Wide-field fluorescence lifetime imaging system and imaging method based on streak camera

Abstract

This invention relates to the field of fluorescence lifetime imaging, and discloses a wide-field fluorescence lifetime imaging system and method based on a streak camera. The system includes an excitation source, a wide-field excitation module, a synchronous detection module, and a synchronous matching module. The wide-field excitation module is positioned along the transmission path of a pulsed laser. The synchronous detection module includes a collection and beam splitting component, a reference camera, and a streak camera component. The input end of the collection and beam splitting component receives the fluorescence beam emitted by the sample, and the output end of the component splits the fluorescence beam into a first detection optical path and a second detection optical path. The reference camera is positioned in the first detection optical path, and the streak camera component is positioned in the second detection optical path. The synchronous matching module is electrically connected to the excitation source, the reference camera, and the streak camera component, respectively. This invention avoids the complex optical encoding scheme and resulting fluorescence photon loss problems of traditional DMD systems, and eliminates the need for complex reconstruction algorithms, thus improving imaging efficiency.

Description

Technical Field

[0001] This invention relates to the field of fluorescence lifetime imaging, and more specifically to a wide-field fluorescence lifetime imaging system and imaging method based on a streak camera. Background Technology

[0002] Fluorescence lifetime imaging (FLIM) is an important technique in biomedical research for label-free and quantitative analysis of the tissue microenvironment. It precisely measures the average time it takes for a fluorophore to return from an excited state to its ground state (i.e., fluorescence lifetime) and utilizes the sensitivity of this parameter to microenvironmental factors such as pH, ion concentration, and oxygen content to achieve multi-parameter quantitative characterization of physiological states. In the field of time-resolved fluorescence lifetime imaging (FLIM), streak cameras are commonly used. Streak cameras are ultrafast detectors capable of achieving picosecond or even sub-picosecond time resolution. Compared to single-photon counting techniques such as TCSPC, streak cameras offer advantages such as high temporal resolution, acquisition of complete temporal waveforms in a single exposure, and a higher dynamic range, making them suitable for lifetime measurement in fast-dynamic processes and high-signal scenarios.

[0003] In existing technologies, such as CN114705661A, a hyperspectral fluorescence lifetime imaging system and method based on a streak camera is disclosed. This method uses a streak camera FLIM to unfold the time dimension into one-dimensional spatial coordinates and map the photon arrival time into another-dimensional spatial coordinates, thereby directly obtaining the fluorescence decay curve and achieving ultra-high temporal resolution fluorescence decay measurement. To achieve wide-field FLIM imaging with the streak camera, this prior art also discloses a pulse light encoding module, specifically a digital micromirror array (DMMA), used to encode the pulse light processed by the pulse light coupling module. However, adding a long and complex DMD encoding optical path at the detector end significantly depletes fluorescence photon energy during the encoding process, leading to reduced fluorescence signal utilization. Furthermore, the above scheme relies on complex reconstruction algorithms, limiting system stability and robustness. Overall, the prior art disclosed in this prior art sacrifices photon efficiency for spatial dimension recovery, presenting a significant trade-off between efficiency and performance. Summary of the Invention

[0004] In view of this, the present invention provides a wide-field fluorescence lifetime imaging system and imaging method based on a streak camera to solve the problems of high photon loss and low imaging efficiency in existing fluorescence lifetime imaging systems using DMD encoders.

[0005] In a first aspect, the present invention provides a wide-field fluorescence lifetime imaging system based on a streak camera, comprising: Excitation light source, used to generate pulsed laser; The wide-field excitation module is positioned on the transmission path of the pulsed laser and is used to provide wide-field illumination to the sample. The synchronous detection module is set on the fluorescence emission path of the sample. The synchronous detection module includes a collection and beam splitting component, a reference camera and a stripe camera component. The light-incident end of the collection and beam splitting component is used to receive the fluorescence beam emitted by the sample. The light-outcident end of the collection and beam splitting component splits the fluorescence beam into a first detection optical path and a second detection optical path. The reference camera is set in the first detection optical path and the stripe camera component is set in the second detection optical path. The synchronization matching module is electrically connected to the excitation source, the reference camera, and the stripe camera assembly, respectively, and is used to achieve synchronous matching between the pulse emission timing of the excitation source and the scanning timing of the stripe camera assembly.

[0006] Beneficial effects: By adopting the above technical solution, the wide-field fluorescence lifetime imaging system based on a streak camera in this embodiment of the invention introduces a reference camera and a streak camera to work in parallel. The real-time two-dimensional spatial intensity image acquired by the reference camera provides a natural "physical spatial reference" for the system, effectively solving the problem of spatial and temporal dimension overlap caused by the streak camera in the fully open slit wide-field imaging mode, and achieving high-precision lifetime reconstruction without relying on complex compressed sensing algorithms.

