A fast fluorescence lifetime imaging method and apparatus

Abstract

The present application relates to the field of fast fluorescence lifetime imaging, in particular to a fast fluorescence lifetime imaging method and device. The method and device comprise: passing a clock signal sine wave through power distribution to generate two sine waves with a phase difference of 0.5pi; passing a fluorescence signal through low-pass filtering to generate a sine wave, and then passing the sine wave through power distribution to divide it into two sine waves with equal amplitude and equal phase; mixing the two sine waves generated by the clock signal with the sine wave generated by the fluorescence signal, and then passing the mixed signal through low-pass filtering to generate a direct current signal; and obtaining the size of the fluorescence lifetime according to the direct current signal. The present application realizes the function of fast fluorescence lifetime imaging by demodulating the fluorescence signal, and the system does not need to calibrate the phase.

Description

Technical Field

[0001] This invention relates to the field of rapid fluorescence lifetime imaging, and more specifically, to a rapid fluorescence lifetime imaging method and apparatus. Background Technology

[0002] Fluorescence microscopy has wide applications in bioimaging because, within reasonable limits, it is non-invasive, non-destructive, and highly sensitive, providing structural and functional information about the investigated object. In biological systems, fluorescence intensity data can be acquired in three spatial dimensions, wavelengths, and light polarization. However, fluorescence intensity data is easily affected by changes in excitation intensity, probe concentration, and photobleaching. Therefore, quantitative measurement based solely on intensity is difficult. Furthermore, distinguishing between different fluorescent probes with very similar fluorescence spectra, and between different parts of a fluorophore with different interaction states with its molecular environment, is also very challenging. Fluorescence lifetime is the time required for the emitted fluorescence intensity of a fluorescent molecule to decay exponentially to 1 / e of its initial intensity after being excited by a very short pulse of laser light. Fluorescence lifetime is generally unaffected by factors such as excitation light intensity, fluorophore concentration, and photobleaching, but is closely related to the microenvironment in which the fluorophore resides. Therefore, fluorescence lifetime imaging microscopy (FLIM), which measures the fluorescence lifetime of a sample, can quantitatively measure many biophysical parameters (such as oxygen pressure and solution hydrophobicity) and biochemical parameters (such as pH and ion concentration) in the microenvironment of the target molecule.

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

[0004] Frequency domain detection techniques use modulated excitation light to excite a sample and calculate fluorescence lifetime by measuring the phase shift and modulation depth 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 depth is reduced. Both the changes in phase and modulation depth depend on the fluorescence lifetime τ and the modulation frequency ω.

[0005] Time-correlated single-photon counting (TCSPC) is a typical example of time-domain detection technology. TCSPC is suitable for detecting low-intensity, high-repetition-frequency fluorescence signals, such as two-photon excitation fluorescence in biological tissues. By recording the occurrence time of a single photon pulse within a signal period and accumulating the number of signal periods with photon pulses at each time point, a histogram of the photon number distribution over time can be constructed, thus obtaining the fluorescence decay curve and calculating the fluorescence lifetime. TCSPC can record fluorescence signals with extremely high temporal resolution and near-ideal efficiency, and is currently the only fluorescence lifetime detection technology capable of reliably distinguishing the components of tissue autofluorescence lifetime.

[0006] Yide Zhang of the University of Colorado Boulder has developed INSTANT-FLIM, a two-photon point scanning technique based on analog signal processing, which can display real-time images of fluorescence intensity, fluorescence lifetime, and phasor plots. They split the fluorescence signal detected by the PMT into four paths, mixed them with pulsed laser signals that underwent four phase shifts (0.5π intervals), and then demodulated the difference frequency term to obtain fluorescence lifetime information.

[0007] In summary, fluorescence lifetime imaging microscopy (FLIM) is increasingly being used in biomedical, materials science, and chemistry research fields due to its high specificity and sensitivity in areas such as cellular microenvironment monitoring, protein-protein interaction studies, metabolic state analysis, drug screening and efficacy analysis, new material characterization, and early cancer diagnosis. However, existing FLIM techniques are slow and costly. Summary of the Invention

[0008] This invention provides a rapid fluorescence lifetime imaging method and apparatus to at least solve the technical problems of slow imaging speed and high cost of existing fluorescence lifetime imaging technologies.

[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 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, and then split into two sine waves with equal amplitude and equal 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 determined based on the DC signal.

