Spectral resolution parallel fluorescence lifetime scanning imaging system

Abstract

The invention discloses a spectral resolution parallel fluorescence lifetime scanning imaging system, and aims to solve the problems of lack of spectral resolution and slow imaging speed in the conventional fluorescence lifetime scanning imaging technology. The system comprises an exciting light module, a scanning imaging module and a spectral resolution life detection module, the exciting light module generates exciting light with specific repetition frequency; the scanning imaging module is used for realizing relative position mobile scanning of exciting light and a sample and separating to obtain a fluorescence signal; the spectral resolution lifetime detection module adopts a multi-channel photoelectric detector to detect fluorescence of different wavelengths at the same time so as to realize parallel fluorescence lifetime measurement. According to the invention, rapid measurement of spectral resolution fluorescence lifetime imaging is realized, the system has the advantages of high time resolution, high spatial resolution and multifunctional measurement, the system structure is simple, the cost is low, and an ideal tool is provided for imaging research such as biomedicine, chemical analysis and environmental monitoring.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of fluorescence imaging technology, in particular to a spectrum-resolved parallel fluorescence lifetime scanning imaging system. BACKGROUND

[0002] Fluorescence imaging technology has important and extensive applications in biomedical, drug development, food safety, chemical analysis, environmental monitoring and other fields. Most fluorescence imaging technologies only measure the intensity of fluorescence, but fluorescence lifetime imaging also obtains more information of the sample by detecting the decay time of the excited state of the fluorescent molecule. Compared with intensity imaging, fluorescence lifetime is not sensitive to probe concentration and excitation light intensity, and can quantitatively reflect biophysical parameters such as molecular conformation change, energy transfer, pH value and ion concentration, which has irreplaceable advantages in many fields such as live cell metabolism monitoring, protein interaction analysis and pathological diagnosis.

[0003] However, traditional fluorescence lifetime scanning imaging has some deficiencies in practical application. On the one hand, the time-dependent single-photon counting or time-gated method based on the existing technology usually adopts single-band detection, resulting in the lack of spectral resolution, so that when there are multiple fluorescent groups in the sample (such as multi-color labeled organelles or autofluorescence interference), the lifetime signals with different spectral characteristics cannot be effectively separated. On the other hand, fluorescence lifetime scanning imaging is slow, and the method of filter wheel rotation to realize single-digit spectral channel measurement significantly reduces the imaging speed and cannot support dynamic process observation.

[0004] In recent years, instruments and technologies that can record time and spectral information simultaneously have been developed: Patent application CN116148232A and CN118518643A introduce spectral resolution fluorescence lifetime measurement devices, aiming to measure fluorescence lifetime spectrum quickly, but they do not cover fluorescence lifetime scanning imaging systems. Patent CN114705661A proposes a hyperspectral fluorescence lifetime imaging system and method based on a stripe camera, which to some extent solves the problem of slow acquisition speed of existing spectral fluorescence lifetime imaging technology. However, stripe cameras are very expensive and difficult to popularize, and the single-pixel reconstruction algorithm based on them makes the spatial resolution and display real-time of the measured image insufficient.

[0005] Therefore, there is an urgent need for a highly integrated multifunctional fluorescence lifetime scanning imaging technology to realize fast parallel fluorescence lifetime detection with high time accuracy and high spectral resolution, and to provide a technical basis for real-time multi-parameter quantitative analysis of complex biological systems. SUMMARY

[0006] The present application aims to provide a spectral resolution parallel fluorescence lifetime scanning imaging system which can effectively overcome the shortcomings of the prior art and realize fast, high time resolution, high spatial resolution and multi-functional measurement of spectral fluorescence lifetime imaging.

[0007] To achieve the above-mentioned purpose, the specific technical scheme of the present application is:

[0008] A spectral resolution parallel fluorescence lifetime scanning imaging system comprises an excitation light module, a scanning imaging module and a spectral resolution lifetime detection module.

[0009] The excitation light module generates excitation light by a laser and guides the excitation light to the scanning imaging module through a collimating mirror, wherein the excitation light has a uniform light intensity distribution.

[0010] In the scanning imaging module, the excitation light passes through a light filtering component, a scanning moving component and an objective lens on an objective lens displacement stage, is guided to focus on the measured sample, and realizes relative position moving scanning of the excitation light and the sample; wherein the scanning moving component is a one-dimensional scanning mirror group, a two-dimensional scanning mirror or a sample displacement stage.

[0011] When the scanning moving component is a one-dimensional scanning mirror group, the excitation light passes through the light filtering component, the one-dimensional scanning mirror group and the objective lens on the objective lens displacement stage in sequence, is guided to focus on the measured sample, and realizes relative position moving scanning of the excitation light and the sample.

[0012] When the scanning moving component is a two-dimensional scanning mirror, the excitation light passes through the light filtering component, the two-dimensional scanning mirror and the objective lens on the objective lens displacement stage in sequence, is guided to focus on the measured sample, and realizes relative position moving scanning of the excitation light and the sample.

