Time-gated wide-field fluorescence lifetime imaging device and method

Abstract

This invention relates to the field of optical imaging technology, specifically to a time-gated wide-field fluorescence lifetime imaging device and method, applicable to fluorescence lifetime imaging of biological tissues. The wide-field fluorescence lifetime imaging device includes an excitation module, a sample stage, a detection module, and a timing controller. The excitation module includes a laser, a homogenizing fiber, and a laser collimating lens; the sample stage is used to mount the imaging target; the detection module includes a filter switching assembly, a high-transmittance imaging lens, and an area array detector; the timing controller outputs a trigger signal to the laser to control the laser to emit pulsed laser light, and outputs a gating trigger signal to the area array detector to control the area array detector to open the gating exposure window at one or more preset delay time points after the laser stops emitting pulsed laser light. Thus, the area array detector and the timing controller can be controlled synchronously, thereby expanding the detection band, increasing imaging speed, and improving system stability in in vivo deep tissue fluorescence lifetime imaging.

Description

Technical Field

[0001] This invention relates to the field of optical imaging technology, specifically providing a time-gated wide-field fluorescence lifetime imaging device and method, which is suitable for fluorescence lifetime imaging of biological tissues, and especially suitable for fluorescence lifetime imaging of in vivo deep tissues in the near-infrared to short-wave infrared bands. Background Technology

[0002] Fluorescence lifetime imaging (FLI), as an important optical imaging method, can reflect the microenvironment parameters of a sample by detecting the time characteristics of fluorophore luminescence decay. It is unaffected by probe concentration and excitation light intensity and is widely used in life sciences, materials science and chemical analysis.

[0003] Time-correlated single photon counting (TCSPC) is currently the most commonly used fluorescence lifetime imaging method. Although it has high temporal resolution, it requires pixel-by-pixel scanning to collect data. Fitting the fluorescence intensity decay curve of a single pixel requires at least several or even thousands of photons, so the imaging speed is usually relatively slow.

[0004] In contrast, time-gated wide-field imaging can significantly shorten imaging time. However, when using a chopper to segment the fluorescence signal, the introduction of high-speed rotating mechanical components leads to unnecessary vibrations, noise, and potential mechanical failure points. Long-term stability and timing accuracy can decrease due to mechanical wear, requiring frequent calibration and resulting in low system integration.

[0005] Furthermore, the aperture of a chopper typically requires a complex relay optical path system containing multiple lenses to accurately image the image onto the modulation plane. During this process, in order to match the size of the chopper aperture and the beam cross-section, beam contraction or expansion transformation is required, which inevitably introduces aberrations, causes additional lens transmission losses and interface reflection losses, resulting in a significant attenuation of the light flux finally reaching the detector. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a time-gated wide-field fluorescence lifetime imaging device and method that can synchronously control an array detector and a timing controller, thereby expanding the detection band, increasing imaging speed, and improving system stability in in vivo deep tissue fluorescence lifetime imaging.

[0007] This invention provides a time-gated wide-field fluorescence lifetime imaging device, specifically comprising an excitation module, a sample stage, a detection module, and a timing controller. The excitation module includes a laser, a homogenizing fiber, and a laser collimating lens. The homogenizing fiber is located in the output optical path of the laser, and the laser collimating lens is located in the output optical path of the homogenizing fiber. The sample stage is located in the transmission optical path of the laser collimating lens and is used to mount the imaging target. The detection module includes a filter switching assembly, a high-transmittance imaging lens, and an area array detector. The filter switching assembly is located in the output optical path of the sample stage, the high-transmittance imaging lens is located in the transmission optical path of the filter switching assembly, and the area array detector is located in the transmission optical path of the high-transmittance imaging lens. The timing controller is signal-connected to both the laser and the area array detector. The timing controller outputs a trigger signal to the laser to control the laser to emit pulsed laser light, and outputs a gating trigger signal to the area array detector to control the area array detector to open the gating exposure window at one or more preset delay time points after the laser stops emitting pulsed laser light.

