Living body fluorescence imaging system based on phase locking technology

Abstract

The utility model discloses a living body fluorescence imaging system based on a phase locking technology, which comprises an objective table, a laser and a dodging device, and the dodging device is connected with the laser and is used for adjusting laser to uniformly irradiate a sample; the refrigeration type near-infrared camera receives fluorescence, excited by the laser, of the sample, and an objective lens of the refrigeration type near-infrared camera is connected with an optical filter; the water cooling structure comprises a cooling-water machine, the refrigeration type near-infrared camera is provided with a water inlet and a water outlet, the water inlet of the refrigeration type near-infrared camera is connected with the water outlet of the cooling-water machine through a water inlet pipe, and the water outlet of the refrigeration type near-infrared camera is connected with the water return port of the cooling-water machine through a water outlet pipe; the phase locking module is used for modulating the pulse of the laser to be synchronous with the exposure time of the refrigeration type near-infrared camera and collecting the data of the refrigeration type near-infrared camera for phase locking data processing, and the phase locking module is connected with the refrigeration type near-infrared camera and the laser. According to the utility model, by using the phase locking technology, the background noise can be greatly reduced, the signal-to-noise ratio and the signal-to-background ratio of imaging can be improved, and low-scattering and high-definition imaging results can be obtained in a long wave band.

Description

Technical Field

[0001] The utility model relates to the technical field of optics, in particular to a living body fluorescence imaging system based on phase-locked technology. Background Art

[0002] In vivo fluorescence imaging is an advanced biomedical imaging technique that utilizes fluorescent markers to observe and track biological processes in living tissue. Traditional in vivo fluorescence imaging relies on single-photon imaging, which involves injecting a contrast agent into the organism, stimulating it with a laser to emit fluorescence, and finally capturing it with a camera. However, before reaching the camera, the fluorescence signal is inevitably reflected, absorbed, and scattered by the tissue, affecting image quality. Scattering has the greatest impact on imaging. According to the Rayleigh scattering law, longer wavelengths reduce scattering. Therefore, increasing the detection wavelength can effectively suppress the effects of light scattering, thereby improving image contrast. However, due to the short emission peak wavelength of current contrast agents, increasing the detection wavelength weakens the camera imaging signal, necessitating a longer camera integration time to enhance the signal. However, this increased signal also increases noise, causing the camera to easily reach its detection threshold, making further signal enhancement impossible. Consequently, the signal is insufficient to overcome the noise, resulting in a poorly defined image. Summary of the Invention

[0003] To overcome the defects of the existing technology, a living fluorescence imaging system based on phase-locked technology is now provided, which can solve the problem that when single-photon imaging increases the detection band, noise masks the signal and a clear image cannot be obtained.

[0004] To achieve the above technical effects, the present invention provides a living body fluorescence imaging system based on phase lock-in technology, which includes:

[0005] a stage for placing samples;

[0006] a laser for emitting laser light;

[0007] A light homogenizer for adjusting the uniform irradiation of the laser on the sample, connected to the laser, and arranged toward the stage;

[0008] a refrigerated near-infrared camera for receiving fluorescence emitted by the sample when excited by the laser, wherein the objective lens of the refrigerated near-infrared camera is connected to a filter;

[0009] A water cooling structure includes a water chiller. The refrigerated near-infrared camera is provided with a water inlet and a water outlet. The water inlet of the refrigerated near-infrared camera is connected to the water outlet of the water chiller via a water inlet pipe, and the water outlet of the refrigerated near-infrared camera is connected to the return water port of the water chiller via a water outlet pipe.

[0010] A phase-locking module is used to modulate the pulse of the laser to synchronize with the exposure time of the refrigerated near-infrared camera and collect data from the refrigerated near-infrared camera for phase-locked data processing. The phase-locking module is connected to the refrigerated near-infrared camera and the laser.

[0011] The phase-locked module can modulate the pulse of the laser to synchronize with the exposure time of the refrigerated near-infrared camera. That is, it can be achieved by periodically modulating the laser on and off and synchronously starting the near-infrared camera, so that the camera only captures images when the laser pulse occurs. The phase-locked module collects the data of the near-infrared camera in each cycle for phase-locked data processing, and updates the iterative phase-locked diagram after the data processing is completed, while continuously displaying the original imaging image for comparison with the phase-locked diagram.