[0007] Furthermore, since the embodiments of the present invention do not require cumbersome spatial encoding and subsequent iterative reconstruction calculations, the system's data processing pressure is significantly reduced, enabling real-time monitoring at higher frame rates. Simultaneously, the elimination of the expensive and complex DMD component lowers hardware costs and improves the stability and ease of adjustment of the optical path. Moreover, the sub-picosecond-level synchronization control achieved by the synchronization matching module, combined with wide-field excitation and dual-path parallel detection, allows the system to acquire the complete two-dimensional spatial structure and picosecond-level fluorescence decay process in a single exposure, truly achieving code-free wide-field ultrafast imaging.

[0008] In one alternative implementation, the beam-collecting and dispersing component includes: The first objective lens is used to collect the fluorescence beam generated by the sample; The first filter unit is disposed downstream of the first objective lens and is used to allow the fluorescent light beam emitted from the first objective lens to pass through, thereby filtering out the non-fluorescent light beam emitted from the first objective lens. The first tube lens is located downstream of the first filter unit; A beam splitter is positioned downstream of the first tube lens. The transmission path of the beam splitter corresponds to the first detection optical path, and the reflection path of the beam splitter corresponds to the second detection optical path.

[0009] Beneficial effects: By adopting the above technical solution, the embodiments of the present invention achieve purification and convergent imaging of fluorescence signals by setting a first filtering unit downstream of the first objective lens and a first tube lens downstream of the first filtering unit. Combined with the physical splitting effect of the beam splitter, it ensures that the two-dimensional spatial distribution information and time decay information of the sample can be acquired synchronously without coding loss. While completely eliminating photon waste and reconstruction artifacts caused by DMD coding, it significantly improves the detection accuracy and real-time imaging performance of the system for weak fluorescence samples.

[0010] In one alternative implementation, the wide-field excitation module includes: A beam expander and collimator is located at the output end of the excitation source; The second filtering unit is located downstream of the beam expanding and collimating unit; The focusing irradiation unit, located downstream of the second filtering unit, is used to focus irradiation on the sample.

[0011] Beneficial effects: When adopting the above technical solution, the wide-field excitation module of the present invention includes a beam expansion and collimation unit, a second filter unit, and a focusing and irradiation unit. The beam expansion and collimation unit realizes the aperture amplification and collimation of the original pulsed laser, ensuring that the wide-field illumination can cover the entire field of view of the sample. Combined with the second filter unit, the excitation spectrum is deeply purified, effectively suppressing the interference of stray light on weak fluorescence detection from the source. In addition, the precise light guide realized by the focusing and irradiation unit ensures the uniform distribution and efficient excitation of excitation energy on the sample surface, thereby providing a high-quality and high-purity initial light source for the two-dimensional spatial image and picosecond-level fluorescence decay information obtained by the subsequent detection end.

[0012] In one alternative embodiment, the focusing illumination unit includes a second tube lens and a second objective lens; The second tube lens is located downstream of the second filter unit, and the back focal plane of the laser beam emitted from the second tube lens is located at the entrance pupil of the second objective lens. The focal plane of the second objective lens coincides with the surface of the sample.

[0013] Beneficial effects: With the above technical solution, the focusing illumination unit includes a second tube lens and a second objective lens. This allows the collimated laser to be pre-focused onto the entrance pupil position of the second objective lens via the second tube lens, thereby constructing a highly uniform wide-field illumination path on the sample surface. This pupil-surface matching structural design ensures that the excitation light can vertically and uniformly cover the entire detection field in a parallel wide-field form. While effectively exciting the two-dimensional spatial fluorescence signal of the sample, it minimizes the risk of lifetime detection impacted by focusing inaccuracies and uneven light fields.

[0014] In one optional embodiment, the second objective lens and the first objective lens are shared objective lenses, the first filter unit is a dichroic mirror, and the dichroic mirror is obliquely placed in the optical path between the excitation light source and the shared objective lens. In this configuration, the reflecting surface of the dichroic mirror faces the excitation light source, while the transmitting surface faces the first tube lens.

[0015] Beneficial Effects: With the above technical solution, the second objective lens shares a single objective lens with the first objective lens. This simplifies the overall system structure and reduces the complexity of optical path adjustment, achieving a "contrast-type" coaxial integrated design for the excitation and detection optical paths. This shared objective lens architecture ensures that the wide-field excitation region and the detection imaging field of view can automatically achieve high spatial overlap and precise alignment, eliminating the focusing errors caused by unavoidable mechanical installation errors in dual-objective systems. Simultaneously, in conjunction with the dichroic mirror for the physical separation of excitation light and fluorescence, while ensuring efficient transmission of excitation energy, it maximizes the extraction of the weak fluorescence emitted by the sample, significantly enhancing the system's integration and the long-term stability of the optical path.