[0008] Preferably, the response band of the area array detector covers at least the near-infrared spectral region.

[0009] Preferably, the area array detector is a shortwave infrared area array detector; the response band of the shortwave infrared area array detector covers 0.85μm to 2.5μm.

[0010] Preferably, the shortwave infrared array detector is an HgCdTe array detector.

[0011] Preferably, the timing controller is a programmable logic device (PLD). The PLD controls the laser to turn on and off by outputting high and low level signals, and controls the exposure window of the area array detector to turn on and off by outputting another high and low level signal.

[0012] Preferably, the filter switching component is an electric filter wheel, which is connected to a timing controller to switch filters of different wavelengths at a preset delay time point according to the instructions of the timing controller.

[0013] Preferably, the high-transmittance imaging lens is a near-infrared high-transmittance lens; the near-infrared high-transmittance lens has a transmittance of more than 90% in the 0.85μm to 2.5μm band.

[0014] The present invention provides a time-gated wide-field fluorescence lifetime imaging method, applied to the time-gated wide-field fluorescence lifetime imaging device provided in the first aspect embodiment, specifically including the following steps S100, S200, S300, and S400: Step S100: The timing controller outputs a trigger signal to the laser, controlling the laser to emit pulsed laser light and irradiate the surface of the imaging target to excite the fluorescent probe; Step S200: The timing controller stops triggering the laser; Step S300: After the first preset delay time, the timing controller outputs the first gating trigger signal to the area array detector, controls the area array detector to open the gating exposure window, and acquires the first fluorescence intensity image. Step S400: After the Nth preset delay time, the timing controller outputs the Nth gated trigger signal to the area array detector, controlling the area array detector to open the gated exposure window again and acquire the Nth fluorescence intensity image. Step S500: Reconstruct the fluorescence lifetime image of the imaging target based on the first fluorescence intensity image to the Nth fluorescence intensity image.

[0015] Preferably, the time-gated wide-field fluorescence lifetime imaging device further includes a data processing unit; the data processing unit reconstructs the fluorescence lifetime image of the imaging target based on the first fluorescence intensity image to the Nth fluorescence intensity image through a fitting algorithm.

[0016] Preferably, the timing controller determines the trigger signal and the first gated trigger signal to the Nth gated trigger signal based on the imaging system parameters; the imaging system parameters include the imaging frame rate of the area array detector, the exposure time, and the first preset delay time to the Nth preset delay time, and also include the laser pulse width of the laser.

[0017] Compared with the prior art, the present invention can achieve the following beneficial effects: A time-gated wide-field fluorescence lifetime imaging device includes an excitation module, a sample stage, a detection module, and a timing controller. The excitation module includes a laser, a homogenizing fiber, and a laser collimating lens. The homogenizing fiber is located in the output optical path of the laser, and the laser collimating lens is located in the output optical path of the homogenizing fiber. The sample stage is located in the transmission optical path of the laser collimating lens and is used to mount the imaging target. The detection module includes a filter switching assembly, a high-transmittance imaging lens, and an area array detector. The filter switching assembly is located in the output optical path of the sample stage, the high-transmittance imaging lens is located in the transmission optical path of the filter switching assembly, and the area array detector is located in the transmission optical path of the high-transmittance imaging lens. The timing controller is signal-connected to both the laser and the area array detector. The timing controller outputs a trigger signal to the laser to control the laser to emit pulsed laser light, and outputs a gating trigger signal to the area array detector to control the area array detector to open the gating exposure window at one or more preset delay time points after the laser stops emitting pulsed laser light.

[0018] The present invention provides a time-gated wide-field fluorescence lifetime imaging device that can synchronously control the area array detector and the timing controller, thereby expanding the detection band, improving the imaging speed, and enhancing the system stability in fluorescence lifetime imaging of in vivo deep tissues.

[0019] In some embodiments, the present invention uses an HgCdTe (MCT) area array detector as the detection core, which can respond to a spectrum with wavelengths from 0.85μm to 2.5μm, breaking through the depth limitations of visible light and near-infrared region I in live imaging, and realizing dynamic observation of deep tissue imaging targets such as cells and tissues in living organisms.

