CN119310057A - A fast fluorescence lifetime imaging method and device - Google Patents
A fast fluorescence lifetime imaging method and device Download PDFInfo
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- CN119310057A CN119310057A CN202411349759.0A CN202411349759A CN119310057A CN 119310057 A CN119310057 A CN 119310057A CN 202411349759 A CN202411349759 A CN 202411349759A CN 119310057 A CN119310057 A CN 119310057A
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Abstract
本发明涉及快速荧光寿命成像领域，具体涉及一种快速荧光寿命成像方法及装置。该方法及装置包括：将时钟信号正弦波经过功率分配产生相位差为0.5π的两个正弦波；将荧光信号经过低通滤波后产生正弦波，再经过功率分配后分为两路幅值相等相位相等的正弦波；将时钟信号产生的两个正弦波与荧光信号产生的正弦波经过混频和低通滤波后产生直流信号；再根据直流信号得出荧光寿命的大小。本发明通过对荧光信号进行解调,系统无需校准相位，实现快速荧光寿命成像功能。
The present invention relates to the field of rapid fluorescence lifetime imaging, and in particular to a rapid fluorescence lifetime imaging method and device. The method and device include: generating two sine waves with a phase difference of 0.5π by power distribution of a clock signal sine wave; generating a sine wave by low-pass filtering a fluorescence signal, and then dividing it into two sine waves with equal amplitude and phase after power distribution; generating a direct current signal by mixing and low-pass filtering the two sine waves generated by the clock signal and the sine wave generated by the fluorescence signal; and then obtaining the size of the fluorescence lifetime according to the direct current signal. The present invention realizes the rapid fluorescence lifetime imaging function by demodulating the fluorescence signal, and the system does not need to calibrate the phase.
Description
Technical Field
The invention relates to the field of rapid fluorescence lifetime imaging, in particular to a rapid fluorescence lifetime imaging method and device.
Background
Fluorescence microscopy has wide application in biological imaging because, to a reasonable extent, it is non-invasive, non-destructive and highly sensitive, providing structural and functional information of the object under investigation. In biological systems, fluorescence intensity data can be acquired in three spatial dimensions, wavelength, and polarization of light. However, fluorescence intensity data is susceptible to excitation intensity variations, probe concentration variations, and optical drift. Therefore, it is difficult to perform quantitative measurement based on intensity alone. In addition, it is also very difficult to distinguish between different fluorescent probes having very similar fluorescence spectra, and different portions of fluorophores that interact differently with the molecular environment. The fluorescence lifetime is the time required for the emitted fluorescence intensity to decay exponentially to 1/e of the initial intensity after excitation of the fluorescent molecule by an extremely short pulse laser. The fluorescence lifetime is generally not affected by factors such as the intensity of stimulated luminescence, the concentration of the fluorophore, and photobleaching, but is closely related to the microenvironment in which the fluorophore is located. Therefore, fluorescence Lifetime Imaging Microscopy (FLIM) for measuring the fluorescence lifetime of a sample can quantitatively measure a number of biophysical parameters (e.g., oxygen pressure, solution hydrophobicity, etc.) and biochemical parameters (e.g., pH, ion concentration, etc.) in the microenvironment where the target molecule is located.
From the point of view of signal processing, fluorescence lifetime imaging techniques can be broadly divided into two types, namely frequency domain detection techniques and time domain detection techniques.
The frequency domain detection technique excites a sample with modulated excitation light and calculates the fluorescence lifetime by measuring the phase shift and modulation of the fluorescence signal relative to the modulated excitation light at one or more modulation frequencies. The fluorescence signal generated by the sample has the same frequency as the modulated excitation light, but the phase is delayed and the modulation degree is reduced. Both the phase and modulation degree are varied depending on the fluorescence lifetime τ and the modulation frequency ω.
Time-dependent single photon counting (TCSPC) is a typical representation of time-domain detection techniques. TCSPC techniques are suitable for detecting low intensity, high repetition frequency fluorescent signals, such as two photon excited fluorescence of biological tissue. By recording the time of occurrence of a single photon pulse in a signal period and accumulating the number of signal periods of occurrence of the photon pulse at each time point, a histogram of photon number distribution over time can be established, thereby obtaining a fluorescence decay curve and calculating fluorescence lifetime. TCSPC technology is capable of recording fluorescent signals with extremely high temporal resolution and near-ideal efficiency, and is currently the only fluorescence lifetime detection technology that can reliably resolve tissue autofluorescence lifetime components.
Zhang Yide (Yide Zhang) of bordeaux university developed a two-photon spot scan INSTANT-FLIM based on analog signal processing capable of displaying in real time the image results of fluorescence intensity, fluorescence lifetime and phasor plot. The fluorescent signals detected by the PMT are divided into four paths, mixed with the pulse laser signals subjected to four-step phase shift (interval of 0.5 pi), and then the difference frequency term is demodulated from the mixed signals to obtain the fluorescent lifetime information.
