging device.

[0025] Two near-infrared femtosecond pulse lasers scan the tissue under the action of an XY scanning galvanometer, and the imaging device obtains the second harmonic image, third harmonic image, two-photon fluorescence image and three-photon fluorescence image of the tissue under test.

[0026] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0027] The present invention adopts two femtosecond pulse lasers, which respectively emit near-infrared femtosecond pulse lasers. The two near-infrared femtosecond pulse lasers can excite the measured tissue and its fluorescent material to produce second harmonics, third harmonics, two-photon fluorescence and three-photon fluorescence. Finally, the second harmonic image, third harmonic image, two-photon fluorescence image and three-photon fluorescence image of the measured tissue can be obtained through the imaging device. By combining harmonics and multi-photon fluorescence, a joint imaging diagnosis of the diseased tissue is performed, thereby improving the accuracy of diagnosis. The endoscope system applies the harmonic and multi-photon fluorescence effects to the endoscope system, enabling it to perform diagnosis of internal body cavity lesions such as brain blood vessel imaging, brain glioma diagnosis, and early diagnosis of lung cancer, thereby avoiding the harm to the patient caused by extracting tissue biopsy. In addition, the illumination and imaging optical paths of the endoscope system share part of the optical path, namely the optical path between the first long-wave pass dichroic mirror and the endoscope, and the scanning device of the harmonic and multiphoton fluorescence imaging system is designed to be outside the endoscope, which greatly reduces the size of the endoscope and enables this technology to observe lesions in smaller body cavities.

[0028] In addition, the wavelength range of the near-infrared femtosecond pulse laser emitted by the first femtosecond pulse laser in the present invention is 850-1250nm, which is mainly used to excite the measured tissue and its fluorescent substance to produce second harmonic and two-photon fluorescence, while the wavelength range of the near-infrared femtosecond pulse laser emitted by the second femtosecond pulse laser is 1300-1350nm, which is mainly used to excite the measured tissue and its fluorescent substance to produce third harmonic and three-photon fluorescence. The two femtosecond pulse lasers in the specific wavelength range can make the imaging device obtain the second harmonic image, third harmonic image, two-photon fluorescence image and three-photon fluorescence image of the measured tissue better; the achromatic objective lens designed in the present invention has a wide working band and can be compatible with excitation light, second harmonic, third harmonic, and multi-photon fluorescence at the same time to obtain second harmonic / third harmonic / multi-photon fluorescence images, and the images can be analyzed or fused according to usage requirements to increase the accuracy of distinguishing diseased tissues and lesion types. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of the structure of an endoscope system based on harmonic imaging and multiphoton fluorescence imaging;

[0030] Figure 2 The following is a workflow diagram of the endoscope system based on harmonic imaging and multiphoton fluorescence imaging;

[0031] Figure 3 This is the optical path diagram of the achromatic objective.

[0032] In the figure: 1. first femtosecond pulse laser; 2. second femtosecond pulse laser; 3. prism; 4. XY scanning galvanometer; 5. first long-wave pass dichroic mirror; 6. coupling objective lens; 7. optical fiber bundle; 8. endoscope; 9. measured tissue; 10. second long-wave pass dichroic mirror; 11. first narrow-band filter; 12. first converging lens; 13. first photomultiplier tube; 14. third long-wave pass dichroic mirror; 15. second narrow-band filter; 16. second converging lens; 17. second photomultiplier tube; 18. fourth long-wave pass dichroic mirror; 19. third narrow-band filter; 20. third converging lens; 21. third photomultiplier tube; 22. fourth narrow-band filter; 23. fourth converging lens; 24. fourth photomultiplier tube; 25. control system. DETAILED DESCRIPTION

[0033] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only intended to illustrate the present invention and are not intended to limit the present invention. In addition, the technical features involved in the various embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.