[0016] In one alternative implementation, the beam expander and collimator is a 4f optical system; The 4f optical system includes a first lens and a second lens arranged in a confocal configuration, and an aperture stop is provided at the common focal plane between the first lens and the second lens; The first lens, the aperture, and the second lens are arranged sequentially along the direction of pulsed laser propagation.

[0017] In one alternative implementation, the synchronization detection module further includes a synchronization trigger line electrically connected to the external trigger interfaces of the reference camera and the streak camera assembly, respectively.

[0018] Beneficial effects: By adopting the above technical solution, the synchronous start and stop control of the reference camera and the streak camera components at the hardware level is realized through the synchronous triggering circuit. This ensures that the two-dimensional spatial intensity information and fluorescence decay time information captured by the system are strictly aligned on the time scale. This "one-to-two" hardware synchronization mechanism not only completely eliminates the timing misalignment error caused by software triggering delay or asynchronous acquisition, but also ensures that the reconstruction of each lifetime spectrum is based on the same transient physical process, especially for fluorescence samples with dynamic evolution characteristics. This greatly improves the accuracy of spatiotemporal data fusion and the measurement robustness of the system.

[0019] In one alternative implementation, the synchronization matching module includes: A photodetector, whose photosensitive surface receives the light signal of a pulsed laser; The digital delay generator has its signal input terminal electrically connected to the output terminal of the photodetector, and its signal output terminal electrically connected to the trigger input terminal of the streak camera assembly.

[0020] Beneficial Effects: By employing the above technical solution, an ultrafast timing control chain based on physical signal feedback is constructed through the combination of a photodetector and a digital delay generator. The transient starting point of the laser pulse is captured in real time using photoelectric conversion, and the picosecond-level high-precision delay adjustment provided by the digital delay generator achieves temporal alignment between the excitation pulse emission and the strobe camera's sweeping window. This hardware-level synchronization mechanism effectively compensates for the inherent delays caused by optical path differences and circuit processing, ensuring that the strobe camera can capture the complete fluorescence decay dynamics curve with extreme stability, providing effective synchronization assurance for sub-picosecond temporal resolution.

[0021] In one optional embodiment, the wide-field fluorescence lifetime imaging system further includes a sample control module, which includes a sample holder and a triaxial precision stage. The sample holder is fixed to the loading end of the triaxial precision stage and placed at the focusing position of the wide-field excitation optical path.

[0022] Secondly, the present invention also provides an imaging method for a wide-field fluorescence lifetime imaging system based on a streak camera, comprising the following steps: Emit pulsed laser; The timing information of the pulsed laser is acquired, and the delay processing is performed based on the transmission delay of the pulsed laser and the fluorescence beam. The trigger signal is then output to the stripe camera component to achieve synchronous matching of the laser pulse emission and stripe scanning timing. Obtain the two-dimensional spatial intensity information of the fluorescence beam in the first detection optical path; The fluorescence beam in the second detection optical path is time-spread to obtain fluorescence decay time information; The two-dimensional spatial intensity information obtained by the reference camera is used as the basis for spatial positioning. Combined with the fluorescence decay time information obtained by the stripe camera component, the sample's wide-field fluorescence lifetime spectrum is reconstructed. Attached Figure Description

[0023] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0024] Figure 1 This is a schematic diagram of the structure of a wide-field fluorescence lifetime imaging system based on a stripe camera according to the present invention; Figure 2 This is a flowchart of a wide-field fluorescence lifetime imaging method based on a streak camera according to the present invention.

[0025] Explanation of reference numerals in the attached figures: 1. Excitation source; 2. Wide-field excitation module; 21. Beam expander and collimator unit; 211. 4f optical system; 2111. First lens; 2112. Aperture; 2113. Second lens; 22. Second filter unit; 23. Focusing illumination unit; 231. Second tube lens; 232. Second objective lens; 3. Synchronous detection module; 31. Collection and beam splitting assembly; 311. First objective lens; 312. First filter unit; 313. First tube lens; 314. Beam splitter; 32. Reference camera; 33. Stripe camera assembly; 34. Synchronous trigger circuit; 4. First detection optical path; 5. Second detection optical path. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0027] It should be noted that in the description of this invention, terms such as "inner" and "outer," indicating directional or positional relationships, are based on the directional or positional relationships shown in the accompanying drawings. This is merely for ease of description and does not indicate or imply that the device or element must have a specific orientation, or be constructed and operated in a specific orientation; therefore, it should not be construed as a limitation of this invention. Furthermore, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance. In the description of this invention, "a plurality of" means two or more, unless otherwise explicitly specified.

[0028] Furthermore, it should be noted that, in the description of this invention, unless otherwise explicitly specified and limited, the terms "connected" and "linked" 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 direct connection or an indirect connection through an intermediate medium. Those skilled in the art can understand the specific meaning of the above terms in this invention according to the specific circumstances.

