CN112113939A

CN112113939A - Fluorescence lifetime imaging method and device based on spectral technology

Info

Publication number: CN112113939A
Application number: CN202010762173.2A
Authority: CN
Inventors: 匡翠方; 刘少聪; 董婉潔; 王文生
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2020-07-31
Filing date: 2020-07-31
Publication date: 2020-12-22

Abstract

The invention discloses a fluorescence lifetime imaging device based on a spectral technology, which comprises a light source, a microscope objective, a sample, a light source control unit and a fluorescence detection unit, wherein the microscope objective is used for converging exciting light emitted by the light source and exciting the sample to emit fluorescence; is provided with: the spectrum splitting module is used for splitting the fluorescence collected by the microscope objective; the detection module is used for receiving the fluorescent molecules of different wave bands after the spectrum division; and the counting module is used for counting photons of the fluorescent molecules and calculating fluorescence life information. Meanwhile, the invention also discloses a fluorescence lifetime imaging method based on the spectrum technology. The invention introduces a spectrum splitting technology, and the broadband fluorescence signal is split into different channels by the spectrum splitting module and received by different detectors, thereby greatly relieving the influence of dead time of a single detector on fluorescence life measurement in the traditional method, relieving the problem of photon accumulation effect of fluorescence life measurement, improving the effective detection efficiency of photons and improving the speed of life imaging.

Description

Fluorescence lifetime imaging method and device based on spectral technology

Technical Field

The invention belongs to the field of fluorescence lifetime imaging, and particularly relates to a fluorescence lifetime imaging method and device based on a spectral technology.

Background

In the field of biomedical imaging, super-resolution fluorescence imaging has been the focus of biomedical research, and fluorescence optical microscopy plays an important role in observing subcellular structures through special labeling of biomolecules. For fluorescence information, there are mainly four basic physical dimensions, including intensity information, wavelength information (absorption and emission spectra), lifetime information, and polarization information. Due to the specificity of fluorescent labels, high contrast results of label structures can be obtained only by acquiring intensity information, so most of the current imaging techniques are directed to intensity information. In addition to intensity information, wavelength information is also widely used in the field of super-resolution imaging, such as stimulated emission depletion (STED), single molecule localization (SMS, including PALM and STORM), and utilizes the difference in absorption and emission spectra of fluorescence to achieve "on-off" modulation in intensity. For polarization, there is a method for realizing super-resolution microscopic imaging by relevant polarization modulation. Besides the information, the life information is also the very important physical dimension of the fluorescent molecule, and because the fluorescence life information is the intrinsic information of the fluorescent molecule, the fluorescence life information is not influenced by the density or the excitation light intensity of the fluorescent molecule, and only responds to the imaging microenvironment, such as pH value, calcium ion concentration, temperature, oxygen concentration, refractive index, viscosity and the like, the life imaging of the fluorescent molecule has more research significance than the ordinary intensity imaging when the environmental parameters need to be quantitatively analyzed.

In the current imaging field, the application of fluorescence lifetime imaging mainly includes two aspects, one is to use lifetime information to observe molecular-scale motion, such as detection of Fluorescence Resonance Energy Transfer (FRET) of a fluorescence molecule pair, which can be applied to research of interaction between protein and cells; on the other hand, the fluorescence lifetime can also be combined with the relevant super-resolution imaging technologies of confocal imaging and STED imaging, a new information dimension is added to the common intensity information, and meanwhile, the lifetime information can also be utilized to improve the imaging resolution of the relevant imaging technologies. Lifetime imaging has therefore a very important potential for modern multi-information dimensional imaging techniques.

The current fluorescence lifetime measurement mainly comprises two types, one is time domain lifetime measurement imaging, the other is frequency domain lifetime measurement imaging, and the essence of the two types of methods is the same. For frequency domain lifetime imaging, more applications are currently applied to wide field imaging, and although the speed is high, the imaging is limited by the resolution; for time domain lifetime imaging, the method is mainly applied to point scanning imaging systems, and is suitable for samples with more flexible, large dynamic range and long lifetime, the most widely applied time domain lifetime system at present is a lifetime measurement system based on a confocal imaging system and a TCSPC counting device, as shown in fig. 1, a super-resolution fluorescence lifetime imaging device provided by CN108120702A can provide better lifetime imaging resolution, and can flexibly measure long and short lifetime samples with various dynamic ranges. However, there are also limitations to this lifetime system based on confocal and TCSPC counters, most critical is the imaging speed limitation. Due to the influence of the detector and the dead time of TCSPC, in order to avoid the influence of photon accumulation on the lifetime, the light intensity must be attenuated, and in order to obtain enough photon numbers to obtain the lifetime information, the single-point detection time length must be extended, so that the imaging speed is limited, and the detector and the TCSPC are difficult to apply to real-time lifetime imaging.

Disclosure of Invention

The invention provides a fluorescence lifetime imaging method and a fluorescence lifetime imaging device based on a spectral technology. The device has compact and simple structure, and can be modified based on a common confocal system; the service life is rapidly and accurately calculated, and the influence of the photon accumulation effect on the service life is greatly inhibited; by combining with parallel detection, the imaging resolution and imaging speed of the service life can be further improved.

On the basis of a traditional confocal system (figure 1), the invention provides a method for further subdividing fluorescence into different spectral sections by using a spectral splitting technology, photons of different spectra are received by using different detectors or counters, and since a fluorescence emission spectrum has a certain waveband range and is distributed uniformly (as shown in figure 4), fluorescent molecules can be divided into different channels by using the spectral splitting technology, so that the photon accumulation effect caused by dead time of a single-channel counter or detector can be relieved and inhibited, the detection efficiency of the molecules is improved, and the speed of life imaging is improved.

