Rapid fluorescence lifetime imaging method and device

Abstract

The invention relates to the field of rapid fluorescence lifetime imaging, in particular to a rapid fluorescence lifetime imaging method and device. The method comprises the following steps: carrying out power distribution on a clock signal sine wave to generate two sine waves with a phase difference of 0.5 pi; carrying out low-pass filtering on the fluorescence signal to generate sine waves, and dividing the sine waves into two paths of sine waves with equal amplitudes and equal phases after power distribution; 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 obtaining the fluorescence lifetime according to the direct current signal. By demodulating the fluorescence signal, the system does not need to calibrate the phase, and the rapid fluorescence lifetime imaging function is realized.

Description

Technical Field

[0001] The present invention relates to the field of rapid fluorescence lifetime imaging, and in particular to a rapid fluorescence lifetime imaging method and device. Background Art

[0002] Fluorescence microscopy has a wide range of applications in biological imaging because it is non-invasive, non-destructive, and highly sensitive within reasonable limits, and can provide structural and functional information about 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 are easily affected by changes in excitation intensity, changes in probe concentration, and photobleaching. Therefore, quantitative measurements based on intensity alone are difficult. In addition, it is also very difficult to distinguish between different fluorescent probes with very similar fluorescence spectra, and different parts of fluorophores that interact differently with the molecular environment. Fluorescence lifetime is the time required for the emission fluorescence intensity of a fluorescent molecule to decay exponentially to 1 / e of the initial intensity after being excited by a very short pulse of laser light. Fluorescence lifetime is usually not affected by factors such as excitation light intensity, fluorophore concentration, and photobleaching, but is closely related to the microenvironment in which the fluorophore is located. Therefore, fluorescence lifetime imaging microscopy (FLIM), which measures the fluorescence lifetime of a sample, can quantitatively measure many biophysical parameters (such as oxygen pressure, solution hydrophobicity, etc.) and biochemical parameters (such as pH, ion concentration, etc.) in the microenvironment in which the target molecule is located.

[0003] From the perspective of signal processing, fluorescence lifetime imaging technology can be roughly divided into two categories: frequency domain detection technology and time domain detection technology.

[0004] Frequency domain detection technology uses modulated excitation light to excite the sample 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 is reduced. The changes in phase and modulation are dependent on the fluorescence lifetime τ and the modulation frequency ω.

[0005] Time-correlated single photon counting (TCSPC) is a typical representative of time-domain detection technology. TCSPC technology is suitable for detecting low-intensity, high-repetition-rate fluorescence signals, such as two-photon excitation fluorescence of biological tissues. By recording the time when a single photon pulse appears within the signal cycle and accumulating the number of signal cycles with photon pulses at each time point, a histogram of the distribution of photon numbers over time can be established, thereby obtaining a fluorescence decay curve and calculating the fluorescence lifetime. TCSPC technology can record fluorescence signals with extremely high time resolution and near-ideal efficiency. It is currently the only fluorescence lifetime detection technology that can reliably distinguish the lifetime components of tissue autofluorescence.

[0006] Yide Zhang from the University of Colorado Boulder developed a two-photon spot scanning INSTANT-FLIM based on analog signal processing, which can display the fluorescence intensity, fluorescence lifetime and phasor plot image results in real time. They divided the fluorescence signal detected by the PMT into four paths, mixed it with a pulsed laser signal that had undergone four phase shifts (interval 0.5π), and then demodulated the difference frequency term to obtain the fluorescence lifetime information.

[0007] In summary, fluorescence lifetime imaging microscopy (FLIM) is increasingly being used in biomedicine, materials science, chemistry and other related research fields due to its high specificity and sensitivity in cell microenvironment monitoring, protein interaction research, metabolic state research, drug screening and efficacy analysis, new material characterization, early cancer diagnosis, etc. However, the existing fluorescence lifetime imaging (FLIM) technology has slow imaging speed and high cost. Summary of the invention

[0008] The embodiments of the present invention provide a fast fluorescence lifetime imaging method and device to at least solve the technical problems of slow imaging speed and high cost of the existing fluorescence lifetime imaging technology.

[0009] According to an embodiment of the present invention, a rapid fluorescence lifetime imaging method is provided, comprising the following steps:

[0010] The clock signal sine wave is power-divided to generate two sine waves with a phase difference of 0.5π;

[0011] The fluorescence signal is low-pass filtered to generate a sine wave, which is then divided into two sine waves with equal amplitude and phase after power distribution;

[0012] The two sine waves generated by the clock signal and the sine wave generated by the fluorescence signal are mixed and low-pass filtered to generate a DC signal;

[0013] The fluorescence lifetime is obtained based on the DC signal.

[0014] According to another embodiment of the present invention, there is provided a fast fluorescence lifetime imaging device, comprising: a laser emission module, a signal demodulation module;

[0015] The laser emission module is used to generate clock signals and fluorescence signals;

[0016] The signal demodulation module is used to generate two sine waves with a phase difference of 0.5π from the clock signal sine wave through power distribution; generate a sine wave from the fluorescence signal after low-pass filtering, and then divide it into two sine waves with equal amplitude and phase after power distribution; generate a DC 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 derive the fluorescence lifetime based on the DC signal.