[0020] In some embodiments, the present invention employs a full-field parallel acquisition mode, which generates high-precision synchronous timing through programmable logic devices to directly control the laser pulses and the gated exposure of the area array detector, thereby improving the imaging speed while maintaining high temporal resolution.

[0021] In some embodiments, the present invention employs a fully electronic timing control scheme, which can avoid mechanical wear and vibration sources generated by mechanical choppers, significantly improve the stability and integration of the imaging device, and effectively reduce aberrations and light energy loss by simplifying the optical path structure, thus ensuring the signal-to-noise ratio and sensitivity of imaging. Attached Figure Description

[0022] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 This is a schematic diagram of the structure of a time-gated wide-field fluorescence lifetime imaging device provided in an embodiment of the present invention; Figure 2 This is a schematic diagram of the principle of a time-gated wide-field fluorescence lifetime imaging device provided in an embodiment of the present invention; Figure 3 This is a schematic flowchart of a time-gated wide-field fluorescence lifetime imaging method provided in an embodiment of the present invention; Figure 4 This is a sequence of fluorescence intensity images captured by the first embodiment of the time-gated wide-field fluorescence lifetime imaging method provided by the present invention; Figure 5 It is based on Figure 4 The fluorescence lifetime image reconstructed from the fluorescence intensity image sequence shown; Figure 6 This is a sequence of fluorescence intensity images captured by a second embodiment of the time-gated wide-field fluorescence lifetime imaging method provided by the present invention; Figure 7 It is based on Figure 6 The fluorescence lifetime image reconstructed from the fluorescence intensity image sequence shown; Figure 8 This is a sequence of fluorescence intensity images captured by the time-gated wide-field fluorescence lifetime imaging method provided by the present invention, according to a third embodiment. Figure 9 It is based on Figure 8 The fluorescence lifetime image is reconstructed from the fluorescence intensity image sequence shown.

[0023] The reference numerals in the accompanying drawings include: 1. Laser; 2. Uniform optical fiber; 3. Laser collimating lens; 4. Imaging target; 5. Filter switching assembly; 6. High-transmittance imaging lens; 7. Area array detector; 8. Timing controller. Detailed Implementation

[0024] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and do not constitute a limitation thereof. Similar elements in different embodiments are referred to by associated similar element reference numerals. In the following embodiments, many details are described to facilitate a better understanding of the invention. However, those skilled in the art will readily recognize that some features may be omitted in different situations, or may be replaced by other elements, materials, or methods. In some cases, certain operations related to the invention are not shown or described in the specification. This is to avoid obscuring the core parts of the invention with excessive description. For those skilled in the art, detailed description of these related operations is not necessary; they can fully understand the related operations based on the description in the specification and general technical knowledge in the art.

[0025] It should be noted that, unless otherwise specified, the embodiments and features described in this invention can be combined to form various implementations. Furthermore, the order of the steps or actions in the method description can be changed or adjusted in a manner readily apparent to those skilled in the art. Therefore, the various orders in the specification and drawings are merely for the clear description of a particular embodiment and do not imply a mandatory order, unless otherwise stated that a particular order must be followed.

[0026] In the description of this invention, it should be understood that the terms "center," "longitudinal," "lateral," "length," "width," "thickness," "upper," "lower," "front," "rear," "left," "right," "vertical," "horizontal," "top," "bottom," "inner," "outer," "clockwise," and "counterclockwise," etc., indicating orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings, are only for the convenience of describing the invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation, and therefore should not be construed as a limitation of the invention. Furthermore, the terms "first," "second," etc., are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined with "first," "second," etc., may explicitly or implicitly include one or more of that feature. In the description of this invention, unless otherwise stated, "a plurality of" means two or more.

[0027] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art will understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0028] The present invention will now be described in detail with reference to the accompanying drawings and embodiments.

[0029] The present invention provides a time-gated wide-field fluorescence lifetime imaging device and method that can synchronously control the area array detector 7 and the timing controller 8, thereby expanding the detection band, improving the imaging speed, and enhancing the system stability in in vivo deep tissue fluorescence lifetime imaging.

