CN108387560B - Fluorescence lifetime imaging system and method for synchronously measuring photon arrival time and position - Google Patents
Fluorescence lifetime imaging system and method for synchronously measuring photon arrival time and position Download PDFInfo
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Abstract
The invention relates to a fluorescence lifetime imaging system and method for synchronously measuring photon arrival time and position, comprising a scanning translation table, a sample to be imaged, an excitation light source, a focusing lens, a polychromator and an MCP position-sensitive anode detector, wherein the scanning translation table is used for carrying out scanning translation on the sample to be imaged; the scanning translation table is used for placing a sample to be imaged; the excitation light source is used for irradiating a sample to be imaged; the focusing lens, the polychromator and the MCP position-sensitive anode detector are sequentially arranged in the propagation direction of excited light of a sample to be imaged; the outer side of the MCP position-sensitive anode detector is connected with a time signal leading-out module; the multi-path output of the position-sensitive anode of the MCP position-sensitive anode detector is sequentially connected with a charge-sensitive preamplifier, a photon arrival time and position synchronous measuring circuit and an image reconstruction module; the time signal leading-out module is connected with the photon arrival time and position synchronous measuring circuit; the scanning translation stage is connected with the image reconstruction module. The invention has finer spectral resolution and can acquire more dimensional information.
Description
Technical Field
The invention relates to the technical field of fluorescence lifetime imaging, in particular to a fluorescence lifetime imaging system for synchronously measuring photon arrival time and photon position, and also relates to an imaging method of the system.
Background
Fluorescence Lifetime Imaging (FLIM) is a novel fluorescence imaging technique that uses the length of fluorescence lifetime measured at a pixel point as the corresponding gray-scale value of the pixel, and is generally not affected by fluorescence intensity interference factors such as excitation laser, bleaching of fluorescent dyes, and uneven distribution of fluorescent dyes, as compared to fluorescence intensity-based analysis and imaging techniques. Therefore, the fluorescence lifetime imaging technology can provide clearer contrast for biological tissues in different states or different types of biological tissues, and can quantitatively measure the fluorophore and the microenvironment parameters in which the fluorophore is located. Fluorescence lifetime imaging techniques have been successfully applied in the fields of cell biology, analytical chemistry, clinical diagnosis, and the like.
Fluorescence lifetime imaging techniques mainly fall into two broad categories, frequency domain and time domain. The frequency modulation method is a method of measuring by comparing the phase shift and the modulation degree of a fluorescence signal with respect to a modulated laser signal, and is often used for imaging measurement of fluorescence lifetime on the order of nanoseconds or less. With the biomedical demand, the technology of nanosecond, picosecond and even femtosecond time domain fluorescence lifetime imaging appears. The Time domain mainly comprises Time-Gated Detection (Time-Gated Detection), Time-correlated single photon counting (TCSPC) and scanning camera imaging (stream-FLIM) which are three implementation methods. The gated FLIM has low collection efficiency and is difficult to distinguish a multi-exponential decay function; scanning cameras, while having extremely high sensitivity and time-resolved detectors, are slow to image due to camera integration time limitations. Time-correlated single photon counting (TCSPC) is a highly accurate optical pulse measurement technique, but since it can only record one photon at a time within an excitation period, the remaining photons are ignored, which affects the photon collection efficiency and the measurement accuracy of the pulse signal. Although the time measurement accuracy of the current TCSPC for measuring the arrival time of photons by using the TAC-ADC or the TDC is high, the measurement range is very limited, and the maximum measurement range is only hundreds of microseconds.
In addition, many occasions in biomedical research and clinical diagnosis have provided the requirement of multispectral resolution, and at present, multispectral fluorescence lifetime imaging is mainly based on bichromatic spectroscope and TCSPC technology. In the system, fluorescence passes through a series of dichroic mirrors to be subjected to spectral separation, and is focused by different lenses to be detected by different single-photon detectors. This results in a system with limited spectrally resolved channels, fixed wavelength separation, and the low imaging rate inherent in TCSPC technology, which limits the applicability of this system. Therefore, the development of multispectral fluorescence lifetime imaging technology with high spectral resolution, fast imaging speed and low cost is the current trend.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, adapt to the practical needs and provide a fluorescence lifetime imaging system and method for synchronously measuring the arrival time and the position of photons.