[0034] The present invention provides an endoscope system based on harmonic imaging and multiphoton fluorescence imaging, comprising a femtosecond pulse laser, a photomultiplier tube, an XY scanning galvanometer, an achromatic objective lens, and other key components. The system diagnoses lesions based on second harmonic, third harmonic, and multiphoton fluorescence images. The endoscope system combines harmonics and multiphoton fluorescence for combined imaging diagnosis of lesions, improving diagnostic accuracy. The application of harmonic and multiphoton fluorescence effects to the endoscope system enables it to perform diagnostics of lesions within body cavities, such as imaging of brain blood vessels, diagnosing gliomas, and early diagnosis of lung cancer, thereby avoiding the harm to patients caused by tissue biopsies. The scanning device of the harmonic and multiphoton fluorescence imaging system is designed to be external to the endoscope, significantly reducing its size and enabling the technology to observe lesions within smaller body cavities. The achromatic objective lens designed in the present invention has a wide operating band and is simultaneously compatible with excitation light, second harmonic, third harmonic, and multiphoton fluorescence.

[0035] The present invention provides an endoscope system based on harmonic imaging and multiphoton fluorescence imaging, which miniaturizes the second harmonic / third harmonic / multiphoton fluorescence imaging system and applies it to the field of endoscopy. The light source is a femtosecond pulsed laser. After passing through an XY scanning galvanometer, the femtosecond pulsed laser enters a fiber bundle through a coupling objective lens. The laser light emitted from the fiber bundle is converged to a focal point by an achromatic objective lens. The focal point of the achromatic objective lens coincides with the surface of human tissue, exciting non-centrosymmetric molecules and ordered structures in the tissue being measured to produce second harmonics, such as structural proteins; substances or interfaces with a high refractive index (compared to the surrounding water), such as organelles, red blood cells, or white blood cells, to produce third harmonics; and various endogenous fluorophores to produce multiphoton fluorescence. The second harmonic / third harmonic / multiphoton fluorescence then returns along the original path, is reflected by a long-wavelength dichroic mirror behind the coupling objective lens, and is then split, filtered, and focused onto the photosensitive surface of a photomultiplier tube. The control system combines the movement of the XY scanning galvanometer with the output of the photomultiplier tube to reconstruct, fuse, and analyze the images. The blood vessels, tissue structure, and protein distribution in tumor tissue differ significantly from those in normal tissue. Combining second harmonic generation (SHG) and third harmonic generation (THG) imaging allows visualization of vascular and blood cell distribution within tumor tissue, while multiphoton fluorescence allows visualization of the distribution of various proteins and colloids. Therefore, combining SHG / THG / multiphoton fluorescence imaging enables precise identification of tumor tissue. The shared optical path between the illumination and imaging systems enhances their consistency, and the placement of the scanning system outside the endoscope significantly reduces the endoscope's outer diameter, making it more suitable for non-invasive or minimally invasive surgery.

[0036] Figure 1 This is a schematic diagram of the structure of an endoscope system based on harmonic imaging and multiphoton fluorescence imaging. Figure 2 This is a workflow diagram for an endoscope system based on harmonic imaging and multiphoton fluorescence imaging. As shown, the illumination and imaging optical paths partially overlap, and the confocal optical path improves the accuracy of harmonic and multiphoton fluorescence signal collection, resulting in a higher signal-to-noise ratio.

[0037] like Figure 1 and Figure 2As shown, the near-infrared laser beams emitted by two femtosecond pulsed lasers are reflected twice by an XY scanning galvanometer 4, controlling the position changes along the X and Y axes, respectively, completing a two-dimensional scan of the surface being measured. A first long-wavelength pass dichroic mirror 5 is used to separate the excitation light from the second harmonic / third harmonic / multiphoton fluorescence. The near-infrared laser beam is coupled to a fiber bundle 7 by a coupling lens 6 and focused onto the tissue being measured after passing through an achromatic objective lens. At the focus of the achromatic objective lens, the high-density photons cause the tissue being measured 9 to produce a nonlinear optical effect, namely, second harmonic / third harmonic / multiphoton fluorescence after being excited by the near-infrared femtosecond pulsed laser. Near-infrared light with a wavelength less than 1300nm primarily excites the second harmonic, while near-infrared light with a wavelength greater than 1300nm primarily excites the third harmonic. The wavelength of the multiphoton fluorescence is related to the type of fluorescent dye / receptor protein. Among them, there is no energy loss in the generation process of second harmonic / third harmonic, so the wavelength of the second harmonic is exactly half of the excitation wavelength, and the wavelength of the third harmonic is one-third of the excitation light wavelength; the multi-photon absorption induced by near-infrared light is two-photon absorption and three-photon absorption, and non-radiative energy loss is generated in the process when the molecules in the ground state absorb two / three long-wave photons and release one short-wave photon.