[0029] To address the issues of high photon loss and low imaging efficiency in fluorescence lifetime imaging systems employing DMD encoders, the following section combines... Figure 1 and Figure 2 Embodiments of the present invention are described.

[0030] According to an embodiment of the present invention, in one aspect, a wide-field fluorescence lifetime imaging system based on a streak camera is provided. The system comprises an excitation source 1, a wide-field excitation module 2, a synchronous detection module 3, and a synchronous matching module (not shown). The excitation source 1 generates femtosecond or picosecond-level pulsed laser light. The wide-field excitation module 2 is located in the laser path and converts the point laser light into wide-field illumination light covering the sample. The synchronous detection module 3 includes a collection and beam splitting component 31, a reference camera 32, and a streak camera component 33. The light-incident end of the collection and beam splitting component 31 receives the fluorescence beam excited by the sample and splits it into two detection optical paths, namely a first detection optical path 4 and a second detection optical path 5. The reference camera 32 is disposed in the first detection optical path 4, and the streak camera component 33 is disposed in the second detection optical path 5. The reference camera 32 is used to acquire a two-dimensional spatial intensity image of the first detection optical path, and the streak camera component 33 is used to acquire attenuation information in the time dimension. The synchronous matching module is electrically connected to the excitation source 1, the reference camera 32, and the streak camera component 33, respectively.

[0031] In a specific implementation of this invention, the streak camera assembly 33 includes a streak camera (not shown), a readout sCMOS camera (not shown), and a camera lens (not shown). Specifically, the streak camera integrates a streak tube, and the light-incident end of the streak tube is provided with a photocathode for receiving the fluorescence beam. In this embodiment, the slit of the streak camera is kept fully open to meet the wide-field imaging requirements. During operation, the fluorescence beam generated by the sample is incident on the photocathode and converted into photoelectrons through the photoelectric effect. Under the action of the sweeping electric field generated by the high-voltage deflection plate inside the streak tube, the photoelectrons are deflected and swept in the longitudinal dimension of space according to their arrival time, and then strike the fluorescent screen at the rear end of the streak tube, realizing a linear mapping from time information to spatial position information. The streak camera achieves precise capture of the ultrafast fluorescence decay dynamics process by operating synchronously with the excitation light source 1. The camera lens is disposed between the output end of the streak camera and the readout sCMOS camera. The camera lens serves as a high-resolution optical coupling unit and is used to image the streak image on the fluorescent screen of the streak camera onto the target surface of the readout sCMOS camera. The sCMOS camera is connected to the output of the camera lens and is used to read the stripe image on the fluorescent screen of the stripe camera in order to output fluorescence attenuation information.

[0032] It should be noted that although the stripe camera assembly 33 described above includes a readout sCMOS camera (not shown), this is not limiting. In other embodiments of the present invention, the stripe camera assembly 33 may also include image sensors such as a charge-coupled device (CCD) camera, an electron multiplier CCD (EMCCD) camera, a complementary metal-oxide-semiconductor (CMOS) camera, or a photomultiplier tube array (PMT array). These image sensors are also used to read the image on the stripe camera's phosphor screen to achieve high-speed output of fluorescence decay information.

[0033] Preferably, in a specific embodiment of the present invention, the reference camera 32 includes a readout sCMOS camera (not shown). It should be noted that although the reference camera 32 described above includes a readout sCMOS camera, this is not limiting. In a specific embodiment of the present invention, the reference camera 32 may also include an image sensor such as a charge-coupled device (CCD) camera, an electron multiplier CCD (EMCCD) camera, a complementary metal-oxide-semiconductor (CMOS) camera, or a photomultiplier tube array (PMT array). These image sensors are also used to read the two-dimensional spatial intensity image of the first probe optical path to achieve high-speed output of the two-dimensional spatial intensity image.

[0034] By adopting the above configuration, this embodiment avoids the problem of significant fluorescence photon loss caused by loading a mask pattern in traditional solutions by introducing a reference camera 32. Furthermore, by using the real-time image acquired by the reference camera 32 as a "physical spatial reference," the aliasing of the "spatial information" and "temporal information" carried by photons on the detector in the fully open slit mode of the streak camera is eliminated. Wide-field ultrafast imaging under a single exposure can be achieved without DMD encoding and complex reconstruction algorithms, significantly reducing hardware costs and algorithmic burden.

[0035] In implementing this invention, the inventors discovered that in traditional fringe imaging, if the slit is fully opened to obtain a wide-field image, the coordinate information on the detector target surface falls into an "information ambiguity" dilemma. That is, each pixel on the longitudinal axis of the detector is actually the result of coupling the original spatial position (y) of the sample with the fluorescence emission time (t). This spatiotemporal information coupling makes it impossible for the system to distinguish the source of photons based on a single detection signal, i.e., it is impossible to determine whether a signal comes from a spatial position above the sample or from a later point in time.