In order to realize the purpose of the invention, the invention adopts the technical scheme that:
a fluorescence lifetime imaging system for synchronously measuring photon arrival time and position comprises a scanning translation table, a sample to be imaged, an excitation light source, a focusing lens, a polychromator, an MCP position sensitive anode detector, a time signal leading-out module, a charge sensitive preamplifier, a photon arrival time and position synchronous measuring circuit and an image reconstruction module;
the scanning translation stage is used for placing a sample to be imaged; the excitation light source is used for irradiating a sample to be imaged;
the focusing lens, the polychromator and the MCP position-sensitive anode detector are sequentially arranged in the propagation direction of stimulated luminescence of a sample to be imaged;
the outer side of the MCP position-sensitive anode detector is connected with a time signal leading-out module;
the multi-path output of the position-sensitive anode of the MCP position-sensitive anode detector is sequentially connected with a charge-sensitive preamplifier, a photon arrival time and position synchronous measuring circuit and an image reconstruction module;
the time signal leading-out module is connected with the photon arrival time and position synchronous measuring circuit;
the scanning translation stage is connected with the image reconstruction module.
The MCP position-sensitive anode detector sequentially comprises an incidence window, a photocathode pressurizing electrode, a ceramic tube shell, a cascade MCP input surface pressurizing electrode, a cascade MCP output surface pressurizing electrode, a germanium layer, a ceramic substrate, a position-sensitive anode pressurizing electrode and an anode substrate.
The time signal leading-out module comprises a resistor, a capacitor and a current sensitive preamplifier, wherein the resistor comprises four resistors which are sequentially connected in series, a branch is led out from the upper part of each resistor and is respectively connected with a photocathode pressurizing electrode, a cascade MCP input surface pressurizing electrode, a cascade MCP output surface pressurizing electrode and a potential sensitive anode pressurizing electrode which are arranged in the MCP potential sensitive anode detector, one end of the capacitor is connected with the cascade MCP input surface pressurizing electrode, and the other end of the capacitor is connected with the current sensitive preamplifier; the current sensitive preamplifier outputs photon arrival timing signals to a photon arrival time and position synchronous measuring circuit.
A fluorescence lifetime imaging method with simultaneous photon arrival time and position measurement, comprising the steps of:
1) the scanning translation table fixes scanning points (X, Y) and outputs the coordinate data to the image reconstruction module, and the excitation light source emits laser to irradiate the sample to be imaged so that the sample to be imaged emits fluorescence;
2) the fluorescence photons emitted by the sample to be imaged are converged by the focusing lens and then enter the polychromator for light splitting;
3) the fluorescence photons dispersed by the polychromator enter an MCP position-sensitive anode detector;
4) synchronously measuring the arrival time and the position of photons by using the outer output signal of the MCP position-sensitive anode detector and the multi-path output signal of the position-sensitive anode;
5) and the image reconstruction module carries out image reconstruction according to the input coordinate of the scanning translation stage, the photon arrival time and the position coordinate data.
In step 4), the method for synchronously measuring the arrival time and the position of the photon comprises the following specific steps:
1) when the MCP position-sensitive anode detector detects photons, a cascade MCP input surface pressurizing electrode charges one end of a capacitor, the other end of the capacitor charges along with the photon arrival time information, a generated current pulse signal carrying photon arrival time information is generated, the signal is amplified by a current sensitive preamplifier and then outputs a photon arrival timing signal, and a time signal leading-out module outputs the photon arrival timing signal to a photon arrival time and position synchronous measuring circuit;
2) when the MCP position-sensitive anode detector detects photons, a multichannel pulse signal which is output by the position-sensitive anode and carries photon arrival position information is amplified by a charge sensitive preamplifier and then is output to a photon arrival time and position synchronous measuring circuit;
3) under the synchronous triggering of photon arrival timing signals, the photon arrival time and position synchronous measuring circuit processes the photon arrival timing signals and a multi-channel pulse signal which is input by the charge sensitive preamplifier and carries photon arrival position information to obtain photon arrival time and position coordinates, and transmits the arrival time and the position coordinates of the continuous arriving photons to the image reconstruction module.