[0038] Second harmonic generation is commonly used for imaging non-centrosymmetric molecules and ordered structures, such as collagen fibers, microtubules, myosin, skin tissue, and corneal stroma; third harmonic generation is commonly used for imaging substances or interfaces with a high refractive index (compared to the surrounding water), such as organelles, red blood cells or white blood cells, lipid droplets, adipose tissue, axon myelin, and bone; and multiphoton absorption can stimulate a variety of endogenous fluorophores, such as keratin, melanin, flavin, elastic fibers, collagen, etc.

[0039] The second harmonic / third harmonic / multiphoton fluorescence light sequentially enters the achromatic objective lens, optical fiber bundle 7, and coupling objective lens 6 in the opposite direction to the excitation light. After being reflected by the first long-wavelength pass dichroic mirror 5, it is split into two paths by the second long-wavelength pass dichroic mirror 10. One path enters the third harmonic imaging optical path, and the other path is split into two paths by the third long-wavelength pass dichroic mirror 14. One path enters the second harmonic imaging optical path, and the other path is further split by the fourth long-wavelength pass dichroic mirror 18. One path enters the two-photon fluorescence imaging optical path, and the other path enters the three-photon fluorescence imaging optical path. The light signals in the four imaging optical paths are filtered, focused, and amplified. After that, a photomultiplier tube converts the optical signals into electrical signals and outputs them to the control system 25. The control system 25 combines the scanning trajectory of the XY scanning galvanometer 4 with the signals output by the photomultiplier tube to perform image reconstruction, obtaining a second harmonic / third harmonic / multiphoton fluorescence image. The images can be analyzed or fused according to usage requirements to increase the accuracy of distinguishing diseased tissue and lesion type.

[0040] Specifically, such as Figure 1As shown, the endoscope system based on harmonic imaging and multiphoton fluorescence imaging according to an embodiment of the present application includes: a first femtosecond pulse laser 1, a second femtosecond pulse laser 2, a laser optical path, an endoscope 8, a tissue to be measured 9, an imaging device, and a control system 25. The laser optical path includes a prism 3, an XY scanning galvanometer 4, a first long-wavelength pass dichroic mirror 5, a coupling objective lens 6, and an optical fiber bundle 7. The prism 3, the XY scanning galvanometer 4, the first long-wavelength pass dichroic mirror 5, the coupling objective lens 6, and the optical fiber bundle 7 are arranged in sequence. The imaging device includes a second long-wave pass dichroic mirror 10, a first narrow-band filter 11, a first converging lens 12, a first photomultiplier tube 13, a third long-wave pass dichroic mirror 14, a second narrow-band filter 15, a second converging lens 16, a second photomultiplier tube 17, a fourth long-wave pass dichroic mirror 18, a third narrow-band filter 19, a third converging lens 20, a third photomultiplier tube 21, a fourth narrow-band filter 22, a fourth converging lens 23 and a fourth photomultiplier tube 24.

[0041] The first femtosecond pulse laser 1 and the second femtosecond pulse laser 2 are femtosecond pulse lasers and serve as the light sources in the system. A prism 3 allows the light emitted by the two lasers to enter an XY scanning galvanometer 4, which scans the entire endoscope field of view point by point under the control of a control system 25. A first long-wavelength dichroic mirror 5 allows the near-infrared femtosecond pulse laser to pass through, while the second harmonic, third harmonic, and multiphoton fluorescence are reflected. A coupling objective lens 6 allows the near-infrared femtosecond pulse laser to enter an optical fiber bundle 7, or allows the second harmonic, third harmonic, and multiphoton fluorescence in the optical fiber bundle 7 to enter the first long-wavelength dichroic mirror 5. Under the action of the XY galvanometer system, the near-infrared femtosecond pulse laser beam scans the end face of the optical fiber bundle point by point. An endoscope 8 consists of an achromatic objective lens and a portion of the optical fiber bundle. It is equipped with a handle for easy gripping by the user. The outer diameter of the working insertion portion of the endoscope is less than 6 mm, enabling the detection of diseased tissue within the cavity.