[0036] This system, by introducing a reference camera 32, constructs a precise physical spatial reference mechanism to decouple spatiotemporal information from the underlying logic: the fluorescence signal is simultaneously distributed to the reference camera 32 and the streak camera via a beam splitter 314. The reference camera 32 and the streak camera operate synchronously, but the reference camera 32 does not perform time sweeping, and its exposure time is much longer than the timescale of the ultrafast process. Therefore, the reference camera 32 captures a time-averaged two-dimensional spatial image (x, y), equivalent to a precise "physical map," pre-locking the true spatial coordinates (x, y) of each luminescent point on the sample. Based on the spatial distribution information of the sample acquired by the reference camera 32, and using this spatial distribution information as a known spatial coordinate reference, the exact position of each luminescent point is used as a known constraint for subsequent spatiotemporal decoupling calculations, i.e., at a specific coordinate (x, y). i y iThere is indeed a physical luminescent center at (x), whose spatial location is a known constant. Specifically, (x) i y i Y' represents the physical spatial position of the ii-th emitting pixel (or ii-th fluorescence emission point) on the sample surface in the reference camera image. The coupled projection coordinates captured by the fringe camera follow the following physical relationship: Y' = a*y + v sweep *t, where a is the system optical magnification, v sweep Let y be the sweep velocity of the stripe tube, y be the spatial longitudinal coordinate of the sample surface, and t be the photon emission time. In the traditional scheme without a reference camera 32, both y and t are unknowns, and a single observation corresponds to infinitely many sets of (y, t) solutions, forming an underdetermined equation. After introducing the spatial reference provided by the reference camera 32, the coordinates become known constants. The algorithm substitutes the known physical spatial coordinates into the above equation, simplifying the originally coupled underdetermined problem into a univariate linear equation with respect to time, thus obtaining a unique solution: t = (Y' - a*y) / v sweep To eliminate spatiotemporal information aliasing under wide-field detection, pixel-level precise reconstruction of the sample's transient decay process is achieved by mapping the detection coordinates to a time coordinate system. Furthermore, fluorescence lifetime distribution parameters reflecting the sample's microenvironment characteristics are obtained through curve fitting, ultimately generating a wide-field fluorescence lifetime spectrum with both high spatial and temporal resolution.

[0037] As the above analysis shows, this embodiment employs a method of "anchoring the time dimension with a spatial reference," which can accurately reconstruct the coupled charge signal acquired by the streak camera to its true emission time. Compared to the compressed ultrafast imaging (CUP) scheme based on digital micromirror devices (DMDs), this invention does not require loading a spatially encoded mask, avoiding photon loss caused by binary encoding, and exhibits higher sensitivity in weak signal detection scenarios. Moreover, by using direct physical mapping instead of iterative compressed sensing reconstruction, the computational complexity is significantly reduced, thus enabling high frame rate real-time monitoring. Furthermore, the physical prior-based decoupling avoids reconstruction artifacts that may be introduced in compressed sensing reconstruction due to insufficient sparsity assumptions or low signal-to-noise ratios, ensuring the physical authenticity of the fluorescence lifetime spectrum.

[0038] like Figure 1As shown, in this embodiment, the beam-splitting assembly 31 includes a first objective lens 311, a first filter unit 312, a first tube lens 313, and a beam splitter 314. The first objective lens 311 collects the light beam emitted from the sample and directs it through its exit end, allowing the fluorescence beam filtered by the first filter unit 312 to enter the entrance end of the first tube lens 313. The first filter unit 312 is located downstream of the first objective lens 311, allowing the fluorescence beam emitted from the first objective lens 311 to pass through while blocking non-fluorescent beams. The first tube lens 313 is located downstream of the first filter unit 312, and together with the first objective lens 311, forms an infinity-corrected imaging system to converge the fluorescence signal into an image. The beam splitter 314 is located downstream of the first tube lens 313, dividing the imaging beam into a first probe light path 4 and a second probe light path 5, ensuring that both cameras acquire completely identical imaging fields of view.

[0039] With the above setup, fluorescence signal purification and convergent imaging were achieved. Combined with the physical splitting effect of the beam splitter 314, it was ensured that the two-dimensional spatial distribution information and time decay information of the sample could be acquired synchronously without encoding loss. While completely eliminating photon waste and reconstruction artifacts caused by encoding, the system's detection accuracy and real-time imaging performance for weak fluorescence samples were significantly improved.

[0040] Please continue reading. Figure 1 In this embodiment, the wide-field excitation module 2 includes a beam expanding and collimating unit 21, a second filtering unit 22, and a focusing and irradiation unit 23. The beam expanding and collimating unit 21, located at the output end of the excitation source 1, consists of multiple lenses and is used to amplify the narrow-beam laser light from the laser. The second filtering unit 22, located downstream of the beam expanding and collimating unit 21, is used to spatially filter or spectrally purify the laser beam output from the excitation source 1 to remove higher-order stray components and obtain a near-Gaussian beam with a more uniform intensity distribution. The focusing and irradiation unit 23, located downstream of the second filtering unit 22, is used to focus and irradiate the sample.