[0013] When the scanning moving component is a sample displacement stage, the excitation light passes through the light filtering component and the objective lens on the objective lens displacement stage in sequence, is guided to focus on the measured sample on the sample displacement stage, and realizes relative position moving scanning of the excitation light and the sample.

[0014] The excitation light can also pass through the one-dimensional scanning mirror group or the two-dimensional scanning mirror, the light filtering component and the objective lens on the objective lens displacement stage in sequence, is guided to focus on the measured sample, and realizes relative position moving scanning of the excitation light and the sample.

[0015] The objective lens on the objective lens displacement stage collects the fluorescence generated by the measured sample, the fluorescence returns along the original light path, realizes spectral separation of the fluorescence and the excitation light at the light filtering component, is guided through a confocal element, is filtered through a confocal pinhole to remove out-of-focus plane fluorescence, and is finally transmitted to the spectral resolution lifetime detection module; or the separated fluorescence is directly guided to the spectral resolution lifetime detection module through the confocal element.

[0016] The spectrum resolution lifetime detection module, the separated fluorescence is distributed in space by different wavelengths by the light splitting component; the multi-channel photoelectric detection component has n detection channels, which simultaneously detects the fluorescence signals of different wavelengths, wherein n is not less than 32; the multi-channel fluorescence lifetime measurement component transmits the excitation light synchronization signal through the cable line; the multi-channel fluorescence lifetime measurement component is connected with the one-dimensional scanning mirror group, the two-dimensional scanning mirror, the objective lens on the objective lens displacement table and the sample displacement table through the cable line to transmit the scanning synchronization signal; the multi-channel photoelectric detection component and the multi-channel fluorescence lifetime measurement component realize parallel fluorescence lifetime measurement of the n detection channels; the upper computer receives the measurement data results of the multi-channel fluorescence lifetime measurement component, and performs data processing and imaging display.

[0017] Further, the excitation light module uses a single laser to generate excitation light, or couples and collimates multiple lasers to generate excitation light; the generated excitation light has a single or multiple wavelength components in the range of 300-3000 nm, and the wavelength components are adjusted by a light splitting element or an optical filtering element;

[0018] The laser selects a pulse laser with a pulse width of ≤10 ns or a modulated continuous laser, which has a fixed pulse repetition frequency in the range of 1 kHz-200 MHz, or the pulse repetition frequency is adjustable in the range of 1 kHz-200 MHz, but the repetition frequency remains stable during the measurement;

[0019] The excitation light synchronization signal is transmitted through the cable line; the signal is an electrical signal output by the laser to synchronize the excitation light in time to the multi-channel fluorescence lifetime measurement component, or an electrical signal output by the multi-channel fluorescence lifetime measurement component to trigger and control the laser to synchronize the excitation light in time.

[0020] Further, the relative position movement scanning of the excitation light and the sample includes one or a combination of the following modes: the sample is stationary, and the excitation light moves in three-dimensional space; the spatial position of the excitation light does not change, and the sample moves in three-dimensional space;

[0021] The sample is stationary, and the excitation light moves in three-dimensional space, which includes galvanometer scanning, resonant scanning, motor-driven scanning, MEMS micro-mirror scanning, piezoelectric driving scanning, acousto-optic deflector scanning, rotating mirror tube (turntable) confocal scanning, and combinations of the above modes;

[0022] The spatial position of the excitation light does not change, and the sample moves in three-dimensional space, which includes manual knob driving, motor driving, piezoelectric ceramic driving, and combinations of the above modes;

[0023] The light filtering component realizes effective separation of the excitation light and the fluorescent light through the difference of the optical propagation characteristics of different wavelengths, and specifically includes one or a combination of multiple of a dichroic mirror, an optical filter, an electro-optical modulator, an acousto-optic modulator, and a spatial light modulator.

[0024] Further, the light splitting component realizes spatial distribution of the fluorescent light according to different wavelengths, and specifically adopts a single dispersion element or a combination of multiple dispersion elements; the dispersion element adopts a grating or a prism.

[0025] The multi-channel photoelectric detection component adopts a multi-channel photoelectric detector with n detection channels to simultaneously detect fluorescent light of different wavelengths, and each detection channel independently converts the fluorescent light signal of the corresponding wavelength into an electrical signal, where the number n of the detection channels is not less than 32.

[0026] The multi-channel photoelectric detector adopts a single multi-channel detection element, a combination of multiple single-channel detection elements, or a combination of multiple multi-channel detection elements; the position is the focal plane of the separated fluorescent light, and the detection surface size covers the spatial region where the fluorescent light is distributed according to different wavelengths.