The image reconstruction module comprises a coordinate caching unit, a time-space distribution storage matrix unit, a multispectral fluorescence lifetime curve reconstruction unit and a multispectral fluorescence lifetime pseudo-color image reconstruction unit, and the image reconstruction method comprises the following steps:
1) the USB2.0 interface of the computer continuously reads the data of the photon arrival time and position synchronous measurement circuit to the memory of the computer by adopting a ping-pong operation mode;
2) the coordinate caching unit caches photon position coordinates, and the time caching unit caches photon arrival time;
3) the space-time distribution storage matrix unit takes photons as a basic class, classifies the photons into different spectral channels according to the wavelength to which the position coordinates of the photons are directed, and stores the arrival time and the position into a matrix in an object-oriented mode with class attributes;
4) the multispectral fluorescence lifetime curve reconstruction unit divides the arrival time of fluorescence photons at the same position coordinate, namely the same wavelength, into a plurality of parts at equal intervals, respectively counts the number of the photons arriving in each time period, takes the arrival time of the photons as a horizontal coordinate and the number of the photons in each time period as a vertical coordinate, and reconstructs the fluorescence lifetime curve of the waveband of the scanning point (X, Y) by a curve fitting method. The multispectral fluorescence lifetime curve can be reconstructed by using the data of a plurality of spectral channels;
5) the multispectral fluorescence lifetime pseudo-color image reconstruction unit reconstructs a multispectral fluorescence lifetime pseudo-color image by using a multispectral fluorescence lifetime curve of each scanning point obtained after scanning a frame.
The invention has the beneficial effects that:
1. finer spectral resolution. In the invention, the complex color light emitted by a sample to be imaged is received by the area array detector after being split by the polychromator, the spectral resolution channel is not limited, and the higher the resolution of the detector is, the finer the high-energy resolution spectrum is, so the invention can obtain higher spectral resolution;
2. and acquiring more dimensional information. The invention adopts a method for synchronously measuring the arrival time and the position of the photon to synchronously obtain the data of the position, the arrival time, the wavelength and the like of the photon emitted by the sample to be imaged, and the data can be used for obtaining the multispectral fluorescence life curve, also obtaining the photon vertical distribution, the photon coherence and the photon high-order coherence and providing more research values;
3. and the data measurement precision is higher. According to the invention, a photon arrival timing signal is led out by utilizing a charging signal of a working circuit of the detector, photon arrival time and photon position are measured separately, time information is preserved by adopting a wide-bandwidth current sensitive preamplifier, and the measurement precision of electric charge quantity is ensured by adopting charge sensitive front for position measurement;
4. faster data processing speed. The image reconstruction data of the invention adopts a matrix storage mode which takes photons as a basic class and takes the arrival time, position coordinates and wavelength of the photons as class attributes, thereby greatly improving the access speed.
Drawings
FIG. 1 is a block diagram of the system components of the present invention.
FIG. 2 is a timing diagram of peak acquisition for a photon arrival time and position synchronized measurement circuit of the present invention.
FIG. 3 is a software flow diagram of the image reconstruction module of the present invention.
In the figure, 1 is a scanning translation table, 10 is a sample to be imaged, 2 is an excitation light source, 3 is a focusing lens, 4 is a polychromator, 5 is an MCP position-sensitive anode detector, 501 is an incidence window, 502 is a photocathode, 503 is a photocathode pressurizing electrode, 504 is a ceramic tube shell, 505 is a cascade MCP, 506 is a cascade MCP input surface pressurizing electrode, 507 is a cascade MCP output surface pressurizing electrode, 508 is a germanium layer, 509 is a ceramic substrate, 510 is a position-sensitive anode, 511 is a position-sensitive anode pressurizing electrode, 512 is an anode substrate, 6 is a time signal extraction module, 601 is a resistor, 602 is a capacitor, 603 is a current-sensitive preamplifier, 604 is a photon arrival timing signal, 7 is a charge-sensitive preamplifier, 8 is a photon arrival time and position synchronization measurement circuit, 9 is an image reconstruction module, 901 is a coordinate buffer unit, 902 is a time buffer unit, 903 is a space-time distribution storage matrix unit, 904 is a multispectral fluorescence lifetime curve reconstruction unit, 905 is a multispectral fluorescence lifetime pseudo-color image reconstruction unit, and 906 is a USB interface.
Detailed Description
The invention is further illustrated with reference to the following figures and examples:
see fig. 1-3.