[0041] Through the above configuration, this embodiment achieves aperture amplification and collimation of the original pulsed laser through the beam expansion and collimation unit 21, ensuring that the wide-field illumination can cover the entire field of view of the sample; combined with the second filter unit 22 to deeply purify the excitation spectrum, it effectively suppresses the interference of stray light on weak fluorescence detection from the source; and with the precision light guide achieved by the focusing illumination unit 23, it ensures the uniform distribution and efficient excitation of excitation energy on the sample surface, thereby providing a high-quality and high-purity initial light source for the back-end detection.

[0042] Please continue reading. Figure 1In this embodiment, the focusing illumination unit 23 includes a second tube lens 231 and a second objective lens 232. Specifically, the second tube lens 231 is located downstream of the second filtering unit 22, and the back focal plane of the laser beam emitted by the second tube lens 231 is located at the entrance pupil position of the second objective lens 232. This pupil-plane matching structural design ensures that the excitation light can vertically and uniformly cover the entire detection field of view in a parallel wide field, effectively exciting the two-dimensional spatial fluorescence signal of the sample while minimizing the risk of lifetime detection effects due to focusing inaccuracies and uneven light fields.

[0043] Please continue reading. Figure 1 In this embodiment, the second objective lens 232 and the first objective lens 311 are shared objectives, and the first filter unit 312 is a dichroic mirror. This dichroic mirror is obliquely positioned in the optical path between the excitation light source 1 and the shared objective lens. Specifically, the reflecting surface of the dichroic mirror faces the excitation light source 1, and the transmitting surface faces the first tube lens 313. More specifically, the dichroic mirror is located between the second tube lens 231 and the shared objective lens, so that when the laser beam emitted from the second tube lens 231 illuminates the dichroic mirror, it can be reflected and incident at the entrance pupil position of the shared objective lens, and then focused by the shared objective lens to form a wide-field excitation beam at the sample. Simultaneously, the shared objective lens receives the mixed beam emitted from the sample, wherein the fluorescence beam passes through the dichroic mirror and illuminates the first tube lens 313.

[0044] Through the above configuration, the first objective lens 311 and the second objective lens 232 are integrated, simplifying the overall system structure and reducing debugging difficulty, achieving a "contrast-type" coaxial integrated design. This architecture ensures that the excitation region and the detection field of view can automatically achieve a high degree of overlap, eliminating the installation errors that are unavoidable in dual-objective systems. At the same time, in conjunction with a dichroic mirror, physical separation of excitation and fluorescence is achieved, significantly enhancing the system's integration and operational stability while ensuring efficient energy transfer.

[0045] It should be noted that the arrangement of the dichroic mirror is not limiting. In other embodiments of the present invention, the dichroic mirror can also be configured with its transmission surface facing the sample and its reflection surface facing the first tube lens 313, so that the excitation laser passes through the dichroic mirror to irradiate the sample, while the fluorescence is reflected by the dichroic mirror into the detection optical path. Such adjustments do not deviate from the basic principles of the present invention and all fall within the protection scope of the present invention.

[0046] Furthermore, the first filtering unit 312 can also be a long-pass filter. The long-pass filter is positioned on the side of the sample away from the objective lens or on the fluorescence path to filter out the excitation light. In this case, the first objective lens 311 and the second objective lens 232 can be located on opposite sides of the sample. This transmissive arrangement also falls within the scope of this invention.

[0047] Please continue reading. Figure 1In this embodiment, the beam expander and collimator 21 is a 4f optical system 211. This system includes a first lens 2111 and a second lens 2113 arranged confocally. An aperture stop 2112 is provided at the common focal plane of the two lenses. Stray light is filtered out from the spectral plane of the first lens 2111 to obtain pure and uniform Gaussian light.

[0048] Please continue reading. Figure 1 In this embodiment, the synchronous detection module 3 also includes a synchronous trigger line 34, the physical medium of which can be a standard or SMA cable. This line is electrically connected to the external hardware trigger interfaces of the reference camera 32 and the streak camera assembly 33, respectively. This "hardware-based dual-transmit and dual-receive" mechanism enables the synchronous start and stop of the two cameras at the nanosecond level, ensuring strict alignment of spatiotemporal data on the physical time scale and effectively solving the temporal misalignment problem when capturing dynamic evolution samples.

[0049] In this embodiment, the synchronization matching module (not shown) includes a photodetector (not shown) and a digital delay generator (not shown). The photodetector receives a portion of the pulsed laser and converts it into an electrical signal. The digital delay generator uses this signal to precisely compensate for the optical path difference of the system, ensuring that the sweeping time window of the streak camera perfectly matches the arrival time of the fluorescence.