[0027] The multi-channel photoelectric detector covers a fluorescent wavelength range and achieves a fluorescent wavelength resolution, which changes with the optical path configuration. The fluorescent wavelength range is 300 nm to 3000 nm, and the fluorescent wavelength resolution is 0.1 nm to 10 nm. The detection element used is a multi-pixel photon counter (MPPC), a single photon avalanche diode (SPAD), a single photon avalanche diode array (SPAD array), a photomultiplier tube (PMT), a multi-channel photomultiplier tube (Microchannel PMT), or a hybrid photo detector.

[0028] The multi-channel photoelectric detection component includes a signal processing circuit for processing the photoelectrically converted signals of the multi-channel photoelectric detector. The processing methods include amplification, discrimination, and shaping. The signal processing circuit channel corresponds to each of the n detection channels. The processed signals are suitable for the multi-channel fluorescent lifetime measurement component.

[0029] The multi-channel fluorescence lifetime measurement component realizes multi-channel fluorescence lifetime measurement by using a time-correlated single-photon counting (TCSPC) method, a digital frequency domain (DFD) method or a phase-modulation shift method (PSM or analog frequency domain).

[0030] The multi-channel fluorescence lifetime measurement component transmits a synchronization signal of excitation light to the laser through a cable, inputs an electrical signal synchronized with the excitation light, or outputs an electrical signal to externally trigger the laser to generate synchronized excitation light; the multi-channel fluorescence lifetime measurement component is connected to a displacement stage of a one-dimensional scanning mirror group or a two-dimensional scanning mirror or a sample displacement stage or an objective lens of an objective lens displacement stage through a cable to transmit a scanning synchronization signal.

[0031] The multi-channel fluorescence lifetime measurement component is configured to detect signals after photoelectric conversion and signal processing of the multi-channel photoelectric detection component, has n lifetime measurement channels corresponding to n detection channels, and realizes multi-channel parallel and simultaneous measurement of fluorescence lifetime.

[0032] The lifetime measurement interval of each measurement channel of the multi-channel fluorescence lifetime measurement component is a fixed time interval, or the lifetime measurement interval is adjustable within a 5 ns to 1 ms time interval, but the lifetime measurement interval remains stable during measurement.

[0033] The upper computer is configured to receive data transmission results of the multi-channel fluorescence lifetime measurement component, can save the data, and can process and display measured data in multi-functional fluorescence lifetime scanning imaging six-dimensional data (x, y, z, λ, τ, t), wherein x, y and z are three-dimensional data of sample space scanning, λ is spectral dimension data, τ is fluorescence lifetime dimension data obtained by processing, and t is time sequence dimension data of overall scanning.

[0034] Further, the time-correlated single-photon counting method specifically includes: using a time-to-digital converter integrated circuit chip or being realized based on a field programmable gate array.

[0035] The time-correlated single-photon counting method realized based on the field programmable gate array uses a combination of coarse counting and fine counting, wherein the coarse counting is based on a master clock period to obtain coarse counting, and the fine counting is based on a tapped delay chain method, a multi-phase clock method, a differential time-to-digital conversion method, a pulse shrinking method and a combination thereof to obtain fine counting by dividing the master clock period.

[0036] Furthermore, other components in the excitation light module, scanning imaging module and spectrally resolved lifetime detection module, except for the sample to be measured, the sample displacement stage, the multi-channel fluorescence lifetime measurement component and the host computer, are configured to be integrated in a light-shielding housing; or each module is integrated in a light-shielding housing, and the optical paths between modules or within the modules are connected by optical fiber coupling.

[0037] Furthermore, the imaging modes include single-photon fluorescence imaging, two-photon fluorescence imaging, multi-photon fluorescence imaging or up-conversion fluorescence imaging; the specific imaging light paths in the excitation light module, scanning imaging module and spectrally resolved lifetime detection module are adjusted according to the imaging mode.

[0038] Furthermore, the imaging includes merging adjacent channels of n lifetime measurement channels in the spectrally resolved lifetime detection module to achieve multi-channel fluorescence lifetime scanning imaging, which is less than the number of detection channels; or merging all channels to achieve single-channel fluorescence lifetime scanning imaging.

[0039] Furthermore, the imaging includes merging different time channels of the same detection channel of the spectrally resolved lifetime detection module to achieve spectrally resolved fluorescence scanning imaging.

[0040] Furthermore, the imaging includes merging different time channels of the same detection channel of the spectrally resolved lifetime detection module, and then merging different detection channels in different ways to achieve fluorescence spectrally resolved scanning imaging with a number less than the number of detection channels; or merging all time channels and detection channels to achieve single-channel fluorescence intensity scanning imaging.

[0041] Compared with the prior art, the advantages of the present invention are:

[0042] 1) The spectrally resolved parallel fluorescence lifetime scanning imaging system of the present invention generates stable excitation light through the excitation light module, realizes high-precision scanning imaging through the scanning imaging module, and performs parallel measurement of fluorescence lifetimes at multiple wavelengths through the spectrally resolved lifetime detection module, thereby realizing rapid measurement of spectrally resolved fluorescence lifetimes and obtaining six-dimensional data (x, y, z, λ, τ, t), ultimately achieving high temporal resolution, high spatial resolution, and multifunctional rapid spectrally resolved fluorescence lifetime imaging.

