Fluorescence signal separation method and equipment based on modulation and demodulation and storage medium
By using a modulation and demodulation-based method, multiple pulsed lasers with different frequencies and demodulation devices are used to separate fluorescence signals, solving the problem of multi-target spatiotemporal synchronous detection that cannot be achieved in the existing technology, and realizing efficient fluorescence signal separation and detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-29
- Publication Date
- 2026-03-31
AI Technical Summary
Existing technologies cannot effectively separate multiple fluorescence signals, making it impossible to achieve simultaneous spatiotemporal detection of multiple targets, especially in life science and biomedical research where they cannot meet the needs of real-time multi-target detection.
A modulation-demodulation-based method is adopted, in which multiple pulsed lasers of different frequencies are simultaneously focused on the sample to generate mixed fluorescence signals. The signals are then separated using demodulation devices and reference signals. Specifically, a lock-in amplifier is used for mixing and low-pass filtering to achieve signal separation.
This technology enables simultaneous spatiotemporal detection of fluorescence signals from multiple targets, improving the efficiency and accuracy of signal separation and meeting the requirements for real-time multi-target detection.
Smart Images
Figure CN121762500A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of signal separation technology, and in particular to a method, apparatus and storage medium for separating fluorescence signals based on modulation and demodulation. Background Technology
[0002] Real-time multi-target detection is crucial in life science and biomedical research. Since the core of life science and biomedical research lies in understanding the overall structure and function of biological systems, rather than focusing solely on individual components, multi-target detection technology can provide a high-resolution view of the entire system, helping to reveal the mechanisms by which biological systems operate in various states. The cellular and tissue structures involved in these studies are often highly complex; real-time multi-target detection technology allows for the simultaneous observation and analysis of multiple biological structures, behaviors, and interactions.
[0003] For example, in the field of neuroscience, numerous complex and transient changes occur constantly within neural tissue: alterations in action potentials when neurons generate and transmit electrical signals; the opening and closing of ion channels; the transmission of information from neurons to downstream neurons through the release of neurotransmitters; synaptic transmission; neuronal metabolic activity; synchronous firing, oscillatory activity, and burst firing in neuronal networks; and the regulation and control of neural network activity by neuromodulatory substances. Therefore, developing real-time multi-target detection technology will not only enable researchers to gain a more comprehensive understanding of the structure and function of neural networks in neuroscience, but also promises to achieve high spatiotemporal resolution observation and quantitative analysis of neural network activity, thus advancing the frontiers of neuroscience research.
[0004] For example, in disease research and diagnosis, real-time multi-target detection technology can help identify and analyze various biomarkers, thereby improving the accuracy and efficiency of early disease diagnosis. Taking tumors as an example, since a tumor is a constantly changing three-dimensional dynamic system, the interaction between tumor cells and their microenvironment involves not only direct contact, signal transduction, and material exchange between cells and the matrix, but also delicate and complex cell deformation and individual movement. Tracking and monitoring such complex dynamic behaviors in the tumor microenvironment requires the ability to perform spatiotemporal synchronous multi-target detection.
[0005] Optical microscopy, which relies on fluorescence signals, is widely recognized as an ideal tool for multi-target detection and is used in numerous life science and biomedical research studies. In its practical applications, effectively separating multiple fluorescence signals is crucial for achieving real-time multi-target detection.
[0006] Currently, there are several methods for achieving multi-target detection based on fluorescence signal separation: The first method is based on the spectral characteristics of fluorescent substances. Since fluorescent substances have different excitation and emission spectra, separation is achieved by selectively exciting and detecting signals of different wavelengths. For example, dichroic mirrors or filters of appropriate wavelengths can be used to separate fluorescence signals from background light or other light sources. Alternatively, multiple fluorescence signals can be separated by stacking multiple filters or using bandpass filters. Spectrometers and multi-channel detection systems can also be used to achieve signal separation based on fluorescence emission characteristics. The second method is based on the lifetime characteristics of fluorescent substances. Fluorescence lifetime refers to the time it takes for a fluorescent substance to emit fluorescence and decay back to its original excited state after excitation. It is an inherent characteristic of fluorescent substances, and different fluorescent substances have different fluorescence lifetimes due to differences in molecular structure and chemical environment. Therefore, the different fluorescence lifetimes of fluorescent substances can be utilized to achieve multi-signal separation.