[0050] In this embodiment, the wide-field fluorescence lifetime imaging system based on a streak camera further includes a sample control module (not shown), which consists of a sample holder and a three-axis precision displacement stage. By adjusting the displacement stage, precise focusing on different regions of the sample can be achieved. The three-axis displacement stage is a conventional structure in the mechanical field and will not be described in detail here.

[0051] On the other hand, this embodiment also provides an imaging method for a wide-field fluorescence lifetime imaging system based on a streak camera, including the following steps: S101: Emits pulsed laser; S102: Acquire the timing information of the pulsed laser, perform delay processing based on the transmission delay of the pulsed laser and the fluorescent beam, and output a trigger signal to the stripe camera component to achieve synchronous matching of the laser pulse emission and stripe scanning timing. S103: Obtain the two-dimensional spatial intensity information of the fluorescence beam in the first detection optical path; S104: Perform time-spreading on the fluorescence beam of the second detection optical path to obtain fluorescence decay time information; S105: The acquired two-dimensional spatial intensity information is used as the basis for spatial positioning. Combined with the acquired fluorescence decay time information, the data is reconstructed to obtain the wide-field fluorescence lifetime spectrum of the sample.

[0052] Specifically, the imaging method of the wide-field fluorescence lifetime imaging system based on a streak camera is as follows: In step S101, the control unit controls the excitation source 1 to emit pulsed laser light. The wide-field fluorescence lifetime imaging system based on a streak camera in this embodiment also includes a control unit (not shown). The control unit communicates with the excitation source 1, the digital delay generator, the reference camera 32, and the streak camera via communication interfaces. The control unit has a built-in image processing algorithm used to read the two-dimensional spatial intensity image of the reference camera 32 as a spatial position mapping, and to perform deconvolution or linear decoupling on the aliased signal acquired by the streak camera assembly 33, thereby generating a wide-field fluorescence lifetime spectrum.

[0053] In S102, the photodetector receives the optical signal of the pulsed laser and converts it into an electrical pulse signal corresponding to the pulsed laser emission timing. Subsequently, the photoelectric controller inputs the electrical pulse signal to a digital delay generator, which performs picosecond-level delay processing based on the photoelectric transmission path difference between the pulsed laser and the fluorescent beam within the system. Finally, the digital delay generator outputs a synchronization trigger signal to the streak camera assembly 33, thereby compensating for the inherent transmission delay of the system and achieving physical resonance alignment between the excitation and scanning timings.

[0054] In S103, the control unit acquires the two-dimensional spatial intensity information collected by the reference camera 32.

[0055] In step S104, firstly, the control unit acquires the original two-dimensional pixel intensity matrix output by the stripe camera assembly 33 in full-open slit mode. The matrix is ​​then denoised using a preset background noise algorithm to extract the effective photoelectron stripe distribution signal. Next, the control unit uses a two-dimensional spatial intensity image synchronously acquired by the reference camera 32 as a spatial position constraint. Based on a pre-calibrated coordinate transformation relationship, the spatial coordinates in the reference camera 32 image are mapped to the pixel coordinate system of the stripe camera image. Finally, the control unit extracts the pixel intensity distribution within a specific spatial region along the time axis, constructing an evolution curve of fluorescence intensity over time.

[0056] In step S105, firstly, the control unit performs mathematical fitting on the extracted fluorescence transient decay curve. In one embodiment, the least squares method or maximum likelihood estimation method is used to fit the data according to a single-exponential or multi-exponential decay model. Through fitting, not only fluorescence lifetime distribution data reflecting the characteristics of the sample's molecular microenvironment can be obtained, but also initial intensity distribution data reflecting the sample concentration can be obtained, thus achieving full parametric characterization of the sample's spatiotemporal dimensions. Then, the control unit injects the calculated decay time information at each spatial location into the corresponding pixels of the two-dimensional spatial intensity image, and finally reconstructs and outputs a wide-field fluorescence lifetime spectrum with high spatiotemporal resolution.

[0057] It should be noted that those skilled in the art can combine, split, and rearrange the above steps. If the modified solution does not change the basic concept, it falls within the protection scope of this invention.

[0058] Although embodiments of the invention have been described in conjunction with the accompanying drawings, those skilled in the art can make various modifications and variations without departing from the spirit and scope of the invention, and such modifications and variations all fall within the scope defined by the appended claims.