[0030] like Figure 1As shown, an embodiment of the first aspect of the present invention provides a time-gated wide-field fluorescence lifetime imaging device, specifically including an excitation module, a sample stage, a detection module, and a timing controller 8. The excitation module includes a laser 1, a homogenizing fiber 2, and a laser collimating lens 3. The homogenizing fiber 2 is located in the output optical path of the laser 1, and the laser collimating lens 3 is located in the output optical path of the homogenizing fiber 2. The sample stage is located in the transmission optical path of the laser collimating lens 3, and the sample stage is used to mount the imaging target 4. The detection module includes a filter switching assembly 5, a high-transmittance imaging lens 6, and an area array detector 7. The filter switching assembly 5 is located in the output optical path of the sample stage, the high-transmittance imaging lens 6 is located in the transmission optical path of the filter switching assembly 5, and the area array detector 7 is located in the transmission optical path of the high-transmittance imaging lens 6. The timing controller 8 is connected to the laser 1 and the area array detector 7 respectively. The timing controller 8 is used to output a trigger signal to the laser 1 to control the laser 1 to emit pulsed laser, and to output a gating trigger signal to the area array detector 7 to control the area array detector 7 to open the gating exposure window at one or more preset delay time points after the laser 1 stops emitting pulsed laser.

[0031] Specifically, the excitation module includes a laser 1 for emitting pulsed laser light. The pulsed laser light emitted by the laser 1 passes through a homogenizing fiber 2 located in the output optical path, forming a uniform light spot under the homogenization effect of the homogenizing fiber 2. The uniform light spot passes through a laser collimating lens 3, and under the collimation effect of the laser collimating lens 3, it illuminates the surface of the imaging target 4 mounted on the sample stage to activate the fluorescent probe. The fluorescence emitted by the fluorescent probe passes through a filter switching assembly 5 located in the output optical path, thereby filtering out fluorescence signals of a specific wavelength. The fluorescence signals of the specific wavelength pass through a high-transmittance imaging lens 6, are focused by the high-transmittance imaging lens 6, and are received by an area array detector 7. Thus, within a preset gated exposure window, a fluorescence lifetime image is reconstructed based on the acquired fluorescence signal to facilitate the analysis of the microenvironment parameters of the imaging target 4.

[0032] The timing controller 8 outputs a trigger signal to the laser 1 to control the laser 1 to emit pulsed laser light, and outputs a gating trigger signal to the area array detector 7 at one or more preset delay time points after the laser 1 stops emitting pulsed laser light, to control the area array detector 7 to delay the opening of the gating exposure window. The delay time of the gating trigger signal relative to the trigger signal can be controlled to the microsecond or even nanosecond level, thereby improving the imaging speed and accuracy of wide-field fluorescence lifetime imaging.

[0033] Therefore, the present invention provides a time-gated wide-field fluorescence lifetime imaging device that can synchronously control the area array detector 7 and the timing controller 8, thereby expanding the detection band, improving the imaging speed, and enhancing the system stability in in vivo deep tissue fluorescence lifetime imaging.

[0034] Among them, the uniform optical fiber 2 has a homogenizing effect on the laser, which can avoid the beam distortion caused by the mismatch between the laser wavelength and the fiber size when switching light sources. At the same time, it works with the laser collimating lens 3 to optimize the stability of the optical path, thereby enabling rapid switching between different light sources and avoiding repeated beam adjustment.

[0035] Among them, the high-transmittance imaging lens 6 can be a high-transmittance imaging lens that transmits visible light, near-infrared light, or both visible light and near-infrared light, so that the time-gated wide-field fluorescence lifetime imaging device can be used to perform fluorescence lifetime imaging on different types of imaging targets 4.

[0036] It is understood that the time-gated wide-field fluorescence lifetime imaging device provided by the present invention can employ different types of area array detectors 7 with different response bands, thus making it suitable for fluorescence lifetime imaging of different types of biological tissues.