[0017] Furthermore, the signal demodulation module includes: a power divider, an avalanche photodiode, a low-pass filter, and a power divider; wherein:

[0018] The laser generates a clock signal sine wave, which passes through a 90° power divider to generate two sine wave signals with a phase difference of 0.5π. The avalanche photodiode receives the fluorescent signal and generates a sine wave after passing through a low-pass filter. After passing through the power divider, it is divided into two sine waves with equal amplitude and phase. The sine wave of the clock signal and the sine wave generated by the fluorescent signal pass through a mixer and a low-pass filter to generate a DC signal.

[0019] Furthermore, after the fluorescence signal passes through the avalanche photodiode, it will generate a voltage signal with an exponential e:

[0020]

[0021] τ is the fluorescence lifetime.

[0022] Furthermore, the fluorescence signal is a periodic signal, and the voltage signal is converted into Fourier transform form:

[0023]

[0024] Furthermore, the fluorescence signal is output through a low-pass filter as:

[0025]

[0026] Furthermore, the laser clock signal output is:

[0027]

[0028] Furthermore, the fluorescence signal is mixed with the clock signal and then low-passed and the output is:

[0029]

[0030] M is the loss of fluorescence signal through the mixer.

[0031] Furthermore, the fluorescence lifetime τ is expressed as:

[0032]

[0033] Furthermore, the laser emission module includes a laser.

[0034] The fast fluorescence lifetime imaging method and device in the embodiment of the present invention generates two sine waves with a phase difference of 0.5π after power distribution of the clock signal sine wave; generates a sine wave after low-pass filtering of the fluorescence signal, and then divides it into two sine waves with equal amplitude and phase after power distribution; generates a DC signal after 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 obtains the size of the fluorescence lifetime according to the DC signal. The present invention realizes the fast fluorescence lifetime imaging function by demodulating the fluorescence signal, and the system does not need to calibrate the phase. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The drawings described herein are used to provide a further understanding of the present invention and constitute a part of this application. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0036] Figure 1 This is a circuit module diagram of the fast fluorescence lifetime imaging device of the present invention;

[0037] Figure 2 It is a signal diagram of the rapid fluorescence lifetime imaging method and device of the present invention. DETAILED DESCRIPTION

[0038] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0039] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0040] The present invention proposes a fast fluorescence lifetime imaging method and device, which adopts an analog signal processing method, realizes the fast transmission of fluorescence intensity and lifetime by quickly collecting image data and real-time data processing. The purpose of the present invention is to solve the shortcomings of the existing fluorescence lifetime imaging system, such as high cost, multiple IO ports of the equipment, and structural redundancy, and to provide a solution for a fast fluorescence lifetime imaging method. In view of the above shortcomings, these problems are solved by adopting a frequency conversion and filtering system. The system has low cost and few IO ports. Only two IO ports are required to realize fluorescence lifetime imaging. The overall system is concise and clear.

[0041] The basic contents of the technical solution of the present invention are as follows:

[0042] The invention provides a rapid fluorescence lifetime imaging method and device, which mainly include: a laser emission module and a signal demodulation module.

[0043] Specific implementation plans such as Figure 1-2 As shown, the laser generates a clock signal sine wave, which passes through a 90° power divider to generate two sine wave signals with a phase difference of 0.5π. The avalanche photodiode receives the fluorescence signal and generates a sine wave after passing through a low-pass filter. After passing through the power divider, it is divided into two sine waves with equal amplitude and phase. The sine wave of the clock signal and the sine wave generated by the fluorescence signal pass through a mixer and a low-pass filter to generate DC signals V(φ) and V(φ+0.5π). The size of the fluorescence lifetime can be obtained based on V(φ) and V(φ+0.5π).

[0044] After the fluorescence signal passes through the avalanche photodiode, a voltage signal with an exponential e will be generated:

[0045]

[0046] τ is the fluorescence lifetime.

[0047] Since the fluorescence signal is periodic, it can be converted into Fourier transform form:

[0048]

[0049] The laser clock signal output is:

[0050]

[0051] The fluorescence signal is mixed with the clock signal and then low-passed and the output is:

[0052]

[0053] M is the loss of fluorescence signal through the mixer.

[0054] The fluorescence lifetime τ can be expressed as:

[0055]

[0056] The key points and intended protection points of the present invention are:

[0057] (1) Overall design and structure of the system. The system only needs two IO ports to realize life acquisition. The whole system is simple and neat, without redundancy or complexity.

[0058] (2) Two sinusoidal signals with a phase difference of 0.5π are generated through a 90° power divider. There is no need for a phase shifter to shift the clock signal, and the system does not need to calibrate the phase. By demodulating the fluorescence signal, fast fluorescence lifetime imaging is achieved.