[0007] The disadvantages of separation methods based on the spectral characteristics of fluorescent substances are: first, the spectra of fluorescent substances have overlapping parts, and there is crosstalk between signals, making effective separation impossible; second, when performing signal separation based on spectral characteristics, it is necessary to first filter the excitation light to control the sample to generate fluorescence, and then further filter the emission light to separate the fluorescence emission into narrow bands before signal separation can be achieved. The filtering process leads to a reduction in the available signal, thereby reducing the signal-to-noise ratio, and requires a longer acquisition time.
[0008] The drawback of separation methods based on fluorescence lifetime characteristics is that some fluorescent substances have similar lifetime characteristics, making effective separation impossible.
[0009] If we want to eliminate crosstalk between signals without causing signal attenuation, we can consider using the method of sequentially exciting different fluorescent substances. However, the sequential excitation method cannot achieve real-time multi-target detection, which hinders life science and biomedical research that needs to track dynamic changes.
[0010] In summary, the current technology has the following main drawbacks: it cannot meet the requirement of effective separation of fluorescence signals in multi-band fusion optical microscopy imaging, and it cannot achieve multi-target spatiotemporal synchronous detection of fluorescence signals. Summary of the Invention
[0011] Therefore, it is necessary to address the problem of poor fluorescence signal separation performance in existing technologies by proposing a fluorescence signal separation method, device, and storage medium based on modulation and demodulation.
[0012] In a first aspect, a method for separating fluorescence signals based on modulation and demodulation is provided, the method comprising: Acquire mixed fluorescence signals, wherein the mixed fluorescence signals are generated by multiple pulsed laser beams of different frequencies being simultaneously focused on the sample; Based on the demodulation device, the reference signal, and the mixed fluorescence signal, signal separation is performed on the mixed fluorescence signal.
[0013] In a second aspect, a computer device is provided, including a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor, when executing the computer program, implements the steps of the above-described modulation-demodulation-based fluorescence signal separation method.
[0014] Thirdly, a computer-readable storage medium is provided, the computer-readable storage medium storing a computer program, which, when executed by a processor, implements the steps of the above-described modulation-demodulation-based fluorescence signal separation method.
[0015] This invention proposes a modulation-demodulation-based fluorescence signal separation method. This method acquires a mixed fluorescence signal generated by simultaneously focusing multiple pulsed laser beams of different frequencies onto a sample. Then, based on a demodulation device, a reference signal, and the mixed fluorescence signal, signal separation is performed. This invention enables multi-target spatiotemporal synchronous detection of fluorescence signals by using a demodulation device and a reference signal to separate the mixed fluorescence signal. Attached Figure Description
[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.
[0017] in: Figure 1 This is a flowchart of a fluorescence signal separation method based on modulation and demodulation in one embodiment; Figure 2 This is a schematic diagram of a module in a modulation and demodulation-based fluorescence signal separation method in one embodiment; Figure 3 This is a schematic diagram illustrating the working principle of a lock-in amplifier in a fluorescence signal separation method based on modulation and demodulation in one embodiment. Figure 4 This is a block diagram of a dual-phase lock-in amplifier structure based on a modulation and demodulation-based fluorescence signal separation method in one embodiment. Detailed Implementation
[0018] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application, are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.
[0019] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.
[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0021] Please see Figure 1 As shown, Figure 1 A schematic flowchart of a fluorescence signal separation method based on modulation and demodulation according to an embodiment of the present invention includes the following steps: Step S1: Acquire the mixed fluorescence signal, which is generated by focusing multiple pulsed laser beams of different frequencies simultaneously onto the sample; In this embodiment, multiple pulsed laser beams with different frequencies can be generated by a pulsed laser and a laser modulation module. Then, each pulsed laser beam is simultaneously focused onto the sample by optical microscopy to generate fluorescence. Finally, the fluorescence is collected to obtain a mixed fluorescence signal.