[0043] 2) The spectrally resolved lifetime detection module of the spectrally resolved parallel fluorescence lifetime scanning imaging system of the present invention has multiple spectral channels and does not require expensive devices such as streak cameras. The system structure is simple and universal, and the cost is low, providing a technical foundation for real-time multi-parameter quantitative analysis of complex biological systems.

[0044] 3) The system of the present invention has good scalability and compatibility, supports multiple imaging modes, can be configured and adjusted according to different application requirements, and has broad application prospects. BRIEF DESCRIPTION OF DRAWINGS

[0045] Figure 1 is a structural schematic diagram of a spectral resolution parallel fluorescence lifetime scanning imaging system according to an embodiment of the present application;

[0046] Figure 2 is a data schematic diagram of a spectral resolution parallel fluorescence lifetime scanning imaging system according to an embodiment of the present application;

[0047] Figure 3 is a structural schematic diagram of a spectral resolution parallel fluorescence lifetime scanning imaging system according to an embodiment of the present application;

[0048] Figure 4 is a structural schematic diagram of a spectral resolution parallel fluorescence lifetime scanning imaging system according to an embodiment of the present application;

[0049] Figure 5 is a structural schematic diagram of a spectral resolution parallel fluorescence lifetime scanning imaging system according to an embodiment of the present application;

[0050] Figure 6 is a structural schematic diagram of a spectral resolution parallel fluorescence lifetime scanning imaging system according to an embodiment of the present application;

[0051] In the figure: 100 - excitation light module; 101 - laser; 102 - collimating mirror; 200 - scanning imaging module; 201 - light filtering component; 202 - one-dimensional scanning mirror group; 203 - objective lens; 204 - confocal element; 205 - confocal pinhole; 206 - two-dimensional scanning mirror; 207 - sample displacement stage; 300 - spectral resolution lifetime detection module; 301 - light splitting component; 302 - multi-channel photodetection component; 303 - multi-channel fluorescence lifetime measurement component; 304 - host computer; 2 - sample to be measured. DETAILED DESCRIPTION

[0052] The embodiments of the present application will be described below in conjunction with the accompanying drawings. It should be noted that the embodiments described in the present specification are not exhaustive, and do not represent the only embodiments of the present application. The following embodiments are only for the purpose of clearly illustrating the inventive content of the present application, and are not limiting on the embodiments. Those skilled in the art can make different forms of changes and modifications on the basis of the above embodiment description, and any changes or modifications within the scope of the technical concept and inventive content of the present application are within the protection scope of the present application.

[0053] Embodiment 1

[0054] The structural schematic diagram of a spectral resolution parallel fluorescence lifetime scanning imaging system according to an embodiment of the present application is as shown in Figure 1As shown, the system has the excitation light module 100, the scanning imaging module 200, the spectrally resolved lifetime detection module 300 described in the summary of the invention, and the functions described therein.

[0055] like Figure 1 As shown, the excitation light module 100 uses a pulsed laser light source (such as a picosecond pulse laser) with an adjustable pulse repetition frequency within the range of 1kHz to 200MHz as the laser 101. In this embodiment, it is set to operate stably at a repetition frequency of 20MHz. The laser is collimated and guided to the subsequent scanning imaging module 200 by a collimator 102. The laser 101 outputs laser light via optical fiber, and a collimated beam is obtained by the collimator 102. At the same time, the laser 101 outputs an excitation light synchronization signal to the multi-channel fluorescence lifetime measurement component 303 via a cable. This synchronization signal is synchronized in time with the light pulses of the laser 101.

[0056] The filter component 201 in the scanning imaging module 200 guides the excitation light through the one-dimensional scanning mirror in the one-dimensional scanning mirror group 202, and then focuses it on the sample 2 under test through the objective lens 203 on the objective lens displacement stage. The filter component 201 uses a dichroic mirror and an optical filter combination to achieve effective separation of the excitation light and fluorescence by utilizing the differences in the optical propagation characteristics of different wavelengths. The one-dimensional scanning mirror in the one-dimensional scanning mirror group 202 is a galvanometer scanning mirror or a resonant scanning mirror, and the objective lens 203 on the objective lens displacement stage is mounted on a piezoelectric drive displacement stage. Through this combination, the excitation light is scanned in three dimensions. At the same time, the one-dimensional scanning mirror group 202 and the objective lens 203 on the objective lens displacement stage respectively output scanning synchronization signals to the multi-channel fluorescence lifetime measurement component 303 via cables. This synchronization signal is synchronized with the scanning changes of the excitation light in three dimensions.

[0057] The objective lens 203 on the objective stage, through its focusing action, enables high-resolution scanning imaging of the region and effectively collects the fluorescence emitted by the sample 2 under test. The fluorescence returns along the original excitation light path to the filter component 201, which acts as a filter element, achieving excellent separation from the excitation light. The separated fluorescence is then guided through the confocal element 204, filtered through the confocal aperture 205 to remove out-of-focus fluorescence, and ultimately transmitted to the spectrally resolved lifetime detection module 300.