[0042] The laser is focused on the surface of the tissue being measured 9, exciting the tissue and its fluorescent substances to produce second harmonics, third harmonics, two-photon fluorescence, and three-photon fluorescence. A second long-wavelength pass dichroic mirror 10 reflects the third harmonic. A first narrowband filter 11, a first converging lens 12, and a first photomultiplier tube 13 form a third harmonic channel, forming a third harmonic imaging subassembly. The first narrowband filter 11 has a bandwidth of 10 nm, and the first converging lens 12 focuses the third harmonic onto the photosensitive surface of the first photomultiplier tube 13. The first photomultiplier tube 13 amplifies the signal and outputs it to the control system 25. The third long-wave pass dichroic mirror 14 reflects the second harmonic; the second narrow-band filter 15, the second converging lens 16, and the second photomultiplier tube 17 are the second harmonic channel, constituting a second harmonic imaging sub-device, wherein the bandwidth of the second narrow-band filter 15 is 10 nm, and the second converging lens 16 focuses the second harmonic on the photosensitive surface of the second photomultiplier tube 17, and the second photomultiplier tube 17 amplifies the signal and outputs it to the control system 25. The fourth long-wavelength pass dichroic mirror 18 reflects two-photon fluorescence and transmits three-photon fluorescence. The third narrowband filter 19, the third converging lens 20, and the third photomultiplier tube 21 form a two-photon fluorescence channel, forming a two-photon fluorescence imaging sub-device. The third narrowband filter 19 has a bandwidth of 10 nm. The third converging lens 20 focuses the two-photon fluorescence on the photosensitive surface of the third photomultiplier tube 21, which amplifies the signal and outputs it to the control system 25. The fourth narrowband filter 22, the fourth converging lens 23, and the fourth photomultiplier tube 24 form a three-photon fluorescence channel, forming a three-photon fluorescence imaging sub-device. The fourth narrowband filter 22 has a bandwidth of 10 nm. The fourth converging lens 23 focuses the two-photon fluorescence on the photosensitive surface of the fourth photomultiplier tube 24, which amplifies the signal and outputs it to the control system 25.

[0043] Control system 25 is responsible for controlling the illumination of the femtosecond pulsed laser, controlling the movement of the XY scanning galvanometer 4, receiving signals from the four photomultiplier tubes, and performing second harmonic, third harmonic, and multiphoton fluorescence image reconstruction, image processing, and image fusion. Using harmonic imaging and multiphoton fluorescence imaging, the distribution of blood vessels, blood cells, and proteins in tissues can be observed to determine the presence and extent of tissue pathology.

[0044] Among them, the wavelength range of the near-infrared femtosecond pulse laser emitted by the first femtosecond pulse laser is 850-1250nm, which is used to excite the measured tissue and its fluorescent substances to produce second harmonic and two-photon fluorescence; the wavelength range of the near-infrared femtosecond pulse laser emitted by the second femtosecond pulse laser is 1300-1350nm, which is used to excite the measured tissue and its fluorescent substances to produce third harmonic and three-photon fluorescence.

[0045] The embodiment of the present application further provides an imaging method of an endoscope system based on harmonic imaging and multiphoton fluorescence imaging as described in any one of the above technical solutions, comprising the following steps:

[0046] The first femtosecond pulse laser and the second femtosecond pulse laser each emit near-infrared femtosecond pulse lasers. The two near-infrared femtosecond pulse lasers are combined through a prism and then enter the XY scanning galvanometer. They then pass through a first long-wavelength dichroic mirror and are coupled into a fiber bundle by a coupling objective lens. Finally, they are focused on the tissue being tested through an endoscope.

[0047] Two near-infrared femtosecond pulse lasers excite the tissue under test and its fluorescent substances to generate second harmonics, third harmonics, two-photon fluorescence, and three-photon fluorescence. The second harmonics, third harmonics, two-photon fluorescence, and three-photon fluorescence return along the original path and are reflected by the first long-wavelength dichroic mirror into the imaging device.

[0048] Two near-infrared femtosecond pulse lasers scan the tissue under the action of an XY scanning galvanometer, and the imaging device obtains the second harmonic image, third harmonic image, two-photon fluorescence image and three-photon fluorescence image of the tissue under test.

[0049] Figure 3 This is the optical path diagram of an achromatic objective lens. It can be used with fiber bundles with an end diameter of 4 mm or less. The maximum scanning range on biological tissue is a circular area with a diameter of 1.27 mm. The parameters of the achromatic objective lens are shown in Table 1.