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

[0015] The laser emitting module is used to generate clock 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; to generate a sine wave from the fluorescence signal after low-pass filtering, and then split it into two sine waves with equal amplitude and phase after power distribution; to generate a DC signal by mixing the two sine waves generated from the clock signal and the sine wave generated from the fluorescence signal through low-pass filtering; and to determine 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 is then divided into two sine wave signals with a phase difference of 0.5π by a 90° power divider. The avalanche photodiode receives the fluorescence signal and generates a sine wave after passing through a low-pass filter. After passing through a power divider, the sine wave is split into two sine waves with equal amplitude and phase. The clock signal sine wave and the sine wave generated by the fluorescence signal are then mixed and filtered by a low-pass filter to generate a DC signal.

[0019] Furthermore, the fluorescent signal, after passing through the avalanche photodiode, will generate an exponential voltage signal:

[0020]

[0021] τ This represents the fluorescence lifetime.

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

[0023]

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

[0025]

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

[0027]

[0028] Furthermore, after the fluorescence signal and clock signal are mixed and low-pass filtered, the output is:

[0029]

[0030] M represents the loss of the fluorescence signal after passing through the mixer.

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

[0032]

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

[0034] The rapid fluorescence lifetime imaging method and apparatus in this invention involves generating two sine waves with a phase difference of 0.5π from a clock signal sine wave through power division; generating a sine wave from a fluorescence signal after low-pass filtering, and then splitting it into two sine waves with equal amplitude and phase after further power division; mixing and low-pass filtering the two sine waves generated from the clock signal and the sine wave generated from the fluorescence signal to generate a DC signal; and then determining the fluorescence lifetime based on the DC signal. This invention achieves rapid fluorescence lifetime imaging by demodulating the fluorescence signal, eliminating the need for phase calibration. Attached Figure Description

[0035] The accompanying drawings, which are included to provide a further understanding of the invention and form part of this application, illustrate exemplary embodiments of the invention and, together with their description, serve to explain the invention and do not constitute an undue limitation thereof. In the drawings:

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

[0037] Figure 2 This is a signal diagram of the rapid fluorescence lifetime imaging method and apparatus of the present invention. Detailed Implementation

[0038] To enable those skilled in the art to better understand the present invention, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort 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, claims, and accompanying drawings of this invention are used to distinguish similar objects and are not necessarily used to describe a specific order or sequence. It should be understood that such data can be interchanged where appropriate so that the embodiments of the invention described herein can be implemented in orders other than those illustrated or described herein. Furthermore, the terms "comprising" and "having," and any variations thereof, are intended to cover a non-exclusive inclusion; for example, a process, method, system, product, or apparatus that comprises a series of steps or units is not necessarily limited to those steps or units explicitly listed, but may include other steps or units not explicitly listed or inherent to such processes, methods, products, or apparatus.

[0040] This invention proposes a rapid fluorescence lifetime imaging method and apparatus. Employing analog signal processing, it achieves rapid transmission of fluorescence intensity and lifetime data through fast image data acquisition and real-time data processing. The purpose of this invention is to address the shortcomings of existing fluorescence lifetime imaging systems, such as high cost, numerous I / O ports, and redundant structure, by providing a solution for rapid fluorescence lifetime imaging. To address the aforementioned drawbacks, frequency conversion and filtering systems are used to solve these problems. This system is low-cost, requires only two I / O ports to achieve fluorescence lifetime imaging, and has a simple and clear overall design.

[0041] The basic content of the technical solution of this invention:

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

[0043] The specific implementation plan is as follows: Figure 1-2 As shown, a laser generates a clock signal sine wave, which, after passing through a 90° power divider, produces two sine wave signals with a phase difference of 0.5π. An avalanche photodiode receives a fluorescence signal, which, after passing through a low-pass filter, generates a sine wave. This sine wave, after passing through a power divider, is split into two sine waves with equal amplitude and phase. The clock signal sine wave and the sine wave generated by the fluorescence signal, after passing through a mixer and a low-pass filter, produce DC signals V(φ) and V(φ+0.5π). The fluorescence lifetime can be determined from V(φ) and V(φ+0.5π).

[0044] The fluorescent signal, after passing through the avalanche photodiode, generates an exponential voltage signal:

[0045]

[0046] τ This represents the fluorescence lifetime.