The invention discloses a fluorescence lifetime imaging system for synchronously measuring photon arrival time and position, which comprises a scanning translation table 1, a sample to be imaged 10, an excitation light source 2, a focusing lens 3, a polychromator 4, an MCP position sensitive anode detector 5, a time signal extraction module 6, a charge sensitive preamplifier 7, a photon arrival time and position synchronous measuring circuit 8 and an image reconstruction module 9, as shown in figure 1.
The scanning translation stage 1 is used for placing a sample 10 to be imaged, and the excitation light source 2 is used for irradiating the sample 10 to be imaged, and the scanning translation stage is characterized in that: the focusing lens 3, the polychromator 4 and the MCP position-sensitive anode detector 5 are sequentially arranged in the transmission direction of excited light of a sample 10 to be imaged, the outer side of the MCP position-sensitive anode detector 5 is connected with a time signal leading-out module 6, and the position-sensitive anode multiplexed output of the MCP position-sensitive anode detector 5 is sequentially connected with a charge sensitive preamplifier 7, a photon arrival time and position synchronous measurement circuit 8 and an image reconstruction module 9; the time signal leading-out module 6 is connected with the photon arrival time and position synchronous measuring circuit 8, and the scanning module of the scanning translation table 1 is connected with the image reconstruction module 9.
The MCP position-sensitive anode detector 5 sequentially comprises an entrance window 501, a photocathode 502, a photocathode pressurizing electrode 503, a ceramic tube shell 504, a cascade MCP 505, a cascade MCP input surface pressurizing electrode 506, a cascade MCP output surface pressurizing electrode 507, a germanium layer 508, a ceramic substrate 509, a position-sensitive anode 510, a position-sensitive anode pressurizing electrode 511 and an anode substrate 512.
After the photons enter the MCP position-sensitive anode detector 5, the photocathode 502 generates a photoelectric effect to emit photoelectrons. The photoelectrons are multiplied by the cascade MCP 505 to form a charge cloud, and the charge cloud is accelerated by an electric field and then received by the potential sensitive anode. The amount of charge received by each electrode, which is insulated from each other, on the potential sensitive anode can be used to solve for the centroid of the charge cloud, which corresponds to the two-dimensional coordinates of the incident photons. Therefore, the multi-channel charge pulse signals output by each electrode of the position-sensitive anode are used as photon arrival position signals, and in order to ensure the measurement accuracy, the multi-channel charge pulse signals output by the position-sensitive anode are amplified by a charge-sensitive preamplifier 7 and then input into a photon arrival time and position synchronous measurement circuit 8. In the case, the position-sensitive anode can be a cursor anode, a resistance anode, a wedge-shaped anode, a delay line anode, etc., and taking the cursor anode as an example, nine mutually insulated electrodes of the cursor position-sensitive anode output nine charge pulse signals carrying photon arrival position information to the photon arrival time and position synchronization measuring circuit 8.
In addition, after the electron cloud is emitted in the photon detection process, the MCP is charged by the MCP working circuit due to the charge conservation principle, and the charging signal is led out by the time signal leading-out module 6 and can be input into the photon arrival time and position synchronization measuring circuit 8 as a photon arrival timing signal. The time signal leading-out module 6 comprises a resistor 601, a capacitor 602 and a current sensitive preamplifier 603, wherein the resistor 601 comprises four resistors which are sequentially connected in series and are sequentially defined as 6011, 6012, 6013 and 6014, a branch is led out from the upper part of each resistor and is respectively connected with a photocathode pressurizing electrode 503, a cascade MCP input surface pressurizing electrode 506, a cascade MCP output surface pressurizing electrode 507 and a potential sensitive anode pressurizing electrode 511 in the MCP potential sensitive anode detector 5, the capacitor 602 is connected with the cascade MCP input surface pressurizing electrode 502, and a charging signal of the capacitor 602 is amplified by the current sensitive preamplifier 603 and then is output to a photon arrival time and position synchronization measuring circuit 8.
