Multi-mode wide-field microscopic imaging system for lymphatic vessel-vein anastomosis

Abstract

The invention discloses a multi-modal wide-field microscopic imaging system for lymphatic vessel-vein anastomosis, and aims to solve the problems of low imaging contrast and insufficient positioning precision of bright-field imaging to tiny veins and near-infrared I-region imaging to lymphatic vessels in clinical microsurgery. The positive wide-field microscopic imaging system, the single silicon-based detector, the switchable optical module and the like are adopted to realize lymphatic vessel fluorescence imaging, vein negative development and bright field imaging under an eyepiece in a near-infrared II region. On the basis of absorption curves of hemoglobin and blood and in combination with the characteristic that a microscope objective collects light under the condition of a wide-field microscopic system, vein negative development of 416-456nm wave band light is innovatively introduced, the contrast ratio of veins and surrounding tissues is remarkably improved, the depth and signal background ratio of lymphatic vessel imaging are improved by adopting near-infrared II-region fluorescence imaging, and the imaging accuracy of the lymphatic vessel is improved. The device is simple in structure, low in cost, simple in principle, suitable for microsurgery such as lymphatic vessel-vein anastomosis and capable of providing finer and more reliable imaging support for clinic.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical microscopic imaging, and relates to a multimodal wide-field microscopic imaging system for lymphatic-venous anastomosis, which is suitable for accurate positioning and visualization of lymphatic vessels and veins in microsurgery. Background Art

[0002] Fluorescence imaging can reveal information about the structure and function of organisms and has been widely used in life science research and clinical diagnosis. This technology injects fluorescent dyes into biological tissues, or introduces fluorescent protein markers into samples, then excites these fluorescent probes with a light source of a specific wavelength to produce fluorescent signals, and finally uses an optical system to focus the fluorescent signals onto a detector to generate a fluorescent image. In recent years, fluorescence imaging technology based on the near-infrared II band (900-1880nm) has become the research frontier in this field. Compared with the visible light (380-760nm) and near-infrared I band (760-900nm), this band has lower scattering and appropriate water absorption in biological tissues, and its imaging has a greater penetration depth and a higher signal-to-background ratio, thus achieving better imaging effects.

[0003] In addition, hemoglobin's absorption of light in the 416-456nm band is significantly stronger than that of skin and fat tissue. This feature is sufficient to overcome the problem that microscope objectives are greatly affected by scattered light. It can make blood vessels show an obvious negative development effect under the irradiation of light in this band, significantly enhancing the contrast between blood vessels and surrounding tissues, providing strong support for vascular imaging and positioning.

[0004] In clinical microsurgery, it is crucial to be able to observe two tissue structures simultaneously and in real time. For example, in lymphatic-venous anastomosis surgery, the two catheters need to be imaged in real time. Before performing the operation, it is first necessary to use fluorescence imaging combined with ultrasound angiography or other imaging techniques to preliminarily locate the two catheters on the body surface and determine the incision position, and then use a traction line to temporarily pull open the incision or surrounding tissue to better expose the surgical area. During the operation, the microscope will focus on the incision area and use near-infrared zone I fluorescence imaging mode to image and locate the lymphatic vessels in the incision; at the same time, the bright field imaging mode is used to observe the overall condition of the veins and incisions. However, the existing imaging technology has the following shortcomings in actual surgery:

[0005] 1. Difficulty in venous visualization: Since the veins to be anastomosed are usually terminal veins, their diameter is usually less than 0.8 mm, making them difficult to locate. In addition, the veins and the overall background of the incision (such as fat, muscle, etc.) are similar in color, resulting in low image contrast. In other words, in the positive development mode, it is difficult to effectively distinguish between tiny veins and surrounding soft tissues, especially in complex incision environments, where background interference is more obvious, making it difficult for tiny veins to be clearly presented in bright field images.

[0006] 2. Insufficient accuracy in lymphatic vessel positioning: Compared with near-infrared II band light, near-infrared I band light is more strongly scattered by biological tissues, has limited penetration depth, and has a relatively low imaging signal background. Therefore, it is difficult to clearly present the position of deep lymphatic vessels. Even the resolution of shallow lymphatic vessels is very limited, which affects the precise positioning of lymphatic vessels.