[0058] In the spectral resolution lifetime detection module 300, the light splitting component 301 uses a grating as a dispersion element, and the separated fluorescence input is distributed in space by the light splitting component 301 according to different wavelengths. The multi-channel photoelectric detection component 302 uses a multi-channel photoelectric detector with n detection channels to simultaneously detect fluorescence of different wavelengths. Each detection channel is equivalent to a single-channel photoelectric detector and can independently convert the corresponding wavelength fluorescence signal to an electrical signal. The n detection channels work in parallel, and the number of detection channels n is not less than 32. The multi-channel photoelectric detector of the embodiment uses a multi-channel detection element with n detection channels. The position is the focal plane of the separated fluorescence, and the detection surface size covers the spatial region where the fluorescence is distributed according to different wavelengths. The fluorescence wavelength range is 300 nm to 3000 nm, and the fluorescence wavelength resolution is 0.1 nm to 10 nm. The multi-channel photoelectric detection component 302 also includes a signal processing circuit for processing the photoelectric converted signal of the multi-channel photoelectric detector. The processing methods include amplification, discrimination, and shaping. The signal processing circuit channel corresponds to the n detection channels one by one, and the processed signal is transmitted to the multi-channel fluorescence lifetime measurement component 303.

[0059] The multi-channel fluorescence lifetime measurement component 303 uses a time-correlated single-photon counting method to realize multi-channel lifetime measurement corresponding to the n detection channels. At the same time, the excitation light synchronization signal and the scanning synchronization signal transmitted by the cable are input into the multi-channel fluorescence lifetime measurement component 303 to realize synchronization of the multi-channel fluorescence lifetime measurement results and the spatial scanning position during the scanning process. The multi-channel fluorescence lifetime measurement component 303 realizes multi-channel fluorescence lifetime measurement based on a field programmable gate array for time-to-digital conversion. Specifically, the field programmable gate array uses a combination of coarse counting and fine counting methods. The coarse counting method obtains coarse counts based on a master clock period, and the fine counting method uses a tapped delay chain method to divide the master clock period to obtain fine counts.

[0060] The host computer 304 receives the measurement data from the multi-channel fluorescence lifetime measurement component 303, can save the data, and can process and display multi-functional fluorescence lifetime scanning imaging results. The results include measured data in the six-dimensional data (x, y, z, λ, τ, t) of multi-functional fluorescence lifetime scanning imaging, where x, y, and z are three-dimensional data of the spatial region scanning of the measured sample 2, λ is spectral dimension data, τ is fluorescence lifetime dimension data obtained by processing, and t is time sequence dimension data of the overall scanning. One data schematic diagram of multi-functional fluorescence lifetime scanning imaging is shown in Figure 2 As shown, the left side is the x, y, z cell measured sample 2 spatial region scanning result, and the right side is the λ spectral dimension and τ fluorescence lifetime dimension data in the circle area on the left side. Figure 2 The schematic diagram is a group of data in the time sequence, and multiple groups of data constitute t time sequence dimension data.

[0061] The other components of the excitation light module 100, the scanning imaging module 200, and the spectral resolution lifetime detection module 300 of the spectral resolution parallel fluorescence lifetime scanning imaging system, except the measured sample 2, the multi-channel fluorescence lifetime measurement component 303, and the host computer 304, are integrated in a light-shielded housing.

[0062] Embodiment 2

[0063] The structure of the spectral resolution parallel fluorescence lifetime scanning imaging system of Embodiment 2 of the present application is basically the same as that of Embodiment 1, and the scanning imaging module 200 and the spectral resolution lifetime detection module 300 used are the same, and only the excitation light module 100 is modified and adjusted. Specifically, the structure is still as shown in Figure 1 The laser 101 in the excitation light module 100 is replaced by a wide-spectrum excitation light source (such as a super-continuous picosecond pulsed laser), and the collimating mirror 102 is replaced by a light splitting element or an optical filtering element to achieve selection of a single or multiple wavelengths as the required wavelength excitation light; the required wavelength range of the excitation light is 300-3000 nm. The other components of the excitation light module 100, the scanning imaging module 200, and the spectral resolution lifetime detection module 300 of the spectral resolution parallel fluorescence lifetime scanning imaging system, except the measured sample 2, the multi-channel fluorescence lifetime measurement component 303, and the host computer 304, are integrated in a light-shielded housing, and the optical paths between the modules are coupled and connected by optical fibers.