[0037] Preferably, in combination with the above scheme, as an embodiment of the present invention, the response band of the area array detector 7 at least covers the near-infrared spectral region.

[0038] In some possible embodiments, the time-gated wide-field fluorescence lifetime imaging device employs an area array detector 7 whose response band covers the near-infrared spectral region, or an area array detector 7 whose response band covers the visible to near-infrared spectral region (e.g., 0.4 μm–2.5 μm). For example, when an InGaAs area array detector is used, it can effectively respond to visible light and part of the near-infrared band (wavelengths from 0.9 μm to 1.7 μm).

[0039] Preferably, in conjunction with the above scheme, as an embodiment of the present invention, the area array detector 7 is a shortwave infrared area array detector 7; the response band of the shortwave infrared area array detector 7 covers 0.85μm to 2.5μm.

[0040] Specifically, when the response band of the short-wave infrared array detector 7 covers 0.85μm to 2.5μm, it can effectively cover the "optical transparency window" of biological tissue wavelengths from 1.3μm to 2.5μm, making the short-wave infrared array detector 7 suitable for detecting fluorescence lifetime imaging of deep tissues in living organisms, thus avoiding limitations in its application in live detection.

[0041] Preferably, in combination with the above scheme, as an embodiment of the present invention, the shortwave infrared array detector 7 is an HgCdTe area array detector 7.

[0042] Specifically, the HgCdTe (MCT) array detector 7 has higher infrared response speed and sensitivity in the near-infrared to short-wave infrared spectral region, and can accurately capture fluorescence lifetime distribution in the band range of 0.85μm to 2.5μm, thereby achieving efficient acquisition of fluorescence signals, and is suitable for dynamic observation of imaging targets 4 of living deep tissues such as cells and tissues.

[0043] Preferably, in combination with the above schemes, such as Figure 2 As shown, in one embodiment of the present invention, the timing controller 8 is a programmable logic device. The programmable logic device controls the laser 1 to turn on and off by outputting high and low level signals, and controls the exposure window of the area array detector 7 to turn on and off by outputting another high and low level signal.

[0044] Understandably, using programmable logic devices to directly control the opening and closing of the exposure windows of laser 1 and area array detector 7 by outputting high and low level signals has advantages such as fast response speed, low timing jitter (down to microsecond level), and flexible programming. It can simplify the complexity of time gating, reduce hardware costs and acquisition time, realize integrated control, and thus meet the needs of fluorescence lifetime detection in living deep tissues.

[0045] In some possible embodiments, a programmable microcontroller is used as the programmable logic device. Specifically, the general purpose input / output (GPIO) ports of the programmable microcontroller are electrically connected to the trigger interface of the laser 1 and the trigger interface of the area array detector 7, respectively. The programmable microcontroller has an internally stored imaging timing program that can output precise TTL high and low level signals to directly control the opening and closing of the exposure windows of the laser 1 and the area array detector 7, respectively. It has higher synchronization accuracy (up to 10 μs) and a delay time setting range (typically 10 μs to 100 ms) to achieve precise control of wide-field fluorescence lifetime imaging.

[0046] Example: like Figure 2 As shown, the programmable microcontroller is programmed according to the following rules: When laser 1 needs to emit laser light, the corresponding GPIO port pin of the programmable microcontroller outputs a high-level (5V) trigger signal to the trigger terminal of laser 1, turning laser 1 on; it outputs a low-level (0V) trigger signal to the trigger terminal of laser 1, turning laser 1 off. Similarly, when the area array detector 7 needs to collect fluorescence signals, another GPIO port pin of the programmable microcontroller outputs a high-level signal to the trigger terminal of area array detector 7, opening the exposure window of area array detector 7; it outputs a low-level signal to the trigger terminal of area array detector 7, closing the exposure window of area array detector 7.

[0047] Preferably, in conjunction with the above scheme, as an embodiment of the present invention, the filter switching component 5 is an electric filter wheel, which is signal-connected to the timing controller 8 to switch filters of different wavelengths at a preset delay time point according to the instructions of the timing controller 8.