[0007] In view of the above problems, the present invention proposes a multimodal wide-field microscopic imaging system for lymphatic-venous anastomosis. Summary of the invention

[0008] Aiming at the problems of difficulty in venous visualization and insufficient lymphatic vessel positioning accuracy in existing imaging systems during lymphatic-venous anastomosis, the present invention proposes a multimodal wide-field microscopic imaging system for lymphatic-venous anastomosis.

[0009] The specific technical solution of the present invention is as follows: a multimodal wide-field microscopic imaging system for lymphatic-venous anastomosis, the system comprising:

[0010] LED light source, used to provide two light sources: white light and near-infrared I zone light;

[0011] Silicon-based detectors, used to provide imaging targets;

[0012] The switchable optical cube module includes three optical cubes for near-infrared II fluorescence imaging, venous negative imaging, and bright field imaging.

[0013] Furthermore, near-infrared II fluorescence imaging achieves visualization of lymphatic vessels by exciting fluorescent probes in the lymphatic vessels with near-infrared I light; venous negative development utilizes the strong absorption characteristics of hemoglobin to visible light in a specific band to enhance the contrast between veins and surrounding tissues; and bright field imaging achieves real-time observation of the tissue incision area.

[0014] Furthermore, the white light in the LED light source is used for illumination of bright field imaging and venous negative development, and the near-infrared I region light is used to excite the fluorescent probe, and the two light sources can be quickly switched through a controller.

[0015] Furthermore, the venous negative imaging is based on the absorption curves of hemoglobin and blood, combined with the fact that the microscope objective is greatly affected by scattering when collecting light, and introduces 416-456nm band light to achieve venous negative imaging and enhance the contrast between veins and surrounding tissues.

[0016] Furthermore, optical cube No. 1 for realizing near-infrared II zone fluorescence imaging includes a 900nm long-wave pass dichroic mirror, a 900nm long-wave pass filter and an 850nm short-wave pass filter; near-infrared I zone light uniformly excites the fluorescent probe in the lymphatic vessels, and the emitted near-infrared II zone fluorescence is emitted as parallel light through the objective lens, and after passing through the 900nm long-wave pass dichroic mirror and the 900nm long-wave pass filter, it is focused on the target surface of the silicon-based detector by the sleeve lens, forming a near-infrared II zone fluorescence image of the lymphatic vessels.

[0017] Furthermore, the optical cube No. II for achieving venous negative development includes a 436nm bandpass filter and a semi-transparent, semi-reflective mirror; the white light enters the optical cube after being shaped by the shaping mirror group, and the light passes through the 436nm bandpass filter and is reflected by the semi-transparent, semi-reflective mirror, achieving uniform wide-field illumination on the surface of the biological tissue incision, and hemoglobin absorbs the light in this band. The signal light passes through the objective lens, the semi-transparent, semi-reflective mirror, the 436nm bandpass filter, and the sleeve lens and is captured by the silicon-based detector to form venous negative development.

[0018] Furthermore, the No. III optical cube used to achieve bright field imaging includes a semi-transparent and semi-reflective mirror. The white light is shaped by the shaping mirror group and then reflected by the semi-transparent and semi-reflective mirror in the optical cube, achieving uniform wide-field illumination on the surface of the biological tissue incision. At this time, the bright field image of the tissue under the sample incision can be directly observed through the eyepiece.

[0019] Furthermore, the switchable optical cube module comprises a bracket and a slide rail, the slide rail is fixed on the bracket, and three optical cubes are slidably connected to the slide rail. After sliding, one of the optical cubes is aligned with the microscope objective lens and the sleeve lens to receive the excitation light shaped by the shaping lens group.

[0020] Furthermore, the effective response band of the silicon-based detector is 400-1180nm, and its quantum efficiency in the band of 900nm and above can reach up to 40%, which can effectively detect the near-infrared II zone fluorescence tailing signal with a wavelength of fluorescence signal emission above 900nm; the silicon-based detector has a resolution of 1440×1152 pixels, and supports binning function to adjust the resolution to 720×576 to improve image brightness.