[0064] Embodiment 3

[0065] The structure of the spectral resolution parallel fluorescence lifetime scanning imaging system of Embodiment 3 of the present application is shown in Figure 3 The scanning imaging module 200 and the spectral resolution lifetime detection module 300 used are the same as those of Embodiment 1, and only the excitation light module 100 is modified and adjusted. In this embodiment, the excitation light module 100 uses multiple lasers 101 for combination, and the output excitation light is coupled and aligned by the mirror 103 and the dichroic mirror 104 element, and then collimated by the collimating mirror 102 to the scanning imaging module 200. Specifically, as shown in Figure 3 Three 1 kHz-200 MHz repetition frequency adjustable wavelength different pulsed laser light sources (such as picosecond pulsed lasers) are used as the laser 101, and the required excitation wavelength channel number of polychromatic excitation can be achieved by increasing, decreasing, and recoupling and aligning the excitation light. At the same time, the laser 101 has a cable line connected to the multi-channel fluorescence lifetime measurement component 303, and respectively outputs an excitation light synchronization signal, which is synchronized in time with the optical pulse of the laser 101.

[0066] Embodiment 4

[0067] The structural schematic diagram of a spectral resolution parallel fluorescence lifetime scanning imaging system of embodiment 4 is shown in Figure 4 As shown, the excitation light module 100 and the spectral resolution lifetime detection module 300 used in embodiment 1 are the same, and only the scanning imaging module 200 is modified and adjusted. Specifically, as shown, Figure 4 The light filtering component 201 in the scanning imaging module 200 guides the excitation light to pass through the objective lens 203 on the objective lens displacement stage in turn, and is finally focused on the measured sample 2. The two-dimensional scanning mirror 206 adopts a micro-electro-mechanical system (MEMS) micro-mirror scanning or a piezoelectric driving scanning mode, and the objective lens 203 on the objective lens displacement stage is installed on a piezoelectric driving displacement stage, which are combined to realize three-dimensional spatial position scanning of the excitation light. At the same time, a three-dimensional spatial position scanning output scanning synchronization signal is output, which is synchronized with the scanning change of the excitation light in space.

[0068] Embodiment 5

[0069] The structural schematic diagram of a spectral resolution parallel fluorescence lifetime scanning imaging system of embodiment 5 is shown in Figure 5 As shown, the excitation light module 100 and the spectral resolution lifetime detection module 300 used in embodiment 1 are the same, and only the scanning imaging module 200 is modified and adjusted. Specifically, as shown, Figure 5 The light filtering component 201 in the scanning imaging module 200 guides the excitation light to pass through the objective lens 203 on the objective lens displacement stage, and is finally focused on the measured sample 2. The measured sample 2 is fixed on the sample displacement stage 207 to realize the spatial position movement of the measured sample 2. The device used by the sample displacement stage 207 is a piezoelectric ceramic driving movement. The spatial position movement of the measured sample 2 is the two-dimensional movement of the sample displacement stage 207, and the objective lens is installed on the piezoelectric driving displacement stage to realize the movement of the laser in the longitudinal axis direction. At the same time, a three-dimensional spatial position scanning output scanning synchronization signal is output, which is synchronized with the scanning change of the excitation light in space.

[0070] Embodiment 6

[0071] The structural schematic diagram of a spectral resolution parallel fluorescence lifetime scanning imaging system of embodiment 6 is shown in Figure 6 Specifically, for realizing multiphoton fluorescence imaging. The excitation light module 100 and the spectral resolution lifetime detection module 300 used in embodiment 6 are consistent with the structure of embodiment 1, but the laser 101 used in the excitation light module 100 adopts a femtosecond pulse laser to realize multiphoton fluorescence excitation. The scanning imaging module 200 is also adjusted to match the multiphoton fluorescence imaging. Specifically, as shown, Figure 6As shown, the excitation light of the input scanning imaging module 200 is guided to sequentially scan the one-dimensional scanning mirror in the one-dimensional scanning mirror group 202, and then passes through the light filtering component 201 and the objective lens 203 on the objective lens displacement stage, and finally focuses on the measured sample 2. The one-dimensional scanning mirror in the one-dimensional scanning mirror group 202 is a galvanometer scanning mirror or a resonant scanning mirror, and the objective lens 203 on the objective lens displacement stage is installed on a piezoelectric driving displacement stage, and the combination realizes the scanning of the excitation light in the three-dimensional space position.

[0072] The measured sample 2 is excited by multi-photon fluorescence, and the emitted fluorescence returns along the original excitation light path until the light filtering component 201 as a light filtering element, so as to realize good separation from the excitation light. The separated fluorescence is sequentially guided to the spectral resolution lifetime detection module 300 through the confocal element 204. The selected optical device matches the excitation and emission wavelength of the multi-photon fluorescence imaging.