Claims

1. A wide-field fluorescence lifetime imaging system based on a streak camera, characterized in that, include: Excitation source (1) is used to generate pulsed laser; A wide-field excitation module (2) is set on the transmission path of the pulsed laser and is used to illuminate the sample with a wide field. A synchronous detection module (3) is set on the fluorescence emission path of the sample. The synchronous detection module (3) includes a collection and beam splitting component (31), a reference camera (32), and a stripe camera component (33). The light-incident end of the collection and beam splitting component (31) is used to receive the fluorescence beam emitted by the sample. The light-outcident end of the collection and beam splitting component (31) splits the fluorescence beam into a first detection light path (4) and a second detection light path (5). The reference camera (32) is set on the first detection light path and is used to acquire a two-dimensional spatial intensity image of the first detection light path. The stripe camera component (33) is set on the second detection light path. The synchronization matching module is electrically connected to the excitation light source (1), the reference camera (32) and the stripe camera assembly (33) respectively, and is used to realize the synchronization matching of the pulse emission timing of the excitation light source (1) and the scanning timing of the stripe camera assembly (33).

2. The wide-field fluorescence lifetime imaging system according to claim 1, characterized in that, The collecting and dispersing component (31) includes: The first objective lens (311) is used to collect the fluorescence beam generated by the sample; The first filter unit (312) is disposed downstream of the first objective lens (311) and is used to allow the fluorescent light beam emitted by the first objective lens (311) to pass through, so as to filter out the non-fluorescent light beam emitted by the first objective lens (311). The first tube lens (313) is disposed downstream of the first filter unit (312); A beam splitter (314) is disposed downstream of the first tube lens (313). The transmission path of the beam splitter (314) corresponds to the first detection optical path, and the reflection path of the beam splitter (314) corresponds to the second detection optical path.

3. The wide-field fluorescence lifetime imaging system according to claim 2, characterized in that, The wide-field excitation module (2) includes: A beam expansion and collimation unit (21) is provided at the output end of the excitation light source (1); The second filter unit (22) is located downstream of the beam expanding and collimating unit (21); A focusing irradiation unit (23) is located downstream of the second filtering unit (22) and is used to focus irradiation on the sample.

4. The wide-field fluorescence lifetime imaging system according to claim 3, characterized in that, The focusing illumination unit (23) includes a second tube lens (231) and a second objective lens (232); The second tube lens (231) is located downstream of the second filter unit (22), and the back focal plane of the laser beam emitted by the second tube lens (231) is located at the entrance pupil position of the second objective lens (232), and the focal plane of the second objective lens (232) coincides with the surface of the sample.

5. The wide-field fluorescence lifetime imaging system according to claim 4, characterized in that, The second objective lens (232) and the first objective lens (311) are shared objective lenses. The first filter unit (312) is a dichroic mirror. The dichroic mirror is obliquely placed in the optical path between the excitation light source (1) and the shared objective lens. The reflective surface of the dichroic mirror faces the excitation light source (1), and the transmission surface faces the first tube lens (313).

6. The wide-field fluorescence lifetime imaging system according to any one of claims 3-5, characterized in that, The beam expanding and collimating unit (21) is a 4f optical system (211); The 4f optical system (211) includes a first lens (2111) and a second lens (2113) arranged in a confocal configuration, and an aperture stop (2112) is provided at the common focal plane between the first lens (2111) and the second lens (2113). The first lens (2111), the aperture (2112), and the second lens (2113) are arranged sequentially along the propagation direction of the pulsed laser.

7. The wide-field fluorescence lifetime imaging system according to any one of claims 1-5, characterized in that, The synchronous detection module (3) further includes a synchronous trigger line (34), which is electrically connected to the external trigger interface of the reference camera (32) and the stripe camera assembly (33), respectively.

8. The wide-field fluorescence lifetime imaging system according to any one of claims 1-5, characterized in that, The synchronization matching module includes: A photodetector, the photosensitive surface of which receives the optical signal of the pulsed laser; The digital delay generator has its signal input terminal electrically connected to the output terminal of the photodetector and its signal output terminal electrically connected to the trigger input terminal of the stripe camera assembly (33).

9. The wide-field fluorescence lifetime imaging system according to any one of claims 1-5, characterized in that, The wide-field fluorescence lifetime imaging system also includes a sample control module, which includes a sample holder and a triaxial precision displacement stage. The sample holder is fixed to the loading end of the triaxial precision displacement stage and is positioned at the focusing position of the wide-field excitation optical path.

10. An imaging method based on a wide-field fluorescence lifetime imaging system based on a streak camera according to any one of claims 1 to 9, characterized in that, Includes the following steps: Emit the pulsed laser; The timing information of the pulsed laser is obtained, and a delay processing is performed based on the transmission delay of the pulsed laser and the fluorescent beam. A trigger signal is output to the stripe camera component (33) to achieve synchronous matching of the laser pulse emission and stripe scanning timing. Obtain the two-dimensional spatial intensity information of the fluorescent beam in the first detection optical path (4); The fluorescence beam in the second detection optical path (5) is time-spread to obtain fluorescence decay time information; The acquired two-dimensional spatial intensity information is used as the basis for spatial positioning, and combined with the acquired fluorescence decay time information for reconstruction to obtain the wide-field fluorescence lifetime spectrum of the sample.

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