Although the multiple output signals of the position-sensitive anode of the MCP position-sensitive anode detector carry the arrival time and position information of the photon at the same time, in order to ensure the accuracy, the photon arrival time and position measurement must be separated, and the output signals of the position-sensitive anode of the detector cannot be used for measurement at the same time. Therefore, the photon arrival timing signal is led out by the MCP working circuit through the time signal leading-out module, and the time information is preserved through the wide-bandwidth current sensitive preamplifier; the position measurement is carried out by utilizing a position-sensitive anode output signal of the detector, and the measurement precision of the electric charge quantity is ensured by adopting a charge sensitive preamplifier, so that the synchronous high-precision measurement of the photon arrival time and the position is realized.
The invention also discloses an imaging method of the fluorescence lifetime imaging system for synchronously measuring the arrival time and the position of photons, which comprises the following steps:
1) the scanning translation table 1 fixes scanning points (X, Y) and outputs the coordinate data to the image reconstruction module 9, and the excitation light source 2 emits laser to irradiate the sample 10 to be imaged, so that the sample 10 to be imaged emits fluorescence;
2) fluorescence photons emitted by a sample 10 to be imaged are converged by a focusing lens 3 and then enter a polychromator 4 for light splitting;
3) the fluorescence photons dispersed by the polychromator 4 enter an MCP position-sensitive anode detector 5;
4) the external output signal of the MCP position-sensitive anode detector 5 and the multi-path output signal of the position-sensitive anode carry out synchronous measurement of photon arrival time and position, and the method comprises the following specific steps:
4.1) when the MCP position-sensitive anode detector 5 detects photons, the cascade MCP input surface pressurizing electrode charges one end of a capacitor 602, the other end of the capacitor 602 is charged, a generated current pulse signal carrying photon arrival time information is generated, the signal is amplified by a current sensitive preamplifier 603, a photon arrival timing signal 604 is output, and a time signal leading-out module 6 outputs the photon arrival timing signal 604 to a photon arrival time and position synchronous measuring circuit 8;
4.2) when the MCP position-sensitive anode detector 5 detects photons, a multichannel pulse signal which is output by the position-sensitive anode and carries photon arrival position information is amplified by a charge sensitive preamplifier 7 and then is output to a photon arrival time and position synchronous measuring circuit 8;
4.3) under the synchronous triggering of the photon arrival timing signal 604, the photon arrival time and position synchronous measuring circuit 8 processes the photon arrival timing signal 604 and a multi-channel pulse signal which is input by the charge sensitive preamplifier 7 and carries photon arrival position information to obtain photon arrival time and position coordinates, and transmits the arrival time and the position coordinates of the continuous arriving photons to the image reconstruction module 9.
5) The image reconstruction module 9 reconstructs an image according to the input coordinate of the scanning translation stage 1, the photon arrival time and the position coordinate data.
The photon arrival time and position synchronous measuring circuit 8 processes a multi-channel pulse signal which is input by the charge sensitive preamplifier 7 and carries photon arrival position information to obtain photon arrival time and position coordinates, and the method comprises the following steps: and triggering synchronous acquisition of multipath pulse peaks and photon arrival time measurement by utilizing photon arrival timing signals, and processing photon arrival time and pulse peak data to solve photon arrival time and position coordinates. The timing diagram of the multi-pulse synchronous peak acquisition is shown in fig. 2, which illustrates nine pulse peak acquisitions for a cursor anode. When a photon arrival timing signal is detected, 9 paths of A/D converters are synchronously started to carry out peak value acquisition on a plurality of paths of pulse signals, 9 paths of pulse peak value data can calculate the position coordinates of the arrival of the photons according to a Vernier anode decoding formula, and specifically, a reference document [1] Qiurong Yan, Baosheng Zhao, Yongan Liu, Hao Yang, Lizhi Sheng, Two-dimensional photon counting imaging detector base on a Vernier position sensing anode readout [ J ]. Chinese Physics C, 2011, 35(4): 368 is calculated.