[0012] Noise has a wide frequency range and generally includes noise generated by ambient light captured by the camera, as well as the camera's own internal noise, namely thermal noise and dark current noise. However, because the phase-locked module modulates the laser to a target frequency, it achieves co-frequency modulation of the fluorescence excited by the laser. The camera captures the modulated fluorescence at the target frequency, and the image data is phase-locked. This ensures that the final phase-locked result contains only the signal at the target frequency, effectively eliminating the influence of non-target frequency noise (such as noise generated by ambient light), thereby significantly reducing imaging noise. Furthermore, compared to air-cooled or uncooled near-infrared cameras, water-cooled near-infrared cameras achieve lower dark current noise and thermal noise. This results in a lower noise floor in the resulting image, allowing for longer exposure times to improve image quality. Furthermore, the camera's water cooling system circulates water to remove heat generated by the cooler's hot end in real time, enabling the internal cooler to maintain efficient cooling and ensure continuous and stable camera operation.

[0013] Preferably, the filter is a longpass filter. Longpass filters, also known as high-pass filters, operate by allowing light longer than a selected wavelength to pass while blocking light shorter than that wavelength. Longpass filters maximize the passage of fluorescence (i.e., emission light) while minimizing excitation light, thereby achieving an optimal signal-to-noise ratio.

[0014] Preferably, the filter is reflective.

[0015] Preferably, the number of the optical filters is at least two, and the plurality of optical filters are installed in a retaining ring, which is connected to the objective lens.

[0016] Preferably, the objective lens is a telephoto lens. Stacking two or more reflective filters and using a telephoto lens can increase the proportion of linear incident photons, reduce the collision between photons and the lens barrel, and thus effectively suppress "glare".

[0017] Preferably, there are two homogenizers, which are symmetrically arranged above the stage. The laser is connected to the input port of the fiber optic splitter through a first optical fiber, and each homogenizer is connected to the output port of the fiber optic splitter through a second optical fiber.

[0018] Preferably, the phase-locked module is an FPGA board or a single-chip microcomputer.

[0019] The beneficial effects of the present invention are that the present invention, a living fluorescence imaging system based on phase-locked technology, can increase the detection band through a long-wave pass filter to suppress the influence of scattering, and significantly reduce background noise through a phase-locked module, allowing many weak signals "swamped" by noise to appear, thereby improving the signal-to-noise ratio and signal-to-background ratio of imaging, achieving high-precision, distortion-free imaging for in situ measurements, and improving the ultimate accuracy of the fluorescence imaging system, so that people can obtain low-scattering and high-definition imaging results in the long-wave band. At the same time, it can also reduce experimental costs and reduce drug toxicity and phototoxicity. For example, using phase-locked technology, under the condition of constant light intensity, the injection dose can be reduced to 1 / 20 without deteriorating the imaging quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] In order to more clearly illustrate the technical solutions in the embodiments of the present invention, the following briefly introduces the drawings required for use in the description of the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0021] Figure 1 This is a schematic diagram of the overall structure of the living body fluorescence imaging system based on the phase-locked technology of the present invention.

[0022] Figure 2 This is a schematic diagram of the clamping ring structure of the living body fluorescence imaging system based on the phase-locked technology of the utility model.

[0023] The corresponding relationship of the labels in the figure is as follows:

[0024] 1. Stage; 2. Laser; 3. Homogenizer; 4. Refrigerated near-infrared camera; 401. Objective lens; 5. Filter; 6. Chiller; 7. Water inlet pipe; 8. Water outlet pipe; 9. Fiber optic beam splitter; 10. Optical fiber; 11. Data cable; 12. Computer; 13. Mouse; 14. Snap ring. DETAILED DESCRIPTION

[0025] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0026] See also Figures 1 to 2 As shown, the embodiment of the present invention provides a living body fluorescence imaging system based on phase-locked technology, including a stage 1, a laser 2, a homogenizer 3, a refrigerated near-infrared camera 4, a phase-locked module, and a chiller 6. Among them:

[0027] Stage 1 is used to place a sample. In this embodiment, the sample is a mouse 13 injected with a contrast agent. Laser 2 is used to emit laser light. Homogenizer 3 is connected to laser 2 and positioned toward stage 1. It is used to adjust the laser light emitted by laser 2 so that it is evenly irradiated on mouse 13 on stage 1. This allows the laser light to penetrate mouse 13 and stimulate the contrast agent therein to emit fluorescence of different wavelengths. Filter 5 is attached to the objective lens 401 of the cooled near-infrared camera 4, which is the camera lens. The fluorescence passes through the tissue of mouse 13 again and is captured by the cooled near-infrared camera 4.