The invention adopts the following specific technical scheme:

a fluorescence lifetime imaging device based on a spectral technology comprises a light source, a microscope objective for converging exciting light emitted by the light source and exciting a sample to emit fluorescence, and is provided with:

the spectrum splitting module is used for splitting the fluorescence collected by the microscope objective;

the detection module is used for receiving the fluorescent molecules of different wave bands after the spectrum division;

and the counting module is used for counting photons of the fluorescent molecules and calculating fluorescence life information.

Further, the light source and the microscope objective are sequentially provided with:

the polarization maintaining optical fiber is used for ensuring the emergent polarization direction of the exciting light to be unchanged;

the collimating mirror is used for collimating and expanding the exciting light output by the polarization maintaining optical fiber;

1/2 wave plate and 1/4 wave plate for adjusting the excitation light to circularly polarized light;

the galvanometer system is used for scanning a sample;

4f system for achieving beam expansion and spot scanning.

In the device of this application, still be provided with:

a dichroic mirror for reflecting the excitation light and transmitting the fluorescence;

the narrow-band filter is used for filtering background noise and improving the imaging signal-to-noise ratio;

the spectrum splitting module is used for realizing spectrum splitting of the fluorescence;

the detection module is used for collecting and detecting fluorescence, and the detector is a photomultiplier tube (PMT) or an Avalanche Photodiode (APD);

a counting module consisting of a time dependent single photon counter (TCSPC) for counting photons, the TCSPC calculating fluorescence lifetime;

and a control system for controlling the laser light source, the galvanometer system, the light splitting module, the detection module and the counting module is arranged.

Optionally, the spectrum splitting module is a spectrum splitter, a beam splitter prism or a beam splitter grating.

Optionally, the collected fluorescence enters the spectral module after passing through the multimode fiber.

Optionally, the detection module is a detector array including a plurality of detectors, and the detectors are photomultiplier tubes or avalanche photodiodes.

Optionally, the counting module is a time-dependent single photon counter or a time-dependent single photon counter array;

and each time-correlated single photon counter in the time-correlated single photon counter array is respectively connected with a detector.

Optionally, the collected fluorescence enters the spectrum splitting module after passing through the multimode fiber bundle, the multimode fiber bundle includes a central fiber and a plurality of edge fibers, the fluorescence signal collected by the central fiber is sent to the spectrum splitting module, and the fluorescence collected by the edge fibers is directly sent to the detection module.

In the device of the application, the specific steps and processes are as follows:

1) collimating a laser beam emitted by a laser after passing through a polarization maintaining optical fiber;

2) the light beam passes through 1/2 wave plate and 1/4 wave plate and becomes circularly polarized light;

3) the light beam enters a galvanometer system after being reflected by the dichroic mirror, so that the final scanning of the light beam on an object plane is realized;

4) after light beams come out of the galvanometer system and are expanded by a 4f system consisting of a scanning lens and a field lens, the light beams are converged on the surface of a sample to excite the fluorescent sample to generate fluorescence after passing through an oil immersion objective lens with large numerical aperture;

5) the excited fluorescence returns along the original light path, passes through the dichroic mirror, is filtered by the narrow-band filter, then enters the spectrum division module, and is further subdivided into different waveband channels; the spectrum splitting module can directly use a spectrum splitter of a related company, can adopt a spectrum splitting prism to split fluorescence according to spectra, and can also realize spectrum splitting by utilizing a grating; fluorescent molecules subdivided into different wave bands are received by corresponding detectors, photons of different spectral channels are sent to a counting module TCSPC to record time information (the time information can be received by one TCSPC or a plurality of TCSPCs), and the collection of the life information is realized.

6) The multiband fluorescence time information is integrated to obtain the integral fluorescence life information, so that the photon accumulation effect caused by dead range time in a single channel is relieved, and the fluorescence life imaging speed is improved. And the fluorescence information is further combined with parallel detection, so that the service life imaging speed and resolution can be further improved.

In addition, the invention also provides a fluorescence lifetime imaging method based on the spectral technology, which comprises the following steps:

1) converging exciting light emitted by a light source on the surface of a sample to excite a fluorescent sample to generate fluorescence;

2) collecting the fluorescence and splitting the spectrum according to the wave band;

3) receiving fluorescent molecules of different wave bands by using a detector, and calculating the service life information of each wave band;

4) and integrating the multiband fluorescence time information to obtain integral fluorescence lifetime information.

Optionally, excitation light emitted by the light source is converted into circularly polarized light through collimation, and the circularly polarized light is converged on the surface of the sample after beam expansion.

The principle of the invention is as follows:

in a traditional time domain lifetime measurement imaging system (fig. 1) based on a confocal system and TCSPC, fluorescent molecules are directly received by a detector after being converged by a converging lens, photon information is sent to the TCSPC by the detector, the TCSPC records time information of photons relative to an original synchronous pulse of excitation light, a distribution curve of photon arrival time is constructed, and final fluorescence lifetime information is obtained by fitting the curve. In the case of a single detector, single TCSPC, the fluorescence lifetime curve can be expressed as a decay function of the number of photons I with respect to the arrival time of the photons (the time the photons are identified by the detector with respect to the synchronization pulse), in an ideal case without taking into account the photon pile-up effect:

I(t)＝I₀e^-t/τ，

wherein I₀The number of photons when t is 0, τ is the fluorescence lifetime, and IRF is the time-dependent Function (IRF) of the lifetime measurement system. As mentioned above, because the detector (APD or PMT) and the counter (TCSPC) have dead time, that is, the detector or the counter cannot process the next photon for a while after processing the first photon, photons arriving at the detector or the counter within the dead time range are lost to cause photon accumulation effect, so that the photon detection efficiency is reduced, the lifetime curve is distorted, and due to the characteristics of the decay function, there are more photons with shorter arrival time, and therefore there are more photons lost, which results in the distortion of the fluorescence lifetime towards the shorter lifetime (as shown in fig. 5), which can be expressed as:

where μ is the average photon rate and is higher

The influence of different photon rates on the fluorescence lifetime curve is reflected, and the lifetime curve is distorted more when the photon rate is larger. Considering that the exciting light in the life measuring system is pulse light and the pulse period is far larger than the life of the measured fluorescence, the effective photon detection counting efficiency alpha can be obtained by integration (actual detection and counting)Number photons/total photons arriving at the detector):

α＝1-e^-u/μ

therefore, under the condition of high photon rate, the effective photon detection counting efficiency is reduced, so that the fluorescence lifetime curve is distorted. Therefore, in the conventional fluorescence lifetime measurement system, in order to avoid the distortion caused by the photon accumulation effect, the light intensity is low (the photon rate is about 1% of the light pulse frequency, that is, about 100 pulses obtain one photon) and the photon receiving time is prolonged, so that the lifetime measurement time is long, and the system is difficult to be applied to high-speed real-time lifetime imaging.

The fluorescence spectrum distribution is utilized to further separate the fluorescence into different channels, so that the photon accumulation effect under the condition of a single channel is relieved. In the confocal system, the excited fluorescence is not monochromatic light but broadband fluorescence with a certain spectral range, taking the fluorescent dye Alexa Fluor488 as an example, as shown in fig. 4, the fluorescence spectrum covers the band range of 480-. In traditional confocal fluorescence life-span measurement system, the wavelength information of fluorescence is not utilized, and the wavelength information that this patent was then make full use of broadband brought utilizes spectral technique with this part fluorescence further subdivide to the detection passageway that different wave bands correspond, and the photon wavelength that arrives the detector is relevant with the relative probability size of each subdivision wave band of its fluorescence spectrum, consequently can be very big through the subdivision wave band alleviate but the photon under the detector condition piles up the effect, promotes effective photon and surveys the count rate:

p_i＝S_i/S,

wherein p is_iThe probability of the fluorescence signal being spectrally separated into different channels, S and S_iThe area of the spectral curve behind the filter and the area of the spectral curve of the spectral band corresponding to a single channel are respectively shown, and alpha' is the effective photon detection counting rate after the spectral splitting technology is utilized. As shown in fig. 5, after photons are divided into multiple channels by the spectroscopic technique, the same counter TCSPC (i.e., the time information of photons received by multiple detectors is processed by the same TCSPC) is used for counting, and the distortion of the fluorescence lifetime curve is well relieved under the condition of a high photon rate; although the effective photon detection counting rate is not improved so much in some cases, the distribution of the fluorescence spectrum is relatively even, and the distribution of photons in each wave band is relatively uniform in probability, so that the utilization rate of multiple channels is very high, and the accumulation effect brought by the dead time of the detector is remarkably relieved. In the conventional life measurement system, the dead-range time of the detector is usually longer than that of the TCSPC, so that the effect of inhibiting the stacking effect of the detector on improving the life imaging speed is very obvious.

In more embodiments of the present disclosure, photons of multiple channels are respectively received by different counters TCSPC, which further alleviates the stacking effect of TCSPC, and can further improve the lifetime imaging speed.

And the multi-channel spectral technology is combined with the parallel detection imaging technology, so that the service life imaging speed and the imaging resolution can be improved simultaneously. The imaging resolution and the lifetime imaging speed can be improved by utilizing parallel detection, namely the photon is received and time is counted by the detector array and the TCSPC array, but at the moment, a limitation still exists, the photon meets Poisson distribution, and the spatial distribution of the detection end surface and the point spread function of the system form a correlation relationship, so that the number of the received photons of the middle detector (the detector positioned on the optical axis) is far more than that of other detectors, the photons cannot be uniformly distributed by an image spectrum, and the accumulation effect generated by the central detector can limit the increase of the lifetime imaging speed. In the embodiment of the patent, the fluorescence signal to enter the central detector is further divided into multiple channels by using a spectral spectroscopy technology, and the photon accumulation effect can be further relieved compared with the parallel detection while the resolution is improved by using the parallel detection, so that the service life imaging speed is further improved.

Compared with the prior art, the invention has the following beneficial technical effects:

(1) the system is simple and is suitable for all fluorescence lifetime imaging systems;

(2) the photon accumulation effect is relieved by utilizing the spectral technology, and the service life imaging speed is improved;

(3) and the service life imaging resolution is improved and the service life imaging speed is further improved by combining with parallel detection.