[0050] Table 1: Achromatic objective parameters

[0051]

[0052]

[0053] This invention combines harmonic and multiphoton fluorescence imaging. The external scanning system miniaturizes the illumination path, making it suitable for endoscopy. This technology can observe structural proteins, high-refractive index materials or interfaces, and the distribution and morphology of various endogenous fluorophores within small body cavities to assess tissue pathology and avoid the pain of biopsies. The combined use of harmonic and multiphoton fluorescence imaging increases the accuracy of identifying diseased or pathological tissue.

[0054] To help understand the present invention, let's take 920nm and 1300nm femtosecond lasers as examples. The working principle of the system is as follows:

[0055] 920nm and 1300nm femtosecond pulsed lasers provide illumination for the optical path. After passing through the XY scanning galvanometer 4, the near-infrared femtosecond pulsed laser is transmitted by the first long-wavelength dichroic mirror 5 and focused by the coupling objective lens 6 onto the end face of the optical fiber bundle 7. The XY scanning galvanometer 4 scans the end face of the optical fiber bundle point by point. The near-infrared femtosecond pulsed laser is transmitted by the optical fiber bundle 7 to the achromatic objective lens of the endoscope 8, which focuses the laser onto the surface of the tissue being measured. At the focal point of the achromatic objective lens, the high-density photons induce the ground-state molecules in the tissue to produce second harmonics, third harmonics, and multiphoton fluorescence.

[0056] Three-photon fluorescence imaging requires the use of fluorescent dyes. For example, the red fluorophore Texas Red is widely used to study neuronal morphology and as a cell-type-selective fluorescent marker for astrocytes. When the excitation wavelength is below 1250nm, the fluorophore only excites a two-photon signal. As the excitation wavelength increases, the excitation signal becomes a mixture of two-photon and three-photon fluorescence. At wavelengths greater than 1300nm, the excitation signal is primarily three-photon fluorescence. Therefore, a 1300nm laser is used to excite the three-photon fluorescence of the fluorescent dye and the third harmonic of materials or interfaces with a high refractive index (compared to the surrounding water).

[0057] The excitation wavelength range used in two-photon imaging is between 700 and 1000 nm. The scattering coefficient of light in this band is small in tissue and it has good penetration. However, near-infrared light of 700 nm to 850 nm can cause autofluorescent substances with shorter excitation wavelengths in tissue to produce fluorescence, interfering with the two-photon fluorescence imaging effect. Therefore, a 920 nm femtosecond pulse laser is used as the excitation light source for two-photon fluorescence. In addition, non-centrosymmetric molecules and ordered structures, such as collagen fibers, microtubules, myosin, skin tissue and corneal stroma, will also generate second harmonics under the action of femtosecond pulse lasers in this band.

[0058] Under the irradiation of the aforementioned 920nm and 1300nm femtosecond pulsed lasers, molecules in the tissue being tested emit 460nm second harmonic, 433nm third harmonic, 615nm three-photon fluorescence, and 520nm two-photon fluorescence. The second harmonic, third harmonic, three-photon fluorescence, and two-photon fluorescence signals return along their original paths through the achromatic objective, fiber bundle, and coupled objective lens. After being reflected by the first long-wavelength dichroic mirror, they enter the imaging optical path. They are then split, filtered, and focused onto the photosensitive surface of a photomultiplier tube (PMT). The PMT converts the optical signal into an electrical signal and outputs it to a control system. The control system reconstructs the scanned signals to produce images of the second harmonic, third harmonic, three-photon fluorescence, and two-photon fluorescence. After image processing, the four images are fused and analyzed. The distribution of blood vessels, blood cells, and proteins within the tissue is compared to determine the tissue's condition, effectively detecting diseased tissue.

[0059] This embodiment combines harmonic and multiphoton fluorescence imaging technologies and miniaturizes their structure, enabling their application in endoscope systems to detect diseased tissue within cavities. While the embodiments illustrate the operating principle of the present invention using only 920nm and 1300nm femtosecond pulsed lasers as examples, the system can be used to excite and receive second harmonic, third harmonic, or multiphoton fluorescence at other wavelengths by changing the excitation wavelength and replacing filters. Such modifications still fall within the scope of the claims. The light source can be a combination of femtosecond pulsed lasers in the near-infrared region I and II (NIR-I and NIR-II: 800-1700nm), and the light waves received by the imaging system can be a combination of one or more of second harmonic, third harmonic, two-photon fluorescence, and three-photon fluorescence. Furthermore, this embodiment externalizes the image scanning system, simplifying the structure of the endoscope portion of the imaging optical path. Furthermore, the illumination and imaging optical paths share a portion of the optical path, resulting in confocal focus on the surface of the tissue being examined, improving the accuracy of the scanning imaging.