[0073] The above embodiments are only preferred embodiments of the present application and are not used to limit the present application. For those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A spectrally resolved parallel fluorescence lifetime scanning imaging system, characterized in that: It comprises an excitation light module (100), a scanning imaging module (200) and a spectral resolution lifetime detection module (300); The excitation light module (100) generates excitation light from a laser (101), and guides the excitation light to the scanning imaging module (200) through a collimating mirror (102), wherein the light intensity distribution of the excitation light is uniform; In the scanning imaging module (200), the excitation light passes through the filter component (201), the scanning moving component and the objective lens (203) on the objective lens displacement stage, guiding the excitation light to focus on the sample (2) to be measured, thereby realizing relative position movement scanning of the excitation light and the sample; wherein the scanning moving component is a one-dimensional scanning mirror group (202), a two-dimensional scanning mirror (206) or a sample displacement stage (207); The objective lens (203) on the objective lens displacement stage collects the fluorescence generated by the sample (2) under test, and the fluorescence returns along the original optical path and is spectrally separated from the excitation light at the filter component (201), guided by the confocal element (204), and then filtered out of the focal plane fluorescence through the confocal aperture (205), and finally transmitted to the spectrally resolved lifetime detection module (300); or the separated fluorescence is guided directly to the spectrally resolved lifetime detection module (300) through the confocal element (204); In the spectrally resolved lifetime detection module (300), the separated fluorescence is spatially distributed according to different wavelengths by the spectroscopic component (301); the multi-channel photoelectric detection component (302) has n detection channels and simultaneously detects fluorescence signals of different wavelengths, wherein n is not less than 32; the multi-channel fluorescence lifetime measurement component (303) and the laser (101) transmit an excitation light synchronization signal via a cable; the multi-channel fluorescence lifetime measurement component (303) is connected to the scanning moving component and the objective lens (203) on the objective lens displacement stage via a cable to transmit a scanning synchronization signal; the multi-channel photoelectric detection component (302) and the multi-channel fluorescence lifetime measurement component (303) realize parallel fluorescence lifetime measurement of n detection channels; the host computer (304) receives the measurement data results of the multi-channel fluorescence lifetime measurement component (303) and performs data processing and imaging display.

2. The spectrally resolved parallel fluorescence lifetime scanning imaging system according to claim 1, wherein: The excitation light module (100) uses a single laser (101) to generate excitation light, or couples and aligns multiple lasers (101) to generate excitation light; the generated excitation light has a single or multiple wavelength components within a wavelength range of 300 to 3000 nm, and the wavelength components are adjusted by a spectroscopic element or an optical filter element; The laser (101) is a pulsed laser with a pulse width of ≤10 ns or a modulated continuous laser, having a fixed pulse repetition frequency within a repetition frequency range of 1 kHz to 200 MHz, or an adjustable pulse repetition frequency within a range of 1 kHz to 200 MHz, but maintaining a stable repetition frequency during the measurement process; The excitation light synchronization signal is transmitted via the cable; the signal is output by the laser (101) as an electrical signal synchronized with the excitation light in time to the multi-channel fluorescence lifetime measurement component (303), or the multi-channel fluorescence lifetime measurement component (303) outputs an electrical signal to trigger the control laser (101) to achieve synchronization in time of outputting the excitation light.

3. The spectrally resolved parallel fluorescence lifetime scanning imaging system according to claim 1, wherein: The method of realizing the relative position movement scanning of the excitation light and the sample includes one or a combination of the following methods: the sample remains stationary and the three-dimensional spatial position of the excitation light is moved; the spatial position of the excitation light remains unchanged and the three-dimensional spatial position of the sample is moved; The sample is kept stationary, and the excitation light is moved in three dimensions, wherein the method includes galvanometer scanning, resonance scanning, motor-driven scanning, micro-electromechanical system (MEMS) micro-mirror scanning, piezoelectric drive scanning, acousto-optic deflector scanning, rotating lens barrel, i.e., turntable confocal scanning, and a combination of the above methods; The spatial position of the excitation light does not change, but the three-dimensional spatial position of the sample is moved, and the method includes manual knob-driven movement, motor-driven movement, piezoelectric ceramic-driven movement, and a combination of the above methods; The filter component (201) achieves effective separation of excitation light and fluorescence by utilizing the difference in optical propagation characteristics of different wavelengths. The filter component (201) specifically includes a single or a combination of multiple components selected from the group consisting of a dichroic mirror, an optical filter, an electro-optic modulator, an acousto-optic modulator, and a spatial light modulator.