FIG. 3 is a software flow diagram of the image reconstruction module of the present invention. The image reconstruction module 9 comprises a coordinate buffer unit 901, a time buffer unit 902, a space-time distribution storage matrix unit 903, a multispectral fluorescence lifetime curve reconstruction unit 904 and a multispectral fluorescence lifetime pseudo-color image reconstruction unit 905; the image reconstruction module 9 is connected to the time and position synchronization measurement circuit 8 via a USB interface 906. The image reconstruction method comprises the following specific steps:
1) the computer USB interface 906 continuously reads the data of the photon arrival time and position synchronization measurement circuit 8 to the computer memory in a ping-pong operation mode;
2) the coordinate buffer unit 901 buffers photon position coordinates, and the time buffer unit 902 buffers photon arrival time;
3) the space-time distribution storage matrix unit 903 uses photons as a basic class, classifies the photons into different spectral channels according to the wavelength to which the position coordinates of the photons are directed, and stores the arrival time and the position into a matrix in an object-oriented manner by using class attributes;
4) the multispectral fluorescence lifetime curve reconstruction unit 904 divides the arrival time of fluorescence photons at the same position coordinate, namely the same wavelength, into a plurality of parts at equal intervals, respectively counts the number of the photons arriving in each time period, reconstructs the fluorescence lifetime curve of the wave band of the scanning point (X, Y) by using the photon arrival time as a horizontal coordinate and the photon number in each time period as a vertical coordinate in a curve fitting manner, and reconstructs the multispectral fluorescence lifetime curve by using the data of a plurality of spectral channels;
5) the multispectral fluorescence lifetime pseudo-color image reconstruction unit 905 reconstructs a multispectral fluorescence lifetime pseudo-color image by using the multispectral fluorescence lifetime curve of each scanning point obtained after scanning a frame. The reconstruction method of the multispectral fluorescence lifetime pseudo-color image comprises the following steps: assuming the scanning plane as an array of pixel cells, if the pixel values are set, where N and M are X-direction pixel values and Y-direction pixel values, respectively, the scanning point coordinates (X, Y) may be converted into pixel coordinate values. And averaging the fluorescence lifetime values of the scanning points in each pixel coordinate, reconstructing a two-dimensional gray level fluorescence lifetime image by taking the fluorescence lifetime average value as a gray value, and fusing the two-dimensional gray level fluorescence lifetime images with different wavelengths to reconstruct a multispectral fluorescence lifetime pseudo-color image.
The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent modifications made by the present invention and the contents of the drawings or directly or indirectly applied to the related technical fields are included in the scope of the present invention.
Claims (4)
1. A fluorescence lifetime imaging system for simultaneous measurement of photon arrival time and position, characterized by: the device comprises a scanning translation table (1), a sample to be imaged (10), an excitation light source (2), a focusing lens (3), a polychromator (4), an MCP position-sensitive anode detector (5), a time signal leading-out module (6), a charge sensitive preamplifier (7), a photon arrival time and position synchronous measurement circuit (8) and an image reconstruction module (9); the scanning translation table (1) is used for placing a sample (10) to be imaged; the excitation light source (2) is used for irradiating a sample (10) to be imaged; the focusing lens (3), the polychromator (4) and the MCP position-sensitive anode detector (5) are sequentially arranged in the propagation direction of excited light of a sample (10) to be imaged; the outer side of the MCP position-sensitive anode detector (5) is connected with a time signal leading-out module (6); the multi-path output of a position-sensitive anode of the MCP position-sensitive anode detector (5) is sequentially connected with a charge-sensitive preamplifier (7), a photon arrival time and position synchronous measurement circuit (8) and an image reconstruction module (9), the time signal extraction module (6) is connected with the photon arrival time and position synchronous measurement circuit (8), the scanning translation stage (1) is connected with the image reconstruction module (9), the MCP position-sensitive anode detector (5) sequentially comprises an entrance window (501), a photocathode (502), a photocathode pressurizing electrode (503), a ceramic tube shell (504), a cascaded MCP (505), a cascaded MCP input surface pressurizing electrode (506), a cascaded MCP output surface pressurizing electrode (507), a germanium layer (508), a ceramic substrate (509), a position-sensitive anode (510), a position-sensitive anode pressurizing electrode (511) and an anode substrate (512), and the time signal extraction module (6) comprises a resistor (601), The device comprises a capacitor (602) and a current sensitive preamplifier (603), wherein the resistor (601) comprises four resistors which are sequentially connected in series, a branch is led out from the upper part of each resistor and is respectively connected with a photocathode pressurizing electrode (503), a cascade MCP input surface pressurizing electrode (506), a cascade MCP output surface pressurizing electrode (507) and a potential sensitive anode pressurizing electrode (511) in the MCP potential sensitive anode detector (5), one end of the capacitor (602) is connected with the cascade MCP input surface pressurizing electrode (506), and the other end of the capacitor (602) is connected with the current sensitive preamplifier (603); the current sensitive preamplifier (603) outputs a photon arrival timing signal (604) to a photon arrival time and position synchronization measurement circuit (8).