[0021] Furthermore, the switchable optical cube module can be replaced with a multimodal imaging module to achieve real-time synchronous imaging of lymphatic vessels and veins;

[0022] The multimodal imaging module includes a 436nm bandpass filter, a 900nm longwave pass filter, a semi-transparent and semi-reflective mirror, an 805DMSP and a near-infrared detector;

[0023] The semi-transparent and semi-reflective mirror is used to reflect the 780nm light shaped by the shaping lens group and focus it on the back focal plane of the objective lens; then, after passing through the 805DMSP and the objective lens, uniform wide-field illumination of the sample cut surface is achieved to excite the fluorescent probe in the lymphatic vessels;

[0024] 805DMSP is placed between the semi-transparent and semi-reflective mirror and the objective lens; the near-infrared II zone fluorescence signal is reflected by 805DMSP after passing through the objective lens, and is focused on the near-infrared detector target surface through a lens, and a 900nm long-wave pass filter is placed in front of the detector target surface; the near-infrared detector captures the near-infrared II zone fluorescence image of the lymphatic vessel;

[0025] The 436nm bandpass filter is placed between the visible light detector and the semi-transparent and semi-reflective mirror; it detects the signal light in the 416-456nm band and captures the negative imaging of the vein.

[0026] The present invention has the following beneficial effects:

[0027] (1) The present invention is aimed at specific microsurgical scenarios. Based on the absorption curves of hemoglobin and blood, combined with the characteristics of light collection by the microscope objective under the conditions of a wide-field microscope system, the present invention innovatively introduces 416-456nm band light for vein negative development, which significantly improves the contrast between veins and surrounding tissues, thereby enhancing the visualization of tiny veins. In addition, the near-infrared II zone fluorescence imaging technology is used to enhance the imaging depth and signal-to-background ratio, and improve the positioning accuracy of tiny lymphatic vessels. Through multimodal imaging, the present invention breaks through the limitations of existing microscope systems that rely solely on bright field images to locate tiny veins and near-infrared I zone images to locate lymphatic vessels, providing more reliable visualization support for minimally invasive surgery.

[0028] (2) The present invention uses only a single silicon-based visible-near infrared extended detector, which has the advantage of low cost. At the same time, the system optical module is simple and fast to switch, easy to operate, and easy to transform into a stereo microscope, which has greater flexibility and clinical application potential.

[0029] (3) The present invention is not only applicable to lymphatic-venous anastomosis, but can also be widely used in the fields of tumor resection, vascular surgery, minimally invasive surgery, etc. The present invention can provide high contrast and depth imaging based on multimodal imaging technology, which can effectively improve the accuracy and safety of surgery and has broad clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 It is a schematic diagram of the system structure of the present invention.

[0031] Figure 2 Schematic diagram of the structure of three optical cubes in the switching optical cube module.

[0032] Figure 3 Schematic diagram of the multimodal imaging system structure.

[0033] Figure 4 The application case of the multimodal wide-field microscopy system for lymphatic-venous anastomosis was demonstrated. DETAILED DESCRIPTION

[0034] The present invention will be further described below in conjunction with the accompanying drawings.

[0035] like Figure 1 As shown, the embodiment of the present invention is built based on an upright wide-field microscopic imaging system. The imaging system includes: a customized LED light source 1, a shaping lens group 2, a switchable optical cube module 3, a microscope objective 4, a stage 5, a tube lens 6, a silicon-based detector 7 and an eyepiece 8, which can achieve the following functions:

[0036] Near-infrared II fluorescence imaging: The fluorescent probes in the lymphatic vessels are excited by near-infrared I light to visualize the lymphatic vessels.

[0037] Negative venous imaging: using the strong absorption characteristics of hemoglobin to visible light in a specific band to enhance the contrast between veins and surrounding tissues;

[0038] Bright field imaging: enables real-time observation of tissue incision areas.

[0039] The switching optical cube module includes a bracket and a slide rail, the slide rail is fixed on the bracket, and three optical cubes are slidably connected to the slide rail. After sliding, one of the optical cubes is aligned with the microscope objective lens and the sleeve lens to receive the excitation light shaped by the shaping lens group. Optical cube No. Ⅰ has a built-in 900nm long-wave pass dichroic mirror, and a specific filter is installed on the incident light side to screen the excitation light that excites the fluorescent probe. A 900nm long-wave pass filter is installed on the outgoing light side to further screen the fluorescence in the near-infrared II band; optical cube No. Ⅱ has a built-in semi-transparent and semi-reflective mirror, a 436nm band-pass filter is installed on the incident light side, and the same band-pass filter is placed on the outgoing light side to filter out stray light; optical cube No. Ⅲ only has a semi-transparent and semi-reflective mirror.