[0048] Specifically, by switching filters of different wavelengths at preset delay time points, fluorescence signals of different wavelengths can be acquired at different time gates, thereby achieving multispectral fluorescence lifetime imaging to obtain more microenvironment parameters of the imaging target 4.

[0049] Preferably, in combination with the above scheme, as an embodiment of the present invention, the high-transmittance imaging lens 6 is a near-infrared high-lens lens; the near-infrared high-lens lens has a transmittance of more than 90% in the 0.85μm to 2.5μm band.

[0050] Specifically, by combining a near-infrared high-lens head with high near-infrared light transmittance with a high-resolution HgCdTe (MCT) area array detector 7, higher signal-to-noise ratio and sensitivity can be achieved in wide-field fluorescence lifetime imaging.

[0051] like Figure 3 As shown, an embodiment of the second aspect of the present invention provides a time-gated wide-field fluorescence lifetime imaging method, applied to the time-gated wide-field fluorescence lifetime imaging device provided in the embodiment of the first aspect, specifically including the following steps S100, S200, S300, S400 and S500: Step S100: The timing controller 8 outputs a trigger signal to the laser 1, controlling the laser 1 to emit pulsed laser light and irradiate the surface of the imaging target 4 to excite the fluorescent probe.

[0052] Specifically, the timing controller 8 outputs a trigger signal to the laser 1 according to a preset program, controlling the laser 1 to emit pulsed laser light. The pulsed laser light passes through the uniform optical fiber 2 and the laser collimating lens 3 in sequence to form a uniform light spot, which then illuminates the surface of the imaging target 4 to excite the fluorescent probe.

[0053] Step S200: Timing controller 8 stops triggering laser 1.

[0054] Specifically, after laser 1 stops emitting laser light, it enters the first delay time.

[0055] Step S300: After the first preset delay time, the timing controller 8 outputs the first gating trigger signal to the area array detector 7, controlling the area array detector 7 to open the gating exposure window and acquire the first fluorescence intensity image.

[0056] Specifically, after a first preset delay time, the timing controller 8 outputs a first gating trigger signal to the area array detector 7 according to a preset program, controlling the area array detector 7 to open the gating exposure window, so that the fluorescence emitted by the fluorescent probe passes sequentially through the filter switching component 5 and the high-transmittance imaging lens 6 to form a fluorescence signal. The area array detector 7 receives the fluorescence signal to acquire the first fluorescence intensity image. Preferably, the first delay time is 10 μs.

[0057] Step S400: After the Nth preset delay time, the timing controller 8 outputs the Nth gated trigger signal to the area array detector 7, controlling the area array detector 7 to open the gated exposure window again and acquire the Nth fluorescence intensity image.

[0058] Specifically, after the Nth delay time, the timing controller 8 outputs the Nth gated trigger signal to the area array detector 7 according to the preset program, and controls the area array detector 7 to open the gated exposure window to receive the fluorescence signal, so as to acquire the Nth fluorescence intensity image for wide-field fluorescence lifetime imaging.

[0059] Step S500: Reconstruct the fluorescence lifetime image of the imaging target 4 based on the first fluorescence intensity image to the Nth fluorescence intensity image.

[0060] Specifically, a fluorescence intensity image sequence is formed based on the first fluorescence intensity image to the Nth fluorescence intensity image, and the fluorescence lifetime image of the imaging target 4 is reconstructed based on the fluorescence intensity image sequence.

[0061] Preferably, in conjunction with the above scheme, as an embodiment of the present invention, the time-gated wide-field fluorescence lifetime imaging device further includes a data processing unit. The data processing unit reconstructs the fluorescence lifetime image of the imaging target 4 using a fitting algorithm based on the first fluorescence intensity image to the Nth fluorescence intensity image.

[0062] Preferably, in combination with the above scheme, as an embodiment of the present invention, the timing controller 8 determines the trigger signal and the first gating trigger signal to the Nth gating trigger signal based on the imaging system parameters.