2. A fluorescence lifetime imaging method for synchronous measurement of photon arrival time and position, characterized by: the method comprises the following steps:
1) the scanning translation table fixes scanning points (X, Y) and outputs the coordinate data to an image reconstruction module (9), and an excitation light source (2) emits laser to irradiate a sample (10) to be imaged, so that the sample (10) to be imaged emits fluorescence;
2) fluorescence photons emitted by a sample (10) to be imaged are converged by a focusing lens (3) and then enter a polychromator (4) for light splitting;
3) the fluorescence photons dispersed by the polychromator enter an MCP position-sensitive anode detector (5);
4) the photon arrival time and position synchronous measurement is carried out by utilizing the outer side output signal of the MCP position-sensitive anode detector (5) and the multi-path output signal of the position-sensitive anode;
5) and the image reconstruction module (9) is used for reconstructing an image according to the input coordinate of the scanning translation table (1), photon arrival time and position coordinate data.
3. The method of fluorescence lifetime imaging with synchronized measurement of photon arrival time and location according to claim 2, wherein: in step 4), the method for synchronously measuring the arrival time and the position of the photon comprises the following specific steps:
1) when the MCP position-sensitive anode detector (5) detects photons, a cascade MCP input surface pressurizing electrode charges one end of a capacitor (602), the other end of the capacitor (602) is charged, a generated current pulse signal carrying photon arrival time information is generated, the signal is amplified by a current sensitive preamplifier (603), a photon arrival timing signal (604) is output, and a time signal leading-out module (6) outputs a photon arrival timing signal (604) to a photon arrival time and position synchronization measuring circuit (8);
2) when the MCP position-sensitive anode detector (5) detects photons, a multichannel pulse signal which is output by the position-sensitive anode and carries photon arrival position information is amplified by a charge sensitive preamplifier (7) and then is output to a photon arrival time and position synchronous measuring circuit (8);
3) under the synchronous triggering of the photon arrival timing signal (604), the photon arrival time and position synchronous measurement circuit (8) processes the photon arrival timing signal (604) and a multi-channel pulse signal which is input by the charge sensitive preamplifier (7) and carries photon arrival position information to obtain photon arrival time and position coordinates, and transmits the arrival time and the position coordinates of the continuous arrival photons to the image reconstruction module (9).
4. The method of fluorescence lifetime imaging with synchronized measurement of photon arrival time and location according to claim 3, wherein: the image reconstruction module (9) comprises a coordinate buffer unit (901), a time buffer unit (902), a space-time distribution storage matrix unit (903), a multispectral fluorescence lifetime curve reconstruction unit (904) and a multispectral fluorescence lifetime pseudo-color image reconstruction unit (905), and the image reconstruction method comprises the following steps,
1) the USB2.0 interface of the computer continuously reads the data of the photon arrival time and position synchronous measuring circuit (8) to the memory of the computer by adopting a ping-pong operation mode;
2) the coordinate buffer unit (901) buffers photon position coordinates, and the time buffer unit (902) buffers photon arrival time;
3) the space-time distribution storage matrix unit (903) takes photons as a basic class, classifies the photons into different spectral channels according to the wavelength to which the position coordinates of the photons are directed, and stores the arrival time and the position into a matrix in an object-oriented mode with class attributes;
4) the multispectral fluorescence lifetime curve reconstruction unit (904) respectively counts the number of photons arriving in each time period by dividing the arrival time of fluorescence photons at the same position coordinate, namely the same wavelength, into a plurality of parts at equal intervals, and reconstructs the fluorescence lifetime curve of the wave band of the scanning point (X, Y) by using the photon arrival time as a horizontal coordinate and the photon number in each time period as a vertical coordinate in a curve fitting way; the multispectral fluorescence lifetime curve can be reconstructed by using the data of a plurality of spectral channels;
5) the multispectral fluorescence lifetime pseudo-color image reconstruction unit (905) reconstructs a multispectral fluorescence lifetime pseudo-color image by using a multispectral fluorescence lifetime curve of each scanning point obtained after scanning a frame.
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