Drawings

FIG. 1 is a schematic diagram of a conventional confocal scanning system and TCSPC based time domain lifetime measurement imaging system setup;

FIG. 2 is a diagram of an example of a time domain lifetime measurement imaging system apparatus based on a spectroscopic detection module according to the present invention, the system is also based on a confocal scanning system, and a TCSPC is used for counting;

FIG. 3 is a system diagram of two different spectral detection modules according to the present invention, wherein (a) is a spectral detection module based on a beam splitter prism, and (b) is a spectral detection module based on a grating;

FIG. 4 is a spectrum diagram of the fluorescence spectrum of the fluorescent dye (Alexa Fluor 488) used in the example, (a) is a spectrum diagram of the fluorescence signal generated by the fluorescent dye through an 8-channel spectrometer;

FIG. 5 is a graph showing the lifetime measurements of the spectrographic lifetime system compared to a conventional confocal lifetime system in an embodiment, wherein (a) is a lifetime graph of the absolute photon count, and (b) is a lifetime graph of the normalized photon count;

FIG. 6 is a diagram of an example of a time domain lifetime measurement imaging system based on a spectroscopic detection parallel counting module according to the present invention, the system being based on a confocal scanning system;

FIG. 7 is a diagram of an example of a time domain lifetime measurement imaging system apparatus based on a spectroscopic detection module and a parallel detection module according to the present invention. The system is based on a confocal scanning system, and the spectrum detection module adopts TCSPC to count;

FIG. 8 shows the distribution of the end faces of the optical fibers in the embodiment, wherein the central optical fiber C is connected to the spectroscopic detection module and the edge optical fiber E is connected to the parallel detection module;

FIG. 9 is a graph showing the lifetime measurements of the spectrographic detection and parallel detection system in comparison with the conventional confocal lifetime detection system and parallel detection system in the embodiment shown in the following drawings, wherein (a) is an absolute photon number lifetime graph and (b) is a photon number normalized lifetime graph;

fig. 10 is a diagram of an example of a time domain lifetime measurement imaging system based on a spectroscopic detection parallel counting module and a parallel detection module according to the present invention, wherein the system is based on a confocal system point scanning system.

Detailed Description

In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, however, the present invention may be practiced in other ways than those specifically described herein, and thus the present invention is not limited to the specific embodiments disclosed below.

Reference will now be made in detail to embodiments of the present invention, examples of which are illustrated in the accompanying drawings, wherein like or similar reference numerals refer to the same or similar elements or elements having the same or similar function throughout. The embodiments described below with reference to the drawings are illustrative and intended to be illustrative of the invention and are not to be construed as limiting the invention.

Example 1

Fig. 2 is a schematic diagram of a time domain lifetime measurement imaging system apparatus based on a spectroscopic detection module, including: the device comprises a 488nm laser 1, a single-mode polarization maintaining optical fiber 2, a collimating lens 3, an 1/2 wave plate 4, a 1/4 wave plate 5, a dichroic mirror 6, a galvanometer scanning system 7, a scanning mirror 8, a field lens 9, a high numerical aperture objective lens 10, a sample stage 12, a reflecting mirror 13, a narrow-band filter 14, a converging lens 15, a small hole 16, a multimode optical fiber 17, a spectrum splitting module 18, a detector array 19, a time-dependent single photon counter (TCSPC)20 and a control system 21.

The 1/2 wave plate 4 and the 1/4 wave plate 5 can adjust the polarization state of the excitation light to circularly polarized light, so that the excitation efficiency of the sample is improved; the scanning mirror 8 and the field lens 9 form a 4f system, so that the size of the light beam of the incident exciting light is matched with the numerical aperture of the objective lens, the entrance pupil position of the objective lens is ensured to be parallel light, and the conjugate of the galvanometer reflecting surface of the galvanometer scanning system 7 and the entrance pupil surface of the objective lens 10 with a high numerical aperture is ensured, so that the minimum distortion caused by light deflection in the scanning imaging process is ensured.

In a detection light path composed of the reflector 13, the narrow-band filter 14, the convergent lens 15, the small hole 16 and the multimode fiber, the light passing band of the narrow-band filter needs to match the effective light band of the spectral module, and the size of the small hole is about 0.8 times of the corresponding airy facula of the system, so that the imaging resolution and the signal-to-noise ratio of the system are ensured.

A schematic diagram of a spectrum detection module composed of the spectrum module 18 and the detector array 19 is shown in fig. 3, and spectrum division can be realized in two ways (a) and (b). The spectral module of fig. 3(a) is composed of a lens 22, a beam splitter prism 23 and a concave mirror 24, wherein fluorescent light is collimated into parallel light by the lens 22 after coming out from the multimode optical fiber 17, the refractive index of the beam splitter prism is different for fluorescent light with different incident wavelengths due to the dispersion characteristic of the light after passing through the beam splitter prism 23, so that the broadband fluorescent light is decomposed into monochromatic light bands, and the fluorescent light with different bands is converged into different detectors in the detector array 19 after being converged by the concave mirror, so that the fluorescent light signals are dispersed into different detectors according to the spectrum (fig. 4 (b)). The principle of the right diagram is similar to that of the left diagram, the spectral module of fig. 3(b) is composed of a lens 25, a spectral grating 26 and a concave mirror 27, the fluorescence coming out from the multimode fiber 17 is collimated into parallel light by the lens 25 and then hits the grating 26, the grating generates a dispersion phenomenon of the light by the principle of multi-slit diffraction, so that the broadband fluorescence is decomposed into monochromatic light bands, and after being reflected and converged by the concave mirror, the fluorescence of different bands is converged to different detectors in the detector array 19 to be collected (fig. 4 (b)). It should be noted that, for the convenience of description, only a four-channel spectroscopy is expressed in fig. 3, and an 8-channel spectroscopy module is used in the embodiment of the present invention to realize the subdivision of the fluorescence band (as shown in fig. 4 (b)).

Under the control of the controller 21, TCSPC 20 is synchronized with the laser 1, and thus can calculate the time difference between the arrival of photons at the counter and the laser pulse, thereby obtaining the time information of fluorescence and the lifetime curve of fluorescence (fig. 5).