[0063] Specifically, the timing controller 8 has an internally stored imaging timing program that can output precise TTL high and low level signals according to the set imaging system parameters, thereby controlling the exposure window of the trigger array detector 7 and the laser 1 in real time. By precisely controlling the duration of the high level (i.e., the laser pulse width and exposure time of the laser emitted by the laser 1) and the preset delay time for switching between high and low levels, high-precision multi-point sampling of the fluorescence lifetime decay curve can be achieved.

[0064] In some possible embodiments, the imaging system parameters include the imaging frame rate of the area array detector 7, the exposure time, and the first preset delay time to the Nth preset delay time, as well as the laser pulse width of the laser 1.

[0065] Example 1: like Figure 4 As shown, after the first preset delay time to the seventh preset delay time (0.01ms, 0.1ms, 1ms, 3ms, 5ms, 10ms, 15ms), the control array detector 7 performs seven fluorescence signal acquisitions on the first imaging target 4, forming the following... Figure 4 The fluorescence intensity image sequence shown is a steady-state image of the first imaging target 4, which is the imaging result under steady-state laser irradiation.

[0066] like Figure 5 As shown, this is based on... Figure 4 The fluorescence intensity image sequence shown is reconstructed into a fluorescence lifetime image using a fitting algorithm.

[0067] Example 2: like Figure 6 As shown, after the first preset delay time to the seventh preset delay time (0.01ms, 0.1ms, 1ms, 5ms, 10ms, 15ms, 20ms), the control array detector 7 performs seven fluorescence signal acquisitions on the second imaging target 4, forming the following... Figure 6 The fluorescence intensity image sequence shown is a steady-state image of the second imaging target 4, with the leftmost image in the sequence being the fluorescence intensity image sequence.

[0068] like Figure 7 As shown, this is based on... Figure 6 The fluorescence intensity image sequence shown is reconstructed into a fluorescence lifetime image using a fitting algorithm.

[0069] Example 3: like Figure 8 As shown, after the first preset delay time to the seventh preset delay time (0.01ms, 0.1ms, 1ms, 2ms, 3ms, 4ms, 5ms), the control array detector 7 performs seven fluorescence signal acquisitions on the third imaging target 4, forming the following... Figure 8 The fluorescence intensity image sequence shown is a steady-state image of the third imaging target 4, with the leftmost image in the sequence being the fluorescence intensity image sequence.

[0070] like Figure 9 As shown, this is based on... Figure 8 The fluorescence intensity image sequence shown is reconstructed into a fluorescence lifetime image using a fitting algorithm.

[0071] The present invention provides a time-gated wide-field fluorescence lifetime imaging device that can synchronously control the area array detector 7 and the timing controller 8, thereby expanding the detection band, improving the imaging speed, and enhancing the system stability in in vivo deep tissue fluorescence lifetime imaging in the near-infrared to short-wave infrared bands.

[0072] In some embodiments, the present invention uses an HgCdTe (MCT) area array detector 7 as the detection core, which can respond to a spectrum with wavelengths from 0.85μm to 2.5μm, breaking through the depth limitations of visible light and near-infrared region I in live imaging, and realizing dynamic observation of the target 4 of deep tissue imaging of living cells, tissues and other living tissues.

[0073] In some embodiments, the present invention employs a full-field parallel acquisition mode, which generates high-precision synchronous timing through programmable logic devices to directly control the pulse of laser 1 and the gating exposure of area array detector 7, thereby improving imaging speed while maintaining high temporal resolution.

[0074] In some embodiments, the present invention employs a fully electronic timing control scheme, which can avoid mechanical wear and vibration sources generated by mechanical choppers, significantly improve the stability and integration of the imaging device, and effectively reduce aberrations and light energy loss by simplifying the optical path structure, thus ensuring the signal-to-noise ratio and sensitivity of imaging.

[0075] Although embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions, and variations to the above embodiments within the scope of the present invention.