[0060] In summary, the present invention uses two femtosecond pulse lasers with different wavelengths as light sources to excite the tissue under test and the fluorescent agent to generate second harmonics, third harmonics, three-photon fluorescence, and two-photon fluorescence. The image sensor fuses and analyzes the four received images to identify specific tissues and structures, assisting doctors in accurately identifying lesions. The illumination light path and the imaging light path share the same light path, and are confocal on the surface of the tissue under test, that is, the focus of the excitation light and the luminous point of the second harmonic, third harmonic, and multiphoton fluorescence are highly coincident, providing accurate position information for subsequent image reconstruction. The illumination light path and the imaging light path share the same light path, which improves the space utilization of the scope. The second harmonic, third harmonic and multiphoton fluorescence are only generated at the focus of the achromatic objective lens, and their light intensity is highly dependent on the photon density of the excitation light. This feature avoids the interference of stray light generated by the tissue around the focus on the system, greatly improving the signal-to-noise ratio. The scanning device of the endoscope system is placed outside the endoscope, so that the outer diameter of the working insertion part of the endoscope is less than 6mm, reducing the size and weight of the handle. At the same time, the external scanning system is less restricted in size, and a higher-precision galvanometer scanning system can be used to scan and image the measured tissue more accurately.

[0061] It should be noted that the size of the serial numbers of the steps in the above embodiments does not mean the order of execution. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0062] It should be pointed out that, according to the needs of implementation, the various steps / components described in this application can be split into more steps / components, or two or more steps / components or partial operations of steps / components can be combined into new steps / components to achieve the purpose of the present invention.

[0063] It will be easily understood by those skilled in the art that the above are merely preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. An endoscope system based on harmonic imaging and multiphoton fluorescence imaging, characterized in that: The endoscope system includes a first femtosecond pulse laser, a second femtosecond pulse laser, a laser optical path, an endoscope, and an imaging device; The laser optical path includes a prism, an XY scanning galvanometer, a first long-wavelength dichroic mirror, a coupling objective lens, and an optical fiber bundle, which are arranged in sequence. The end of the optical fiber bundle is connected to the endoscope. The first femtosecond pulse laser and the second femtosecond pulse laser respectively emit near-infrared femtosecond pulse lasers. The two near-infrared femtosecond pulse lasers merge through the prism and enter the XY scanning galvanometer. After that, they pass through the first long-wavelength dichroic mirror and are coupled into the optical fiber bundle by the coupling objective lens. Finally, they are focused on the tissue to be measured through the endoscope. Two near-infrared femtosecond pulse lasers scan the measured tissue under the action of the XY scanning galvanometer, exciting the measured tissue and its fluorescent substances to produce second harmonics, third harmonics, two-photon fluorescence and three-photon fluorescence. The second harmonics, third harmonics, two-photon fluorescence and three-photon fluorescence return along the original path, are reflected by the first long-wave pass dichroic mirror and enter the imaging device, obtaining the second harmonic image, third harmonic image, two-photon fluorescence image and three-photon fluorescence image of the measured tissue.

2. The endoscope system based on harmonic imaging and multiphoton fluorescence imaging according to claim 1, characterized in that: The wavelength range of the near-infrared femtosecond pulse laser emitted by the first femtosecond pulse laser is 850-1250nm, which is used to excite the tissue under test and its fluorescent substances to produce second harmonic and two-photon fluorescence; the wavelength range of the near-infrared femtosecond pulse laser emitted by the second femtosecond pulse laser is 1300-1350nm, which is used to excite the tissue under test and its fluorescent substances to produce third harmonic and three-photon fluorescence.

3. The endoscope system based on harmonic imaging and multiphoton fluorescence imaging according to claim 2, characterized in that: The wavelength of the near-infrared femtosecond pulse laser emitted by the first femtosecond pulse laser is 920 nm, and the wavelength of the near-infrared femtosecond pulse laser emitted by the second femtosecond pulse laser is 1300 nm.