In this embodiment, the numerical aperture NA of the microscope objective 10 is 1.4; the fluorescence sample used was Alex Fluor488, and the fluorescence spectrum distribution thereof is shown in fig. 4 (a); the narrow-band filter 14 is a narrow-band filter passing 500-550 nm; the spectral splitting module can split the fluorescence in the 500-550nm band into 8 channels from short to long, and the corresponding spectral density of each channel is shown in fig. 4 (b).

The process of using the device shown in fig. 2 to realize super-resolution fluorescence imaging is as follows:

exciting light emitted by the laser passes through the single-mode polarization-maintaining optical fiber 5 and is emitted in parallel through the collimating lens 6, the exciting light passes through the 1/2 wave plate 4 and the 1/4 wave plate 5 and is polarized and modulated into circularly polarized light, and the circularly polarized exciting light can improve the excitation efficiency of a sample. The excitation light enters the galvanometer scanning system 7 after being reflected by the dichroic mirror 6 to realize two-dimensional scanning on the final sample surface. Exciting light passes through a scanning mirror 8 and a field lens 9 after coming out of a vibrating mirror system 7 and then is converged into an exciting light spot limited by a diffraction limit by a high numerical aperture objective lens 10, the converged light spot excites a fluorescent sample 11 fixed on a sample stage 12, generated fluorescent light is collected by the high numerical aperture objective lens 10 again, the generated fluorescent light returns to pass through the field lens 9, the scanning mirror 8 and the vibrating mirror system 7 again according to an original light path, the fluorescent light is reflected by a reflecting mirror 13 after penetrating through a dichroic mirror 6, the fluorescent light is converged into a multimode optical fiber 17 by a converging lens 15 after passing through a narrow-band filter and is collected, and meanwhile, the existence of a small hole 16 ensures the resolution guarantee of confocal imaging. The fluorescent signals are collected by the multimode optical fiber 17, enter the spectral splitting module 18, are then spectrally distributed to different channels to be received by the detector array 19, and the received photons enter the TCSPC 20 in an electric signal mode to be counted, so that a life imaging graph is finally obtained.

The spectrum module adopts 8-channel spectrum to realize the separation of the fluorescence (figure 4(a)) of 500-550nm wave band, the widths of the fluorescence wave bands corresponding to the 8 channels are basically the same, and the probability of the fluorescence photons entering the different channels is determined by the fluorescence spectrum density of the dye Alexa Fluor488 used in the embodiment, as shown in figure 4 (b). As can be seen from fig. 4(b), since the fluorescence spectral density curve is relatively flat, the fluorescence can be relatively uniformly distributed into each channel or detector, therefore, under the condition of higher photon rate, the photon accumulation effect of a single detector can be greatly relieved by the parallel detection of the spectrum, so that the life measurement is more accurate, as shown in figure 5, on the premise of not considering the influence of the dead-range time of the counter TCSPC (the dead-range time of many TCSPC in the current market is far less than that of the detector, so that the dead-range time can be ignored under most time conditions), compared with the condition that the life curve of a single detector is distorted, the embodiment of the invention adopts the spectrum parallel detection to obviously improve the detection efficiency of photons, more obviously obtain an almost ideal fluorescence life curve and improve the accuracy of fluorescence life measurement under the condition of high photon rate. In the simulation of fig. 5, (a) is an absolute photon number lifetime graph and (b) is a photon number normalized lifetime graph; the photon rate was 800MHz, the dead-range time for each detector was 40ns, and the dead-range time for TCSPC was negligible. Therefore, the service life can be measured by adopting the spectrum-division parallel detection module without difficulty, and the speed of service life imaging can be realized by improving the excitation light intensity.

Example 2

Fig. 6 is a schematic diagram of a time domain lifetime measurement imaging system apparatus based on a spectrum detection parallel counting module, including: the device comprises a 488nm laser 1, a single-mode polarization maintaining optical fiber 2, a collimating lens 3, an 1/2 wave plate 4, a 1/4 wave plate 5, a dichroic mirror 6, a galvanometer scanning system 7, a scanning mirror 8, a field lens 9, a high numerical aperture objective lens 10, a sample stage 12, a reflecting mirror 13, a narrow-band filter 14, a converging lens 15, a small hole 16, a multimode optical fiber 17, a spectrum splitting module 18, a detector array 19, a time-dependent single photon counter array (TCSPC array) 20 and a control system 21.

In a detection light path composed of the reflector 13, the narrow-band filter 14, the convergent lens 15, the small hole 16 and the multimode fiber 17, the light passing band of the narrow-band filter needs to match the effective light band of the spectral module, and the size of the small hole is about 0.8 times of the corresponding airy facula of the system, so that the imaging resolution and the signal-to-noise ratio of the system are ensured.

Fig. 3 shows a schematic diagram of a spectrum detection module composed of the spectrum module 18 and the detector array 19. It should be noted that, for the convenience of description, only a four-channel spectroscopy is expressed in fig. 3, and an 8-channel spectroscopy module is used in the embodiment of the present invention to realize the subdivision of the fluorescence band (as shown in fig. 4 (b)). Each detector is connected with each TCSPC in the TCSPC array 20 to form a parallel counting module, and is synchronized with the laser 1 under the control of the controller 21, so that the time difference of the photons reaching the counter relative to the laser pulse can be calculated, and the time information of the fluorescence and the life curve of the fluorescence can be obtained.

In this embodiment, the numerical aperture NA of the microscope objective 10 is 1.4; the spectroscopy module can divide the fluorescence into 8 channels of the same wavelength length from short to long.