[0040] The custom LED light source integrates two light sources: white light and near-infrared I light. White light is used for bright field imaging and venous negative imaging, and near-infrared I light is used to excite fluorescent probes. Through the controller, users can achieve rapid switching between the two light sources. The central wavelength of near-infrared I light is about 780nm.

[0041] The fluorescent probe used to mark lymphatic vessels is indocyanine green (ICG), which has been certified by the U.S. Food and Drug Administration (FDA) and has been widely used in clinical practice. The absorption peak wavelength of ICG is about 780nm, the fluorescence emission peak wavelength is about 810nm, and there is a near-infrared II region fluorescence tailing signal above 900nm.

[0042] The effective response band of the silicon-based detector is 400-1180nm, and its quantum efficiency in the band of 900nm and above can reach up to 40%, which can effectively detect the near-infrared II region fluorescence tailing signal emitted by ICG with a wavelength above 900nm. The detector has a resolution of 1440×1152 pixels, and supports the binning function to adjust the resolution to 720×576 to improve the image brightness. The adjusted resolution is still higher than the commonly used short-wave infrared camera (resolution is 640×512). This design not only greatly reduces costs, but also effectively meets different imaging needs.

[0043] The imaging system of the embodiment of the present invention is applied as follows:

[0044] First, the customized LED light source 1 emits near-infrared zone I light, which is shaped by the shaping lens group 2 and then enters the optical cube No. 1 in the switching optical cube module 3; the excitation light is reflected by the 900nm long-wave pass dichroic mirror and focused on the rear focal plane of the microscope objective 4, and then fills the objective exit pupil, and realizes uniform wide-field illumination of the sample incision surface after passing through the objective; the uniform excitation light excites the fluorescent probe ICG in the lymphatic vessels, and the near-infrared zone II fluorescence emitted by it is emitted as parallel light through the objective lens, and is focused on the target surface of the silicon-based detector 7 by the sleeve lens 6 after passing through the 900nm long-wave pass dichroic mirror and the 900nm long-wave pass filter, forming a near-infrared zone II fluorescence image of the lymphatic vessels.

[0045] Next, switch the LED light source 1 to the white light mode, push the pull rod to switch to the optical cube II, and the light enters the optical cube after being shaped by the shaping lens group; the light passes through the 436nm bandpass filter and is reflected by the semi-transparent and semi-reflective mirror, and is uniformly illuminated on the surface of the biological tissue incision through the microscope objective. Since hemoglobin has a strong absorption of light in this band, the signal light is captured by the silicon-based detector after passing through the objective lens, semi-transparent and semi-reflective mirror, 436nm bandpass filter, and sleeve lens to form venous negative imaging.

[0046] Finally, push the lever to switch to optical cube III, and the white light emitted by LED light source 1 is shaped by the shaping lens group and reflected by the semi-transparent and semi-reflective mirror in the optical cube, and then uniformly illuminates the surface of the biological tissue cut through the microscope objective lens. At this time, switch the microscope to bright field imaging mode, and directly observe the bright field image of the tissue under the sample cut through the eyepiece 8.

[0047] The present invention uses multimodal imaging technology combined with the switching of different optical components to provide clear and comprehensive images of the biological incision area, facilitating the precise operation of lymphatic-venous anastomosis.