4. The endoscope system based on harmonic imaging and multiphoton fluorescence imaging according to claim 1, characterized in that: The imaging device includes a second long-wave-pass dichroic mirror, a third long-wave-pass dichroic mirror, a fourth long-wave-pass dichroic mirror, a third harmonic imaging sub-device, a second harmonic imaging sub-device, a two-photon fluorescence imaging sub-device, and a three-photon fluorescence imaging sub-device; A second long-wavelength pass dichroic mirror, a third long-wavelength pass dichroic mirror, and a fourth long-wavelength pass dichroic mirror are sequentially arranged; the second harmonic, the third harmonic, the two-photon fluorescence, and the three-photon fluorescence entering the imaging device sequentially pass through the second long-wavelength pass dichroic mirror, the third long-wavelength pass dichroic mirror, and the fourth long-wavelength pass dichroic mirror; Among them, the third harmonic is reflected by the second long-wavelength pass dichroic mirror and enters the third harmonic imaging sub-device to obtain a third harmonic image; then the second harmonic is reflected by the third long-wavelength pass dichroic mirror and enters the second harmonic imaging sub-device to obtain a second harmonic image; finally, the two-photon fluorescence is reflected by the fourth long-wavelength pass dichroic mirror and enters the two-photon fluorescence imaging sub-device to obtain a two-photon fluorescence image, while the three-photon fluorescence passes through the fourth long-wavelength pass dichroic mirror and enters the three-photon fluorescence imaging sub-device to obtain a three-photon fluorescence image.

5. The endoscope system based on harmonic imaging and multiphoton fluorescence imaging according to claim 4, characterized in that: Each imaging sub-device includes a narrowband filter, a converging lens and a photomultiplier tube.

6. The endoscope system based on harmonic imaging and multiphoton fluorescence imaging according to claim 5, characterized in that: The endoscope system also includes a control system, which is connected to the XY scanning galvanometer and the photomultiplier tubes of each imaging sub-device, and is used to control the movement of the XY scanning galvanometer and receive signals from each photomultiplier tube, and reconstruct various images by combining the scanning trajectory of the XY scanning galvanometer and the signals of each photomultiplier tube to obtain second harmonic images, third harmonic images, two-photon fluorescence images and three-photon fluorescence images.

7. The endoscope system based on harmonic imaging and multiphoton fluorescence imaging according to claim 6, characterized in that: The control system is also connected to the first femtosecond pulse laser and the second femtosecond pulse laser, and is used for controlling the opening and closing of the first femtosecond pulse laser and the second femtosecond pulse laser.

8. The endoscope system based on harmonic imaging and multiphoton fluorescence imaging according to claim 1, characterized in that: The endoscope includes an achromatic objective lens and a portion of an optical fiber bundle; the portion of the optical fiber bundle is connected to the end of the optical fiber bundle, and two near-infrared femtosecond pulse lasers are focused on the measured tissue through the achromatic objective lens in the endoscope.

9. The endoscope system based on harmonic imaging and multiphoton fluorescence imaging according to claim 1, characterized in that: The endoscope is equipped with a handle, and the outer diameter of the working insertion portion of the endoscope is less than 6 mm.

10. An imaging method for an endoscope system based on harmonic imaging and multiphoton fluorescence imaging according to any one of claims 1 to 9, characterized in that: The following steps are involved: The first femtosecond pulse laser and the second femtosecond pulse laser respectively emit near-infrared femtosecond pulse lasers; Two near-infrared femtosecond pulse lasers merge through a prism and enter the XY scanning galvanometer. They then pass through the first long-wavelength dichroic mirror, are coupled into the fiber bundle by the coupling objective lens, and are finally focused on the tissue being tested through the endoscope. Two near-infrared femtosecond pulse lasers excite the tissue under test and its fluorescent substances to generate second harmonics, third harmonics, two-photon fluorescence, and three-photon fluorescence. The second harmonics, third harmonics, two-photon fluorescence, and three-photon fluorescence return along the original path and are reflected by the first long-wavelength dichroic mirror into the imaging device. Two near-infrared femtosecond pulse lasers scan the tissue under the action of an XY scanning galvanometer, and the imaging device obtains the second harmonic image, third harmonic image, two-photon fluorescence image and three-photon fluorescence image of the tissue under test.

Images