The process of using the device shown in fig. 6 to realize super-resolution fluorescence imaging is as follows:

exciting light emitted by the laser passes through the single-mode polarization-maintaining optical fiber 5 and is emitted in parallel through the collimating lens 6, the exciting light passes through the 1/2 wave plate 4 and the 1/4 wave plate 5 and is polarized and modulated into circularly polarized light, and the circularly polarized exciting light can improve the excitation efficiency of a sample. The excitation light enters the galvanometer scanning system 7 after being reflected by the dichroic mirror 6 to realize two-dimensional scanning on the final sample surface. Exciting light passes through a scanning mirror 8 and a field lens 9 after coming out of a vibrating mirror system 7 and then is converged into an exciting light spot limited by a diffraction limit by a high numerical aperture objective lens 10, the converged light spot excites a fluorescent sample 11 fixed on a sample stage 12, generated fluorescent light is collected by the high numerical aperture objective lens 10 again, the generated fluorescent light returns to pass through the field lens 9, the scanning mirror 8 and the vibrating mirror system 7 again according to an original light path, the fluorescent light is reflected by a reflecting mirror 13 after penetrating through a dichroic mirror 6, the fluorescent light is converged into a multimode optical fiber 17 by a converging lens 15 after passing through a narrow-band filter and is collected, and meanwhile, the existence of a small hole 16 ensures the resolution guarantee of confocal imaging. The fluorescence signals are collected by the multimode optical fiber 17, enter the spectral splitting module 18 and then are received by the detector array 19 after being spectrally distributed to different channels, photons received by each detector in the detector array 19 enter each TCSPC in the TCSPC array 20 respectively in an electric signal mode to be counted, and finally, the life imaging result is obtained.

The embodiment is directed to that when the TCSPC itself has a large photon accumulation effect (that is, the dead-range time of the TCSPC cannot be ignored compared with the dead-range time of the detector), if photons still enter the same TCSPC after the spectroscopic module performs parallel detection at this time, the photon accumulation effect of the TCSPC greatly limits the accumulation effect suppression effect brought by the spectroscopic parallel detection module, and the imaging speed is still limited due to the influence of the photon accumulation effect. In this embodiment, each detector in the detector array 19 is connected to each TCSPC in the TCSPC array 20, and compared with the case that a plurality of detectors are connected to the same TCSPC in embodiment 1, this embodiment can further improve the counting efficiency of each detector and TCSPC after passing through the spectrum, further alleviate the photon accumulation effect of the system, improve the photon detection efficiency, and further improve the lifetime imaging speed.

Example 3

Fig. 7 is a schematic diagram of a time domain lifetime measurement imaging system apparatus based on a spectroscopic detection module and a parallel detection module, including: the device comprises a 488nm laser 1, a single-mode polarization maintaining optical fiber 2, a collimating lens 3, an 1/2 wave plate 4, a 1/4 wave plate 5, a dichroic mirror 6, a galvanometer scanning system 7, a scanning mirror 8, a field lens 9, a high numerical aperture objective lens 10, a sample stage 12, a reflecting mirror 13, a narrow-band filter 14, a converging lens 15, a multimode optical fiber bundle 17, a spectrum splitting module 18, a detector array 19, a time-dependent single photon counter (TCSPC) array 20 and a control system 21.

In a detection light path composed of a reflector 13, a narrow-band filter 14, a converging lens 15 and a multimode fiber bundle 17, the light passing band of the narrow-band filter needs to match the effective light band of a spectral module, the end face of the multimode fiber bundle 17 is as shown in fig. 8, wherein a central fiber C sends a collected fluorescence signal to the spectral module 18, an edge fiber E directly sends the collected fluorescence to a corresponding detector in a detector array 19, the size of each fiber is equivalent to about 0.3 times of the corresponding airy spot of the system, and the parallel detection module composed of the edge fiber E in the multimode fiber bundle 17 and the detector array 19 realizes the improvement of the resolution and the signal-to-noise ratio of fluorescence intensity imaging.

A schematic diagram of a spectral detection module composed of a spectral module 18 to which the central fiber C of the multimode fiber bundle 17 is connected and the relevant detectors in the detector array 19 is shown in fig. 3. It should be noted that, for the convenience of description, only a four-channel spectroscopy is expressed in fig. 3, and an 8-channel spectroscopy module is used in the embodiment of the present invention to realize the subdivision of the fluorescence band (as shown in fig. 4 (b)). The detector in the spectrum splitting module is connected with a single TCSPC in a TCSPC array 20, and the other detectors in the detector array 19 collect fluorescence signals in the edge fibers E of the multimode fiber bundle 17 respectively and are connected with other TCSPCs in the TCSPC array 20 respectively, and are synchronized with the laser 1 under the control of the controller 21, so that the time difference of the photons reaching the counter relative to the laser pulse can be calculated, and the time information of the fluorescence can be obtained, and the lifetime curve of the fluorescence can be obtained, as shown in fig. 9, (a) is an absolute photon number lifetime graph, and (b) is a photon number normalized lifetime graph.