[0028] The water cooling structure includes a chiller 6. A water inlet and a water outlet are provided on the refrigerated near-infrared camera 4. The water inlet of the refrigerated near-infrared camera 4 is connected to the water outlet of the chiller through a water inlet pipe, and the water outlet of the refrigerated near-infrared camera 4 is connected to the return water port of the chiller through a water outlet pipe.

[0029] Specifically, the cooled near-infrared camera 4 is equipped with a refrigerator, which provides a low-temperature environment for the sensor within the camera and removes heat generated by the hot end of the refrigerator in real time through water cooling. The water cooling structure also includes a water-cooling pipe attached to the surface of the refrigerator. One end of the water-cooling pipe passes through the water inlet of the cooled near-infrared camera 4 and is connected to the water inlet pipe 7, and the other end of the water-cooling pipe passes through the water outlet of the cooled near-infrared camera 4 and is connected to the water outlet pipe 8. Preferably, a water-cooling plate can also be installed on the surface of the refrigerator, so that the water inlet port of the water-cooling plate is connected to the water outlet port of the chiller through the water inlet pipe 7, and the water outlet port of the water-cooling plate is connected to the return water port of the chiller 6 through the water outlet pipe 8.

[0030] The cooler can be a Peltier cooler or a Stirling cooler. In this embodiment, a Peltier cooler is used, connected to the camera's power system, and the cold end of the Peltier cooler is brought into contact with the image sensor of the cooled near-infrared camera 4 to cool the sensor. A water-cooled plate is fixed to the hot end of the Peltier cooler using thermally conductive adhesive, which removes heat from the hot end and improves the cooling efficiency of the Peltier cooler. The Peltier cooler and water-cooled plate are integrated with the camera system to ensure the compactness and functionality of the overall design. The cooled near-infrared camera 4 can use the First Light C-RED 2 camera model. Air cooling can also be used for cooling, where a heat sink and a cooling fan are installed at the hot end of the cooler. When the temperature is too high, the fan automatically starts to blow heat away from the heat sink.

[0031] The phase-locked module is connected to the cooled near-infrared camera 4 and the laser 2 via a data line 11, and is used to modulate the pulse of the laser 2 to synchronize with the exposure time of the cooled near-infrared camera 4, and to collect data from the cooled near-infrared camera 4 for phase-locked data processing.

[0032] In this embodiment, the phase-lock module synchronizes the laser 2 pulses with the exposure time of the cooled near-infrared camera 4 by periodically modulating the laser 2's on-off state and simultaneously activating the cooled near-infrared camera 4. Specifically, the phase-lock module is connected to the laser 2 and modulates the laser's on-off state by outputting a specific waveform, making the laser's on-off state periodic. The phase-lock module is also connected to the cooled near-infrared camera 4 to control the camera's on / off state and collect camera data. Specifically, the phase-lock module simultaneously activates the camera and laser modulation at the start of phase-lock, ensuring that the camera captures images only when laser 2 is emitting. The camera collects data during each cycle for phase-locked data processing and updates the iterative phase-locked graph each cycle.

[0033] In a preferred embodiment, filter 5 is a longpass filter. The detection wavelength band is selected by filter 5, and a longpass filter can be used to extend the detection wavelength band. That is, light is transmitted only when its wavelength exceeds the filter's specific starting wavelength. Furthermore, imaging in the near-infrared region II (900nm-1700nm) is superior to imaging in the near-infrared region I (700nm-900nm). The cooled near-infrared camera 4 can detect both near-infrared regions I and II. For example, a 1300nm longpass filter can achieve imaging in the 1300-1700nm wavelength band (only up to 1700nm because current cameras can only capture light up to 1700nm).

[0034] As a preferred embodiment, the filter 5 is reflective.

[0035] As a preferred embodiment, the number of filters 5 is at least two, and the multiple filters 5 are installed in the retaining ring 14, and the retaining ring 14 is connected to the objective lens 401. Figure 2 As shown, in this embodiment, the number of filters 5 is three.

[0036] As a preferred embodiment, the objective lens 401 is a telephoto lens.