[0048] The switchable optical cube module can be replaced by a multimodal imaging module to achieve real-time synchronous imaging of lymphatic vessels and veins; the multimodal imaging module includes a 436nm bandpass filter, a 900nm long-wave pass filter, a semi-transparent and semi-reflective mirror, an 805DMSP and a near-infrared detector; the structure of the multimodal imaging system proposed by the present invention is as follows Figure 4 As shown, the 780nm light is shaped by the shaping lens group and then reflected by the semi-transparent and semi-reflective mirror and focused on the back focal plane of the objective lens. Then, after passing through the 805DMSP and the objective lens, uniform wide-field illumination of the sample incision surface is achieved to excite the fluorescent probe in the lymphatic vessel. The near-infrared II zone fluorescence signal is reflected by the 805DMSP after passing through the objective lens and focused on the near-infrared detector target surface through the lens. A 900nm long-wave pass filter is placed in front of the detector target, and the detector captures the near-infrared II zone fluorescence image of the lymphatic vessel. At the same time, the annular white light illuminates the biological incision. The blood vessels have a strong absorption of the 416-456nm band light. A 436nm bandpass filter is placed in front of the visible light detector to detect the signal light in this band and capture the negative imaging of the vein. At this time, the two detectors can image the two catheters at the same time. After turning off the 780nm LED light source, the user can observe the bright field image of the sample incision surface through the eyepiece.

[0049] Example:

[0050] The mice were anesthetized by injection of 1% sodium pentobarbital 20 minutes before in vivo imaging, and then injected with 1 mg / mL ICG aqueous solution through the foot pad. Then, the mice were fixed in a suitable position under the microscope objective 4 (LSM04, Thorlabs, USA), and the skin tissue on the surface of the thigh area was peeled off. According to the imaging system in the above specific embodiment, the silicon-based detector 7 (THNIR 1450, Shenzhen Tonghao Juhui Optoelectronics Technology Co., Ltd.) first captured the near-infrared II zone fluorescence image of the lymphatic vessels, such as Figure 4 Then, by switching the LED light emission mode and the optical cube, the silicon-based detector 7 captures the negative imaging of the vein, as shown in (a) of FIG. Figure 4 Finally, by switching the optical cube and adjusting the microscope to the bright field imaging mode, the bright field image under the tissue incision can be observed in real time through the eyepiece. In order to record this image, we used a mobile phone camera to take a bright field image of the tissue incision seen under the eyepiece, as shown in Figure 4 As shown in (c) in .

[0051] In summary, the present invention proposes a multimodal wide-field microscopic imaging system for lymphatic-venous anastomosis, which overcomes the limitation of traditional clinical microscopic instruments that it is difficult to clearly locate tiny veins in bright field mode, and significantly improves the contrast of veins. At the same time, the present invention introduces near-infrared II zone fluorescence imaging, which can achieve greater depth and higher signal-to-background ratio of lymphatic fluorescence imaging. In addition, the present invention can be used as a reference for upgrading the corresponding multi-channel microscopic system to observe and fuse the images of each channel in real time. The results of laboratory live animal experiments show that the present invention can clearly and real-time display the distribution and status of two types of pipes in the body. Therefore, the present invention has great clinical application potential and scalability, and provides an important reference for the research and development of medical devices.

[0052] The above embodiment is an example to explain the present invention, rather than limiting the application cases of the present invention. Within the spirit of the present invention and the protection scope of the claims, any improvement and modification based on the present invention belongs to the protection scope of the present invention.

Claims

1. A multimodal wide-field microscopic imaging system for lymphatic-venous anastomosis, characterized by: The system includes: LED light source, used to provide two light sources: white light and near-infrared I zone light; Silicon-based detectors, used to provide imaging targets; The switchable optical cube module includes three optical cubes for near-infrared II fluorescence imaging, venous negative imaging, and bright field imaging.

2. A multimodal wide-field microscopic imaging system for lymphatic-venous anastomosis according to claim 1, characterized in that: Near-infrared II fluorescence imaging achieves visualization of lymphatic vessels by exciting fluorescent probes in the lymphatic vessels with near-infrared I light; venous negative development utilizes the strong absorption properties of hemoglobin for visible light in a specific band to enhance the contrast between veins and surrounding tissues; bright field imaging achieves real-time observation of tissue incision areas.

3. The multimodal wide-field microscopic imaging system for lymphatic-venous anastomosis according to claim 1, characterized in that: The white light in the LED light source is used for bright field imaging and venous negative imaging, and the near-infrared I region light is used to excite the fluorescent probe. The two light sources can be quickly switched through the controller.

4. The multimodal wide-field microscopic imaging system for lymphatic-venous anastomosis according to claim 1, characterized in that: The venous negative development is based on the absorption curve of hemoglobin and blood, combined with the characteristic that the microscope objective is greatly affected by scattering when collecting light, and introduces 416-456nm band light to achieve venous negative development and enhance the contrast between veins and surrounding tissues.