The process of using the device shown in fig. 7 to realize super-resolution fluorescence imaging is as follows:

exciting light emitted by the laser passes through the single-mode polarization-maintaining optical fiber 5 and is emitted in parallel through the collimating lens 6, the exciting light passes through the 1/2 wave plate 4 and the 1/4 wave plate 5 and is polarized and modulated into circularly polarized light, and the circularly polarized exciting light can improve the excitation efficiency of a sample. The excitation light enters the galvanometer scanning system 7 after being reflected by the dichroic mirror 6 to realize two-dimensional scanning on the final sample surface. Exciting light is gathered into the excitation facula that diffraction limit restricted by high numerical aperture objective 10 after going out from mirror system 7 that shakes behind scanning mirror 8 and field lens 9, the fluorescence sample 11 of gathering facula excitation fixing on sample platform 12, the fluorescence of production is collected by high numerical aperture objective 10 again, return once more through field lens 9 according to former light path, scanning mirror 8 and mirror system 7 that shakes, reflect by speculum 13 behind the dichroscope 6 through, gather in multimode fiber bundle 17 by convergent lens 15 behind the narrowband filter, wherein the connection of the other end of multimode fiber bundle is different: as shown in fig. 8, the central fiber C of the multimode fiber bundle 17 transmits the fluorescence signal to the spectral splitting module 18, the spectral splitting module distributes the part of fluorescence to 8 different channels according to the spectrum to be received by corresponding 8 detectors in the detector array 19, and the 8 detectors are connected to a single TCSPC constituent spectrum parallel detection lifetime measurement module in the TCSPC array 20; and the other 6 edge optical fibers E in the multimode optical fiber bundle 17 transmit the fluorescence signals to the corresponding other 6 detectors in the detector array 19 for receiving, and are respectively connected with the 6 TCSPCs in the TCSPC array 20 to form a parallel detection lifetime measurement module. And combining the fluorescence intensity information and the time information obtained by the two modules to obtain a super-resolution fluorescence lifetime imaging result.

In this embodiment, the existing parallel detection life measurement system is further upgraded, and the spectroscopic technology of the present invention is introduced to further increase the fluorescence life imaging speed. In the multimode fiber bundle 17 of the embodiment, the central fiber C will collect the most signal (about 40% of the total fluorescence signal) and the edge fiber E collects about 10% of the fluorescence signal according to the airy spot intensity distribution. Therefore, for the existing parallel detection life-span measuring system, the alleviation degree of the photon accumulation effect is still limited by the central detector, and the photons enter the central detector more intensively to be unfavorable for alleviating the photon accumulation effect. Therefore, in this embodiment, the fluorescence signals collected by the edge optical fiber E still normally enter the corresponding detectors and TCSPC to form a parallel detection life measurement system, the center optical fiber C is connected to the spectrum splitting module, and is split into 8 channels according to the spectrum to be collected by 8 different detectors, and is finally counted by TCSPC, thereby forming a spectrum splitting detection module, the fluorescence signals are further split into different detectors by the spectrometer, and on the other hand, the spectrum density distribution is gentler than the airy spot fluorescence density distribution, so that the fluorescence signals are split into different detectors by the spectrum splitting module and are more even than the spatial parallel detection, and the capability of alleviating the photon accumulation effect is stronger. In this embodiment, the dead time of TCSPC is considered to be much smaller than that of the detector, and therefore can be ignored, so that 8 detectors in the spectroscopic detection module are connected to the same TCSPC. As shown in fig. 9, under the condition of high photon rate, the lifetime curve obtained by the system (parallel detection spectrum lifetime curve) of the embodiment is obviously superior to the lifetime curve obtained by a single detector or by the pure parallel detection. The photon accumulation effect in the system can be further relieved by the embodiment, the influence of dead time on life measurement is reduced, and the photon detection efficiency is improved, so that an accurate fluorescence life curve is obtained under the condition of high photon rate, and the fluorescence life imaging speed can be obviously improved. In the simulation of FIG. 9, the photon rate is 800MHz, the dead-range time of each detector is 40ns, and the dead-range time of TCSPC is negligible.

In summary, the parallel detection in this embodiment can bring the improvement of the fluorescence intensity imaging resolution and the signal-to-noise ratio, and the introduction of the spectroscopic technology and the combination with the parallel detection can significantly improve the accuracy and speed of the lifetime imaging, and have the potential of realizing the real-time super-resolution lifetime imaging.

Example 4

Fig. 10 is a schematic diagram of a time domain lifetime measurement imaging system apparatus based on a spectroscopy detection parallel counting module and a parallel detection module, including: the device comprises a 488nm laser 1, a single-mode polarization maintaining optical fiber 2, a collimating lens 3, an 1/2 wave plate 4, a 1/4 wave plate 5, a dichroic mirror 6, a galvanometer scanning system 7, a scanning mirror 8, a field lens 9, a high numerical aperture objective lens 10, a sample stage 12, a reflecting mirror 13, a narrow-band filter 14, a converging lens 15, a multimode optical fiber bundle 17, a spectrum splitting module 18, a detector array 19, a time-dependent single photon counter (TCSPC) array 20 and a control system 21.

In a detection light path composed of a reflector 13, a narrow-band filter 14, a converging lens 15 and a multimode fiber bundle 17, the light passing band of the narrow-band filter needs to match the effective light band of a spectral module, the end face of the multimode fiber bundle 17 is as shown in fig. 8, wherein a central fiber C sends a collected fluorescence signal to the spectral module 18, other surrounding fibers E directly send the collected fluorescence to corresponding detectors in a detector array 19, the size of each fiber is equivalent to about 0.3 times of the corresponding airy spot of the system, and the parallel detection module composed of an edge fiber E in the multimode fiber bundle 17 and the detector array 19 realizes the improvement of the resolution and the signal-to-noise ratio of fluorescence intensity imaging.