In summary, fluorescence Lifetime Imaging Microscope (FLIM) has higher specificity and sensitivity in the aspects of cell microenvironment monitoring, protein interaction research, metabolic state research, drug screening, drug effect analysis, new material characterization, early cancer diagnosis and the like, and is increasingly applied to relevant research fields of biomedicine, material science, chemistry and the like. However, existing Fluorescence Lifetime Imaging (FLIM) techniques are slow and costly to image.
Disclosure of Invention
The embodiment of the invention provides a rapid fluorescence lifetime imaging method and device, which at least solve the technical problems of low imaging speed and high cost of the existing fluorescence lifetime imaging technology.
According to an embodiment of the present invention, there is provided a rapid fluorescence lifetime imaging method including the steps of:
generating two sine waves with the phase difference of 0.5 pi by distributing power to the sine waves of the clock signal;
the fluorescent signal is subjected to low-pass filtering to generate sine waves, and then is divided into two paths of sine waves with equal amplitude and equal phase after power distribution;
Mixing and low-pass filtering two sine waves generated by the clock signal and a sine wave generated by the fluorescent signal to generate a direct current signal;
and obtaining the fluorescence lifetime according to the direct current signal.
According to another embodiment of the present invention, there is provided a rapid fluorescence lifetime imaging apparatus including a laser emitting module, a signal demodulating module;
The laser emission module is used for generating a clock signal and a fluorescent signal;
The signal demodulation module is used for generating two sine waves with the phase difference of 0.5 pi by power distribution of clock signal sine waves, generating sine waves by low-pass filtering of fluorescent signals, dividing the fluorescent signals into two paths of sine waves with equal amplitude and equal phase after power distribution, generating direct-current signals by mixing the two sine waves generated by the clock signals with the sine waves generated by the fluorescent signals and low-pass filtering, and obtaining the size of fluorescent service life according to the direct-current signals.
Further, the signal demodulation module comprises a power divider, an avalanche photodiode, a low-pass filter and a power divider, wherein:
The laser generates a clock signal sine wave, two sine wave signals with the phase difference of 0.5 pi are generated through a 90-degree power divider, the avalanche photodiode receives a fluorescent signal and generates a sine wave after passing through a low-pass filter, the sine wave is divided into two paths of sine waves with equal amplitude and equal phase after passing through the power divider, and the sine wave of the clock signal and the sine wave generated by the fluorescent signal generate a direct current signal after passing through a mixer and the low-pass filter.
Further, the fluorescence signal, after passing through the avalanche photodiode, generates a voltage signal with an e-index:
τ The fluorescence lifetime is the size.
Further, the fluorescent signal is a periodic signal, and the voltage signal is converted into a fourier transform form:
Further, the fluorescence signal is output through a low-pass filter as:
further, the laser clock signal output is:
Further, the fluorescent signal is mixed with the clock signal and then output as follows:
m is the loss of fluorescent signal through the mixer.
Further, the fluorescence lifetime τ is expressed as:
Further, the laser emitting module includes a laser.
The rapid fluorescence lifetime imaging method and device provided by the embodiment of the invention are characterized in that a clock signal sine wave is subjected to power distribution to generate two sine waves with the phase difference of 0.5 pi, a fluorescence signal is subjected to low-pass filtering to generate a sine wave, the sine wave is further subjected to power distribution to be divided into two paths of sine waves with equal amplitude and equal phase, the two sine waves generated by the clock signal and the sine wave generated by the fluorescence signal are subjected to frequency mixing and low-pass filtering to generate a direct current signal, and the fluorescence lifetime is obtained according to the direct current signal. According to the invention, the fluorescent signal is demodulated, and the system does not need to calibrate the phase, so that the rapid fluorescent lifetime imaging function is realized.
Drawings
The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application and do not constitute a limitation on the application. In the drawings:
FIG. 1 is a circuit block diagram of a fast fluorescence lifetime imaging device of the present invention;
FIG. 2 is a signal diagram of a method and apparatus for rapid fluorescence lifetime imaging in accordance with the present invention.
Detailed Description
In order that those skilled in the art will better understand the present invention, a technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in which it is apparent that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the present invention without making any inventive effort, shall fall within the scope of the present invention.
It should be noted that the terms "first," "second," and the like in the description and the claims of the present invention and the above figures are used for distinguishing between similar objects and not necessarily for describing a particular sequential or chronological order. It is to be understood that the data so used may be interchanged where appropriate such that the embodiments of the invention described herein may be implemented in sequences other than those illustrated or otherwise described herein. Furthermore, the terms "comprises," "comprising," and "having," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, system, article, or apparatus that comprises a list of steps or elements is not necessarily limited to those steps or elements expressly listed but may include other steps or elements not expressly listed or inherent to such process, method, article, or apparatus.