[0076] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A time-gated wide-field fluorescence lifetime imaging device, characterized in that, include: The excitation module includes a laser (1), a uniform optical fiber (2), and a laser collimating lens (3). The uniform optical fiber (2) is located on the output optical path of the laser (1), and the laser collimating lens (3) is located on the output optical path of the uniform optical fiber (2). The sample stage is located on the transmission light path of the laser collimating lens (3) and is used to mount the imaging target (4). The detection module includes a filter switching assembly (5), a high-transmittance imaging lens (6), and an area array detector (7). The filter switching assembly (5) is located on the light output path of the sample stage, the high-transmittance imaging lens (6) is located on the transmission path of the filter switching assembly (5), and the area array detector (7) is located on the transmission path of the high-transmittance imaging lens (6). The timing controller (8) is connected to the laser (1) and the area array detector (7) respectively. The timing controller (8) is used to output a trigger signal to the laser (1) to control the laser (1) to emit pulsed laser, and to output a gating trigger signal to the area array detector (7) to control the area array detector (7) to open the gating exposure window at one or more preset delay time points after the laser (1) stops emitting pulsed laser.

2. The time-gated wide-field fluorescence lifetime imaging device according to claim 1, characterized in that, The response band of the array detector (7) covers at least the near-infrared spectral region.

3. The time-gated wide-field fluorescence lifetime imaging device according to claim 2, characterized in that, The array detector (7) is a shortwave infrared array detector (7); The response band of the shortwave infrared array detector (7) covers 0.85 μm to 2.5 μm.

4. The time-gated wide-field fluorescence lifetime imaging device according to claim 3, characterized in that, The shortwave infrared array detector (7) is an HgCdTe array detector (7).

5. The time-gated wide-field fluorescence lifetime imaging device according to claim 1, characterized in that, The timing controller (8) is a programmable logic device. The programmable logic device controls the opening and closing of the laser (1) by outputting high and low level signals, and controls the opening and closing of the exposure window of the area array detector (7) by outputting another high and low level signal.

6. The time-gated wide-field fluorescence lifetime imaging device according to claim 1, characterized in that, The filter switching component (5) is an electric filter wheel, which is connected to the timing controller (8) to switch filters of different wavelengths at a preset delay time point according to the instructions of the timing controller (8).

7. The time-gated wide-field fluorescence lifetime imaging device according to claim 1, characterized in that, The high-transmittance imaging lens (6) is a near-infrared high-lens lens; The near-infrared high-lens lens has a transmittance of greater than 90% in the 0.85μm to 2.5μm band.

8. A time-gated wide-field fluorescence lifetime imaging method, applied to the time-gated wide-field fluorescence lifetime imaging device according to any one of claims 1 to 7, characterized in that, include: The timing controller (8) outputs a trigger signal to the laser (1) to control the laser (1) to emit pulsed laser light and irradiate the surface of the imaging target (4) to excite the fluorescent probe; The timing controller (8) stops triggering the laser (1); After the first preset delay time, the timing controller (8) outputs a first gate trigger signal to the area array detector (7) to control the area array detector (7) to open the gate exposure window and acquire the first fluorescence intensity image; After the Nth preset delay time, the timing controller (8) outputs the Nth gate trigger signal to the area array detector (7) to control the area array detector (7) to open the gate exposure window again and acquire the Nth fluorescence intensity image; The fluorescence lifetime image of the imaging target (4) is reconstructed based on the first fluorescence intensity image to the Nth fluorescence intensity image.

9. The time-gated wide-field fluorescence lifetime imaging method according to claim 8, characterized in that, The time-gated wide-field fluorescence lifetime imaging device also includes a data processing unit; The data processing unit reconstructs the fluorescence lifetime image of the imaging target (4) based on the first fluorescence intensity image to the Nth fluorescence intensity image using a fitting algorithm.

10. The time-gated wide-field fluorescence lifetime imaging method according to claim 8, characterized in that, The timing controller (8) determines the trigger signal, and the first gated trigger signal to the Nth gated trigger signal based on the imaging system parameters; The imaging system parameters include the imaging frame rate, exposure time, and first preset delay time to Nth preset delay time of the area array detector (7), and also include the laser pulse width of the laser (1).

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