[0022] As an example, the mixed fluorescence signal can be acquired through a laser module, an optical microscopy imaging module, and a fluorescence signal acquisition module. (See reference...) Figure 2 The laser module is used to generate multiple pulsed laser beams with different frequencies. As an example, refer to... Figure 1The laser module comprises two parts: N pulsed lasers 11 numbered 1-N and a laser modulation module 12. The pulsed lasers 11 generate pulsed laser light; the laser modulation module 12 modulates the laser frequency, for example, using an electro-optic modulator (EOM) or an acousto-optic modulator (AOM) pulse selector. A pulse selector is an electrically controlled optical switch used to extract a single pulse from a fast pulse sequence, thus achieving frequency modulation. The principle of the pulse selector is explained using an AOM as an example. An acousto-optic modulator (AOM) is a device that modulates a light beam using the acousto-optic effect. It has optical input and output terminals; the beam enters from one end, is modulated by a medium, and exits from the other end. By changing the frequency of the driving signal, the angle of the diffracted beam can be changed, achieving frequency modulation of the beam. The pulsed lasers 11 have a natural frequency, which is known or can be obtained through measurement. Two scenarios are possible: First, the frequencies of the N pulsed lasers 11 are all different, in which case the laser modulation module 12 is not needed; second, the pulsed lasers have the same frequency, in which case the laser modulation module 12 is used to modulate the frequency so that the laser frequencies are different. Each of the N pulsed lasers 11 emits a pulsed laser, which is then processed by the laser modulation module 12 to obtain N pulsed laser beams with known and different frequencies. These N pulsed laser beams then enter the optical microscopic imaging module 2.
[0023] The optical microscopy imaging module is used to simultaneously focus each pulsed laser onto the sample to generate fluorescence. Specifically, refer to... Figure 2 The function of the optical microscopy imaging module 2 is to generate fluorescence, which is then collected by the fluorescence signal acquisition module 3. The optical microscopy imaging module 2 can be a two-photon microscopy imaging system, a single-photon microscopy imaging system, a three-photon microscopy imaging system, etc.
[0024] The fluorescence signal acquisition module is used to acquire the fluorescence and obtain various fluorescence signals. Specifically, refer to... Figure 2 The fluorescence signal acquisition module 3 consists of a detector. Since the optical microscopy imaging module uses point scanning imaging, the detector needs to be a dot array detector, typically a photomultiplier tube (PMT). The fluorescence signal acquired by the detector in the fluorescence signal acquisition module 3 is used as the input signal and input to the fluorescence signal separation module 4.
[0025] Step S2: Based on the demodulation device, the reference signal, and the mixed fluorescence signal, perform signal separation on the mixed fluorescence signal.
[0026] In this embodiment, signal separation of the mixed fluorescence signals is achieved using a demodulation device and a reference signal. As an example, the reference signal... Figure 2 The function of the fluorescence signal separation module 4 is to demodulate in order to achieve signal separation. It is composed of devices with demodulation function, which can be lock-in amplifiers.
[0027] refer to Figure 3 , Figure 3 This is a schematic diagram illustrating the working principle of a lock-in amplifier. A lock-in amplifier includes a signal channel, a reference channel, a phase-sensitive detector (PSD), and a low-pass filter (LPF). The signal channel receives the input signal (the mixed fluorescence signal); the reference channel receives the reference signal; the PSD performs phase-sensitive detection (multiplication operation), mixing the input signal with the reference signal; and the LPF filters out high-frequency components, retaining the effective low-frequency signal. (See the block diagram of a dual-phase lock-in amplifier for reference.) Figure 4 Given an input signal S(t) and a reference signal R(t), the input signal is multiplied by the reference signal, and a mixer is used to generate a mixed signal with in-phase and quadrature components. The mixed signal is then low-pass filtered to remove high-frequency components and retain only the low-frequency effective signal, resulting in a signal with in-phase and quadrature components after low-pass filtering. The amplitude and phase of the output signal can be calculated from the in-phase and quadrature components.
[0028] In one embodiment, after obtaining the separated fluorescence signal, a separation signal output module can be used for output, as shown in the reference. Figure 2 The signal separation output module 5 includes a data acquisition card 51 and a computer 52. The output terminal of the fluorescence signal separation module 4 is connected to the data acquisition card 51, and then to the computer 52. The function of the data acquisition card 51 is to acquire the output signal from the fluorescence signal separation module 4, and the function of the computer 52 is to perform data storage, processing, and other operations.
[0029] In one embodiment, the signal channel is used to determine an input signal based on each of the fluorescence signals; the reference channel is used to acquire a reference signal; the phase-sensitive detector (PSD) is used to perform frequency mixing processing on the input signal and the reference signal to obtain in-phase and quadrature components; the low-pass filter (LPF) is used to perform low-pass filtering processing on the in-phase and quadrature components to obtain a target in-phase component and a target quadrature component; and the separated signal output module is used to calculate the amplitude and phase information of the output signal based on the target in-phase and target quadrature components.