The invention provides a rapid fluorescence lifetime imaging method and device, which adopt an analog signal processing method to realize rapid transmission of fluorescence intensity and lifetime by rapidly acquiring image data and processing real-time data. The invention aims to solve the defects of high cost, multiple IO ports of equipment, structural redundancy and the like of the traditional fluorescence lifetime imaging system and provides a solution of a rapid fluorescence lifetime imaging method. These problems are addressed by employing a frequency translation and filtering system for the above-mentioned drawbacks. The system has low cost and few IO ports, can realize fluorescence lifetime imaging only by two IO ports, and is simple and clear as a whole.
The technical scheme of the invention comprises the following basic contents:
the invention provides a rapid fluorescence lifetime imaging method and a rapid fluorescence lifetime imaging device, which mainly comprise the following components: the device comprises a laser emitting module and a signal demodulating module.
1-2, The laser generates a sine wave of a clock signal, two sine wave signals with a phase difference of 0.5 pi are generated through a 90-degree power divider, the avalanche photodiode receives a fluorescent signal and generates a sine wave after passing through a low-pass filter, the sine wave is divided into two paths of sine waves with equal amplitude and equal phase after passing through the power divider, and the sine wave of the clock signal and the sine wave generated by the fluorescent signal generate direct current signals V (phi) and V (phi+0.5 pi) after passing through a mixer and the low-pass filter. The magnitude of the fluorescence lifetime can be derived from V (phi) and V (phi +0.5 pi).
The fluorescent signal, after passing through the avalanche photodiode, generates an e-exponential voltage signal:
τ The fluorescence lifetime is the size.
Since the fluorescent signal is periodic, it can be converted into a fourier transform form:
the laser clock signal output is:
The fluorescent signal and the clock signal are mixed and then output as follows:
m is the loss of fluorescent signal through the mixer.
The fluorescence lifetime τ can be expressed as:
The key points and the points to be protected of the invention are as follows:
(1) The overall design and construction of the system. The service life of the system can be acquired by only 2 IO ports, and the whole system is concise and regular, free of redundancy and complex.
(2) Two sinusoidal signals with the phase difference of 0.5 pi are generated through a 90-degree power divider, a phase shifter is not needed to carry out phase shifting operation on the clock signals, a system is not needed to calibrate the phase, and a rapid fluorescence lifetime imaging function is realized through demodulating the fluorescence signals.
Compared with the prior art, the invention has the advantages that:
(1) INSTANT FLIM IO ports are needed for data acquisition, only 2 IO ports are needed, and the whole system is simple in structure, easy to build, not complex and low in cost.
(2) The 90-degree power divider replaces the phase shifter, the system does not need to perform phase calibration, the 90-degree power divider is a passive device, an additional IO port power supply and the like are not needed, and the requirement on the acquisition system is lower.
(3) The INSTANT_FLIM divides the fluorescence signal into four paths, and the invention divides the fluorescence signal into two paths, so that the imaging efficiency and the success rate can be greatly improved.
The invention proves to be feasible through a large number of experiments, simulation and use.
The foregoing embodiment numbers of the present invention are merely for the purpose of description, and do not represent the advantages or disadvantages of the embodiments.
In the foregoing embodiments of the present invention, the descriptions of the embodiments are emphasized, and for a portion of this disclosure that is not described in detail in this embodiment, reference is made to the related descriptions of other embodiments.
In the several embodiments provided in the present application, it should be understood that the disclosed technology may be implemented in other manners. The system embodiments described above are merely exemplary, and for example, the division of units may be a logic function division, and there may be another division manner in actual implementation, for example, multiple units or components may be combined or integrated into another system, or some features may be omitted, or not performed. Alternatively, the coupling or direct coupling or communication connection shown or discussed with each other may be through some interfaces, units or modules, or may be in electrical or other forms.
The units described as separate parts may or may not be physically separate, and parts displayed as units may or may not be physical units, may be located in one place, or may be distributed over a plurality of units. Some or all of the units may be selected according to actual needs to achieve the purpose of the solution of this embodiment.
In addition, each functional unit in the embodiments of the present invention may be integrated in one processing unit, or each unit may exist alone physically, or two or more units may be integrated in one unit. The integrated units may be implemented in hardware or in software functional units.
The integrated units, if implemented in the form of software functional units and sold or used as stand-alone products, may be stored in a computer readable storage medium. Based on such understanding, the technical solution of the present invention may be embodied in essence or a part contributing to the prior art or all or part of the technical solution in the form of a software product stored in a storage medium, comprising several instructions for causing a computer device (which may be a personal computer, a server or a network device, etc.) to perform all or part of the steps of the method of the various embodiments of the present invention. The storage medium includes a U disk, a Read-Only Memory (ROM), a random access Memory (RAM, random Access Memory), a removable hard disk, a magnetic disk, or an optical disk, etc. which can store the program code.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (10)
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