[0037] As a preferred embodiment, Figure 1 As shown, there are two homogenizers 3, symmetrically positioned above the stage 1. The laser 2 is connected to the input port of the fiber optic beam splitter 9 via a first optical fiber, and each homogenizer 3 is connected to the output port of the fiber optic beam splitter 9 via a second optical fiber. The fiber optic beam splitter splits the laser light in the first optical fiber into two laser beams, which are then input into the homogenizer 3 via the second optical fiber. Bilateral symmetrical illumination achieves uniform illumination of the sample, and this illumination method is reflective. Transmissive illumination, i.e., illumination from bottom to top, can also be used. Furthermore, uniform illumination can be selected to eliminate or retain speckle.

[0038] In a preferred embodiment, the phase-lock module is an FPGA board or a single-chip microcomputer. Insert the FPGA board with phase-lock functionality into computer 12, set the phase-lock parameters (camera integration time, phase-lock period, and phase-lock frequency) through software, and click Start. The FPGA board will periodically modulate the laser 2 on and off, simultaneously turning on the camera and importing the camera data.

[0039] The specific implementation steps of the in vivo fluorescence imaging system based on phase lock technology of the present invention are as follows:

[0040] S1, inject 200 μL of 0.05 mg / ml AIE contrast agent into the tail vein of mice;

[0041] S2. After the contrast agent is evenly distributed in the mouse body, turn on laser 2 and set the laser power, for example, to 10 W, so that the laser irradiating the mouse does not affect its physiological state;

[0042] S3. Set the exposure time of the near-infrared camera, for example, 10 seconds, so that it reaches the upper limit of its detection capability, that is, close to overexposure;

[0043] S4. Open the phase-lock software, set the phase-lock frequency, period, and confirmed camera exposure time, and click Start. The built-in FPGA board will synchronously read the camera data and modulate the laser on and off of Laser 2.

[0044] S5 and the FPGA board collect camera data every cycle and perform phase-locked data processing. After the data processing is completed, the software will update the iterative phase-locked diagram every cycle, while continuously displaying the original imaging image, thereby achieving low-scattering and high-definition imaging results in the long-wave band.

[0045] Any portion not described in the present invention is the same as the prior art or can be implemented using the prior art. Although the embodiments of the present invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions, and variations may be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A living fluorescence imaging system based on phase-locked technology, characterized in that: include: a stage for placing samples; a laser for emitting laser light; A light homogenizer for adjusting the uniform irradiation of the laser on the sample, connected to the laser, and arranged toward the stage; a refrigerated near-infrared camera for receiving fluorescence emitted by the sample when excited by the laser, wherein the objective lens of the refrigerated near-infrared camera is connected to a filter; A water cooling structure includes a water chiller. The refrigerated near-infrared camera is provided with a water inlet and a water outlet. The water inlet of the refrigerated near-infrared camera is connected to the water outlet of the water chiller via a water inlet pipe, and the water outlet of the refrigerated near-infrared camera is connected to the return water port of the water chiller via a water outlet pipe. A phase-locking module is used to modulate the pulse of the laser to synchronize with the exposure time of the refrigerated near-infrared camera and collect data from the refrigerated near-infrared camera for phase-locked data processing. The phase-locking module is connected to the refrigerated near-infrared camera and the laser.

2. The in vivo fluorescence imaging system based on phase lock-in technology according to claim 1, characterized in that: The optical filter is a long-wave pass filter.

3. The in vivo fluorescence imaging system based on phase lock technology according to claim 2, characterized in that: The filter is reflective.

4. The in vivo fluorescence imaging system based on phase lock technology according to claim 3, characterized in that: The number of the optical filters is at least two, and the plurality of optical filters are installed in a retaining ring, which is connected to the objective lens.

5. The in vivo fluorescence imaging system based on phase lock-in technology according to claim 1, characterized in that: The objective lens is a telephoto lens.

6. The in vivo fluorescence imaging system based on phase lock-in technology according to claim 1, characterized in that: There are two light homogenizers, which are symmetrically arranged above the stage. The laser is connected to the input port of the fiber optic beam splitter through a first optical fiber, and each light homogenizer is connected to the output port of the fiber optic beam splitter through a second optical fiber.

7. The in vivo fluorescence imaging system based on phase lock-in technology according to claim 1, characterized in that: The phase-locked module is an FPGA board or a single-chip microcomputer.

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