[0059] Compared with the prior art, the advantages of the present invention are:

[0060] (1) INSTANT FLIM requires 9 IO ports for data acquisition, while the present invention only requires 2 IO ports. The entire system structure is simple, easy to build, uncomplicated, and low in cost.

[0061] (2) By replacing the phase shifter with a 90° power divider, the system does not need to perform phase calibration. The 90° power divider is a passive device and does not require additional IO port power supply, etc., which places lower requirements on the acquisition system.

[0062] (3)INSTANT_FLIM divides the fluorescence signal into four paths, while the present invention divides the fluorescence signal into two paths, which can greatly improve the imaging efficiency and success rate.

[0063] The present invention has been proven to be feasible through a large number of experiments, simulations and uses.

[0064] The serial numbers of the above embodiments of the present invention are only for description and do not represent the advantages or disadvantages of the embodiments.

[0065] In the above embodiments of the present invention, the description of each embodiment has its own emphasis. For parts that are not described in detail in a certain embodiment, reference can be made to the relevant descriptions of other embodiments.

[0066] In the several embodiments provided in this application, it should be understood that the disclosed technical content can be implemented in other ways. Among them, the system embodiments described above are only schematic. For example, the division of units can be a logical function division. There may be other division methods in actual implementation. For example, multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be through some interfaces, indirect coupling or communication connection of units or modules, which can be electrical or other forms.

[0067] The units described as separate components may or may not be physically separated, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed over multiple units. Some or all of the units may be selected according to actual needs to achieve the purpose of the present embodiment.

[0068] In addition, each functional unit in each embodiment of the present invention may be integrated into one processing unit, or each unit may exist physically separately, or two or more units may be integrated into one unit. The above-mentioned integrated unit may be implemented in the form of hardware or in the form of software functional units.

[0069] If the integrated unit is implemented in the form of a software functional unit and sold or used as an independent product, it can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of the present invention, in essence, or the part that contributes to the prior art, or all or part of the technical solution can be embodied in the form of a software product, and the computer software product is stored in a storage medium, including a number of instructions for a computer device (which can be a personal computer, a server or a network device, etc.) to perform all or part of the steps of the methods of each embodiment of the present invention. The aforementioned storage medium includes: U disk, read-only memory (ROM, Read-Only Memory), random access memory (RAM, Random Access Memory), mobile hard disk, magnetic disk or optical disk and other media that can store program codes.

[0070] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A rapid fluorescence lifetime imaging method, characterized in that: The following steps are involved: The clock signal sine wave is power-divided to generate two sine waves with a phase difference of 0.5π; The fluorescence signal is low-pass filtered to generate a sine wave, which is then divided into two sine waves with equal amplitude and phase after power distribution; The two sine waves generated by the clock signal and the sine wave generated by the fluorescence signal are mixed and low-pass filtered to generate a DC signal; The fluorescence lifetime is obtained based on the DC signal.

2. A fast fluorescence lifetime imaging device, characterized in that: include: Laser emission module, signal demodulation module; The laser emission module is used to generate a clock signal and a fluorescence signal; The signal demodulation module is used to generate two sine waves with a phase difference of 0.5π from the clock signal sine wave through power distribution; generate a sine wave from the fluorescence signal after low-pass filtering, and then divide it into two sine waves with equal amplitude and phase after power distribution; generate a DC 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 obtain the size of the fluorescence lifetime based on the DC signal.

3. The fast fluorescence lifetime imaging device according to claim 2, characterized in that: The signal demodulation module includes: a power divider, an avalanche photodiode, a low-pass filter, and a power divider; wherein: The laser generates a clock signal sine wave, which passes through a 90° power divider to generate two sine wave signals with a phase difference of 0.5π. The avalanche photodiode receives the fluorescent signal and generates a sine wave after passing through a low-pass filter. After passing through the power divider, it is divided into two sine waves with equal amplitude and phase. The sine wave of the clock signal and the sine wave generated by the fluorescent signal pass through a mixer and a low-pass filter to generate a DC signal.

4. The fast fluorescence lifetime imaging device according to claim 3, characterized in that: After the fluorescence signal passes through the avalanche photodiode, a voltage signal with an exponential e will be generated: τ is the fluorescence lifetime.

5. The fast fluorescence lifetime imaging device according to claim 4, characterized in that: The fluorescence signal is a periodic signal, and the voltage signal is converted into Fourier transform form:

6. The fast fluorescence lifetime imaging device according to claim 5, characterized in that: The fluorescence signal is output after passing through a low-pass filter:

7. The fast fluorescence lifetime imaging device according to claim 6, characterized in that: The laser clock signal output is:

8. The fast fluorescence lifetime imaging device according to claim 7, characterized in that: The fluorescence signal is mixed with the clock signal and then low-passed and the output is: M is the loss of fluorescence signal through the mixer.

9. The fast fluorescence lifetime imaging device according to claim 8, characterized in that: The fluorescence lifetime τ is expressed as:

10. The fast fluorescence lifetime imaging device according to claim 2, characterized in that: The laser emission module includes a laser.

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