4. The spectrally resolved parallel fluorescence lifetime scanning imaging system according to claim 1, wherein: The light splitting component (301) realizes the distribution of fluorescence in spatial regions according to different wavelengths, specifically using a single dispersion element or a combination of multiple dispersion elements; the dispersion element is a grating or a prism; The multi-channel photoelectric detection component (302) uses a multi-channel photoelectric detector with n detection channels to simultaneously detect fluorescence of different wavelengths, and each detection channel independently converts the corresponding wavelength fluorescence signal into an electrical signal, wherein the number of detection channels n is not less than 32; The multi-channel photoelectric detector adopts a single multi-channel detection element, a combination of multiple single-channel detection elements, or a combination of multiple multi-channel detection elements; the position is the focal plane of the separated fluorescence, and the detection surface size covers the spatial area where the fluorescence is distributed according to different wavelengths; The multi-channel photodetector covers a fluorescence wavelength range and achieves a fluorescence wavelength resolution that varies with the optical path configuration. The fluorescence wavelength range is 300 nm to 3000 nm, and the fluorescence wavelength resolution is 0.1 nm to 10 nm. The detection elements used are multi-pixel photon counters, single-photon avalanche diodes, single-photon avalanche diode arrays, photomultiplier tubes, multi-channel photomultiplier tubes, or hybrid detectors. The multi-channel photoelectric detection component (302) includes a signal processing circuit for processing the signal after photoelectric conversion of the multi-channel photoelectric detector, and the processing method includes amplification, identification and shaping. The signal processing circuit channel corresponds to n detection channels one by one, and the processed signal is applicable to the multi-channel fluorescence lifetime measurement component (303); The multi-channel fluorescence lifetime measurement component (303) uses a time-correlated single photon counting method, a digital frequency domain method or a phase shift method to achieve multi-channel fluorescence lifetime measurement; The multi-channel fluorescence lifetime measurement component (303) transmits an excitation light synchronization signal to the laser (101) via a cable, inputs an electrical signal synchronized with the excitation light, or outputs an electrical signal to trigger the laser (101) to generate synchronized excitation light; the multi-channel fluorescence lifetime measurement component (303) is connected to the scanning moving component and the objective lens (203) on the objective lens displacement stage via a cable, respectively, to transmit the scanning synchronization signal; The multi-channel fluorescence lifetime measurement component (303) is configured to detect the signal after photoelectric conversion and signal processing by the multi-channel photoelectric detection component (302), and has n lifetime measurement channels corresponding to n detection channels in a one-to-one manner, thereby realizing multi-channel parallel and simultaneous measurement of fluorescence lifetime; The lifetime measurement interval of each measurement channel of the multi-channel fluorescence lifetime measurement component (303) is a fixed time interval, or is within a time interval range of 5ns to 1ms, the lifetime measurement interval is adjustable, but a stable lifetime measurement interval is maintained during the measurement process; The host computer (304) is configured to receive data transmission results from the multi-channel fluorescence lifetime measurement component, is capable of storing the data, and is capable of processing and displaying the measured data in the six-dimensional data (x, y, z, λ, τ, t) of the multifunctional fluorescence lifetime scanning imaging, wherein x, y, and z are three-dimensional data of the sample spatial area scan, λ is spectral dimension data, τ is the fluorescence lifetime dimension data obtained by processing, and t is the time series dimension data of the overall scan.

5. The spectrally resolved parallel fluorescence lifetime scanning imaging system according to claim 4, characterized in that: The time-correlated single photon counting method specifically includes: using a time-to-digital converter integrated circuit chip, or implementing based on a field programmable gate array; The time-correlated single-photon counting method based on the field programmable gate array is implemented by combining coarse and fine counting methods, wherein the coarse counting method obtains coarse counts based on the main clock cycle, and the fine counting method is based on the tapped delay chain method, the multi-phase clock method, the differential time-to-digital conversion method or the pulse contraction method and their combination to divide the main clock cycle to obtain fine counts.

6. The spectrally resolved parallel fluorescence lifetime scanning imaging system according to claim 1, wherein: In the excitation light module (100), the scanning imaging module (200) and the spectrally resolved lifetime detection module (300), other components except the sample to be measured (2), the multi-channel fluorescence lifetime measurement component (303) and the host computer (304) are configured to be integrated in the light-shielding housing; or each module is separately integrated in the light-shielding housing, and the light paths between the modules or within the modules are connected by optical fiber coupling.

7. The spectrally resolved parallel fluorescence lifetime scanning imaging system according to claim 1, wherein: The imaging mode includes single-photon fluorescence imaging, two-photon fluorescence imaging, multi-photon fluorescence imaging or up-conversion fluorescence imaging; the specific imaging light path is adjusted according to the imaging mode.

8. The spectrally resolved parallel fluorescence lifetime scanning imaging system according to claim 1, wherein: The imaging comprises merging adjacent channels of n lifetime measurement channels in a spectrally resolved lifetime detection module (300) to achieve multi-channel fluorescence lifetime scanning imaging, which is less than the number of detection channels; or merging all channels to achieve single-channel fluorescence lifetime scanning imaging.

9. The spectrally resolved parallel fluorescence lifetime scanning imaging system according to claim 1, wherein: The imaging comprises merging different time channels of the same detection channel of the spectrally resolved lifetime detection module (300) to achieve spectrally resolved fluorescence scanning imaging.

10. The spectrally resolved parallel fluorescence lifetime scanning imaging system according to claim 1, wherein: The imaging comprises merging different time channels of the same detection channel of the spectrally resolved lifetime detection module (300), and then merging the different detection channels in different ways to achieve fluorescence spectrally resolved scanning imaging with a number less than the number of detection channels; or merging all time channels and detection channels to achieve single-channel fluorescence intensity scanning imaging.

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