5. The multimodal wide-field microscopic imaging system for lymphatic-venous anastomosis according to claim 1, characterized in that: Optical cube No. 1 used to achieve near-infrared II zone fluorescence imaging includes a 900nm long-wave pass dichroic mirror, a 900nm long-wave pass filter and an 850nm short-wave pass filter; near-infrared I zone light uniformly excites the fluorescent probe in the lymphatic vessels, and the emitted near-infrared II zone fluorescence is emitted as parallel light through the objective lens, and after passing through the 900nm long-wave pass dichroic mirror and the 900nm long-wave pass filter, it is focused on the target surface of the silicon-based detector by the tube lens, forming a near-infrared II zone fluorescence image of the lymphatic vessels.

6. The multimodal wide-field microscopic imaging system for lymphatic-venous anastomosis according to claim 1, characterized in that: Optical cube No. II for achieving venous negative development includes a 436nm bandpass filter and a semi-transparent, semi-reflective mirror; white light enters the optical cube after being shaped by the shaping mirror group, and the light passes through the 436nm bandpass filter and is reflected by the semi-transparent, semi-reflective mirror to achieve uniform wide-field illumination on the surface of the biological tissue incision. Hemoglobin absorbs the light in this band, and the signal light passes through the objective lens, semi-transparent, semi-reflective mirror, 436nm bandpass filter, and sleeve lens and is captured by the silicon-based detector to form venous negative development.

7. The multimodal wide-field microscopic imaging system for lymphatic-venous anastomosis according to claim 1, characterized in that: The No. III optical cube used to achieve bright field imaging includes a semi-transparent and semi-reflective mirror. The white light is shaped by the shaping mirror group and then reflected by the semi-transparent and semi-reflective mirror in the optical cube, achieving uniform wide-field illumination on the surface of the biological tissue incision. At this time, the bright field image of the tissue under the sample incision can be directly observed through the eyepiece.

8. The multimodal wide-field microscopic imaging system for lymphatic-venous anastomosis according to claim 1, characterized in that: The switchable optical cube module comprises a bracket and a slide rail, the slide rail is fixed on the bracket, three optical cubes are slidably connected to the slide rail, after sliding, one of the optical cubes is aligned with the microscope objective lens and the sleeve lens to receive the excitation light shaped by the shaping lens group.

9. The multimodal wide-field microscopic imaging system for lymphatic-venous anastomosis according to claim 1, characterized in that: The effective response band of the silicon-based detector is 400-1180nm, and its quantum efficiency in the band of 900nm and above can reach up to 40%. It can effectively detect the near-infrared II region fluorescence tailing signal with a wavelength of fluorescence signal emission above 900nm; the silicon-based detector has a resolution of 1440×1152 pixels and supports the binning function to adjust the resolution to 720×576 to improve the image brightness.

10. The multimodal wide-field microscopic imaging system for lymphatic-venous anastomosis according to claim 1, characterized in that: The switchable optical cube module can be replaced with a multimodal imaging module to achieve real-time synchronous imaging of lymphatic vessels and veins; The multimodal imaging module includes a 436nm bandpass filter, a 900nm longwave pass filter, a semi-transparent and semi-reflective mirror, an 805DMSP and a near-infrared detector; The semi-transparent and semi-reflective mirror is used to reflect the 780nm light shaped by the shaping lens group and focus it on the back focal plane of the objective lens; then, after passing through the 805DMSP and the objective lens, uniform wide-field illumination of the sample cut surface is achieved to excite the fluorescent probe in the lymphatic vessels; The 805DMSP is placed between the semi-transparent and semi-reflective mirror and the objective lens; The near-infrared II zone fluorescence signal is reflected by 805DMSP after passing through the objective lens, and is focused on the near-infrared detector target surface through a lens. A 900nm long-wave pass filter is placed in front of the detector target surface. The near-infrared detector captures the near-infrared II zone fluorescence image of the lymphatic vessel. The 436nm bandpass filter is placed between the visible light detector and the semi-transparent and semi-reflective mirror; it detects the signal light in the 415-456nm band and captures the negative imaging of the vein.

Images