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

[0048]

[0049] The laser clock signal output is:

[0050]

[0051] The output after low-pass filtering of the mixed fluorescence signal and clock signal is:

[0052]

[0053] M represents the loss of the fluorescence signal after passing through the mixer.

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

[0055]

[0056] The key points and areas to be protected in this invention are:

[0057] (1) Overall design and structure of the system. The system only requires 2 I / O ports to realize lifetime acquisition. The whole system is simple and well-organized, without redundancy or complexity.

[0058] (2) Two sinusoidal signals with a phase difference of 0.5π are generated by a 90° power divider. No phase shifter is needed to perform phase shifting operation on the clock signal. The system does not need to calibrate the phase. By demodulating the fluorescence signal, the function of rapid fluorescence lifetime imaging is realized.

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

[0060] (1) INSTANT FLIM requires 9 I / O ports for data acquisition, while this invention only requires 2 I / O ports. The entire system structure is simple, easy to build, not complicated, 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 reduces the requirements for the acquisition system.

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

[0063] The invention has been proven feasible through extensive experiments, simulations, and use.

[0064] The sequence numbers of the above embodiments of the present invention are for descriptive purposes only and do not represent the superiority or inferiority of the embodiments.

[0065] In the above embodiments of the present invention, the descriptions of each embodiment have different focuses. For parts not described in detail in a certain embodiment, please refer 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. The system embodiments described above are merely illustrative; for example, the division of units can be a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or direct coupling or communication connection shown or discussed may be through some interfaces; the indirect coupling or communication connection of units or modules may be electrical or other forms.

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

[0068] Furthermore, the functional units in the various embodiments of the present invention can be integrated into one processing unit, or each unit can exist physically separately, or two or more units can be integrated into one unit. The integrated unit can be implemented in hardware or as a software functional unit.

[0069] If the integrated unit is implemented as 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. This computer software product is stored in a storage medium and includes several instructions to cause a computer device (which may be a personal computer, server, or network device, etc.) to execute all or part of the steps of the methods of the various embodiments of the present invention. The aforementioned storage medium includes various media capable of storing program code, such as USB flash drives, read-only memory (ROM), random access memory (RAM), portable hard drives, magnetic disks, or optical disks.

[0070] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A rapid fluorescence lifetime imaging method, characterized in that, Includes the following steps: The clock signal sine wave is 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, and then split into two sine waves with equal amplitude and equal 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 determined based on the DC signal; where: The clock signal is generated by the laser emission module.

2. A rapid fluorescence lifetime imaging device, characterized in that, include: Laser emission module, signal demodulation module; The laser emitting module is used to generate clock signals and fluorescence signals; 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; to generate a sine wave from the fluorescence signal after low-pass filtering, and then split it into two sine waves with equal amplitude and phase after power distribution; to generate a DC signal by mixing the two sine waves generated from the clock signal and the sine wave generated from the fluorescence signal through low-pass filtering; and to determine the fluorescence lifetime based on the DC signal.

3. The rapid 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 is then divided into two sine wave signals with a phase difference of 0.5π by a 90° power divider. The avalanche photodiode receives the fluorescence signal and generates a sine wave after passing through a low-pass filter. After passing through a power divider, the sine wave is split into two sine waves with equal amplitude and phase. The clock signal sine wave and the sine wave generated by the fluorescence signal are then mixed and filtered by a low-pass filter to generate a DC signal.

4. The rapid fluorescence lifetime imaging device according to claim 3, characterized in that, The fluorescent signal, after passing through the avalanche photodiode, generates an exponential voltage signal: This represents the fluorescence lifetime.

5. The rapid 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 a Fourier transform form: 。 6. The rapid fluorescence lifetime imaging device according to claim 5, characterized in that, The output of the fluorescence signal after passing through the low-pass filter is: 。 7. The rapid fluorescence lifetime imaging device according to claim 6, characterized in that, The laser clock signal output is: 。 8. The rapid fluorescence lifetime imaging device according to claim 7, characterized in that, The output after low-pass filtering of the mixed fluorescence signal and clock signal is: M represents the loss of the fluorescence signal after passing through the mixer.

9. The rapid fluorescence lifetime imaging device according to claim 8, characterized in that, The fluorescence lifetime τ is expressed as: 。 10. The rapid fluorescence lifetime imaging device according to claim 2, characterized in that, The laser emitting module includes a laser.

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