A schematic diagram of a spectral detection module composed of a spectral module 18 to which the central fiber C of the multimode fiber bundle 17 is connected and the relevant detectors in the detector array 19 is shown in fig. 3. It should be noted that, for the convenience of description, only a four-channel spectroscopy is expressed in fig. 3, and an 8-channel spectroscopy module is used in the embodiment of the present invention to realize the subdivision of the fluorescence band (as shown in fig. 4 (b)). The corresponding 8 detectors in the detector array 19 in the spectrum detection module are connected with the corresponding 8 TCSPC in the TCSPC array 20 to form a spectrum detection parallel counting module, the other detectors in the detector array 19 collect fluorescence signals in the edge optical fiber E of the multimode optical fiber bundle 17 respectively and are connected with the other TCSPC in the TCSPC array 20 respectively, and the fluorescence signals are synchronized with the laser 1 under the control of the controller 21, so that the time difference of the photons reaching the counter relative to the laser pulse can be calculated, the time information of the fluorescence is obtained, and the life information of the fluorescence is obtained.

The process of using the apparatus shown in FIG. 10 to achieve super-resolution fluorescence imaging is as follows:

exciting light emitted by the laser passes through the single-mode polarization-maintaining optical fiber 5 and is emitted in parallel through the collimating lens 6, the exciting light passes through the 1/2 wave plate 4 and the 1/4 wave plate 5 and is polarized and modulated into circularly polarized light, and the circularly polarized exciting light can improve the excitation efficiency of a sample. The excitation light enters the galvanometer scanning system 7 after being reflected by the dichroic mirror 6 to realize two-dimensional scanning on the final sample surface. Exciting light is gathered into the excitation facula that diffraction limit restricted by high numerical aperture objective 10 after going out from mirror system 7 that shakes behind scanning mirror 8 and field lens 9, the fluorescence sample 11 of gathering facula excitation fixing on sample platform 12, the fluorescence of production is collected by high numerical aperture objective 10 again, return once more through field lens 9 according to former light path, scanning mirror 8 and mirror system 7 that shakes, reflect by speculum 13 behind the dichroscope 6 through, gather in multimode fiber bundle 17 by convergent lens 15 behind the narrowband filter, wherein the connection of the other end of multimode fiber bundle is different: as shown in fig. 8, the central fiber C of the multimode fiber bundle 17 sends the fluorescence signal to the spectrum splitting module 18, the spectrum splitting module distributes the part of fluorescence to 8 different channels according to the spectrum to be received by corresponding 8 detectors in the detector array 19, and the 8 detectors are connected to corresponding 8 TCSPCs in the TCSPC array 20 to form a spectrum parallel detection counting module; and the other 6 edge optical fibers E in the multimode optical fiber bundle 17 transmit the fluorescence signals to the corresponding other 6 detectors in the detector array 19 for receiving, and are respectively connected with the 6 TCSPCs in the TCSPC array 20 to form a parallel detection lifetime measurement module. And combining the fluorescence intensity information and the time information obtained by the two modules to obtain a super-resolution fluorescence lifetime imaging result.

The embodiment aims at the problem that when a large photon accumulation effect exists in TCSPC (that is, the dead-range time of TCSPC cannot be ignored compared with the dead-range time of a detector), if photons obtained by a spectrum splitting detection module still enter the same TCSPC, the photon accumulation effect of TCSPC greatly limits the accumulation effect suppression effect brought by the spectrum splitting parallel detection module, and the imaging speed is still limited due to the influence of the photon accumulation effect. In this embodiment, each detector in the detector array 19 is connected to each TCSPC in the TCSPC array 20, and compared with the case that a plurality of detectors are connected to the same TCSPC in embodiment 3, this embodiment can further improve the counting efficiency of each detector and TCSPC after passing through the spectrum, further alleviate the photon accumulation effect of the system, improve the photon detection efficiency, and further improve the lifetime imaging speed.

The above description is only exemplary of the preferred embodiments of the present invention, and is not intended to limit the present invention, and any modifications, equivalents, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. The utility model provides a fluorescence life-span imaging device based on spectral technique, includes the light source, converges the exciting light that the light source sent and arouses the microscope objective that the sample sent fluorescence, its characterized in that is provided with:

2. The fluorescence lifetime imaging apparatus based on spectroscopic technology as set forth in claim 1, wherein:

the galvanometer system is used for scanning a sample;

4f system for achieving beam expansion and spot scanning.

3. The fluorescence lifetime imaging apparatus based on spectral technique of claim 1, wherein said spectral module is a spectrometer, a beam splitter prism or a beam splitter grating.

4. The fluorescence lifetime imaging apparatus based on spectroscopic technique of claim 1, wherein the collected fluorescence enters said spectroscopic module via a multimode fiber.

5. The fluorescence lifetime imaging apparatus according to claim 1, wherein said detecting means is a detector array comprising a plurality of detectors, and said detectors are photomultiplier tubes or avalanche photodiodes.

6. The fluorescence lifetime imaging apparatus based on spectral technique of claim 5, wherein said counting module is a time-dependent single photon counter or a time-dependent single photon counter array;

7. The fluorescence lifetime imaging apparatus based on spectral technique of claim 1, wherein the collected fluorescence enters said spectral module via a multimode fiber bundle, said multimode fiber bundle comprises a central fiber and a plurality of edge fibers, the fluorescence signal collected by the central fiber is sent to the spectral module, and the fluorescence signal collected by the edge fibers is directly sent to the detection module.

8. A fluorescence lifetime imaging method based on a spectral technology is characterized by comprising the following steps:

9. The fluorescence lifetime imaging method according to claim 8, wherein the excitation light from the light source is converted into circularly polarized light by collimation, and the circularly polarized light is expanded to be converged on the surface of the sample.