[0030] In one embodiment, the demodulation device is a lock-in amplifier, and the step of separating the mixed fluorescence signals based on the demodulation device and the reference signal includes: Step S11: Determine the input signal based on the mixed fluorescence signals; Step S12: Based on the input signal, the reference signal, and the lock-in amplifier, obtain the amplitude and phase information of the output signal. The lock-in amplifier is used to perform frequency mixing processing on the input signal and the reference signal to obtain in-phase and quadrature components, and to perform low-pass filtering processing on the in-phase and quadrature components to obtain target in-phase and target quadrature components. Based on the target in-phase and target quadrature components, calculate the amplitude and phase of the output signal.
[0031] In one embodiment, a mixer generates in-phase and quadrature components, and the mixed fluorescence signal includes frequencies of... The fluorescence signal a and the frequency are The fluorescence signal b, the reference signal The step of obtaining the amplitude and phase information of the output signal based on the input signal, the reference signal, and the lock-in amplifier, having the same frequency as the fluorescence signal a, includes: By using a lock-in amplifier, a reference signal with the same frequency as the fluorescence signal a is utilized. The mixed fluorescence signals are separated to obtain the amplitude and phase of the output signal corresponding to fluorescence signal a; The input signal is represented by the following formula:
[0032] in, , These are the amplitudes of fluorescence signal a and fluorescence signal b, respectively. , These are the phases of fluorescence signal a and fluorescence signal b, respectively. The reference signal As shown in the following formula:
[0033] in, It is the phase of the reference signal; The mixing signal is obtained by multiplying the input signal and the reference signal, and the mixing signal is shown in the following formula: .
[0034] In one embodiment, the in-phase component is the result of directly multiplying the input signal and the reference signal, and the in-phase component is shown in the following formula:
[0035] in, These are in-phase components; The quadrature component is obtained by multiplying the signal that is 90 degrees out of phase with the reference signal and the input signal. The signal that is 90 degrees out of phase with the reference signal is represented as follows: ,Right now ; The orthogonal components are shown in the following equation: .
[0036] In one embodiment, low-pass filtering refers to filtering out high-frequency components and retaining only the effective low-frequency signal, which is achieved by expanding using the trigonometric function multiplication formula. , The expansion is shown below:
[0037] The expanded high-frequency components include and Low-frequency components include and and
[0038] if , If the phase difference is large enough, the low-pass filter will filter out... Item, only retain item.
[0039] Therefore, the target in-phase component It is expressed as follows: .
[0040] Among them, the target in-phase component This refers to the in-phase component. The signal after low-pass filtering.
[0041] Similarly, the orthogonal components The signal after low-pass filtering is the target quadrature component. The target orthogonal component It is expressed as follows: .
[0042] In one embodiment, the amplitude of the output signal As shown in the following formula:
[0043] In one embodiment, the phase of the output signal As shown in the following formula: .
[0044] After the above-described lock-in demodulation process, the output signal of fluorescence signal a can be obtained. Similarly, by setting the reference frequency to be the same as that of fluorescence signal b, the output signal of fluorescence signal b can be obtained, thus realizing fluorescence signal separation.
[0045] In one embodiment, the mixed fluorescence signal includes a frequency of The fluorescence signal a and the frequency are The fluorescence signal b, the reference signal The step of obtaining the amplitude and phase information of the output signal based on the input signal, the reference signal, and the lock-in amplifier, having the same frequency as the fluorescence signal b, includes: By using a lock-in amplifier, a reference signal with the same frequency as the fluorescence signal b is utilized. The mixed fluorescence signals are separated to obtain the amplitude and phase of the output signal corresponding to fluorescence signal b.
[0046] As another example, to better understand the modulation-demodulation-based fluorescence signal separation method proposed in this invention, the method can be understood to include the following steps: Step A: Multiple pulsed lasers emit pulsed lasers, which are then modulated by a frequency modulation device to make the laser frequencies different from each other; Step B: Multiple pulsed lasers of known and different frequencies are simultaneously focused on the sample by an optical microscopy imaging system to generate fluorescence signals; Step C: The mixed fluorescence signals are collected by a detector and input as input signals to the input terminal of the demodulation device; Step D: The mixed fluorescence signals are effectively separated through the demodulation process and then stored and processed; Step E: Multi-target spatiotemporal synchronous detection is achieved.
[0047] In one embodiment, a computer device is provided, which may be a server. The computer device includes a processor, memory, a network interface, and a database connected via a system bus. The processor provides computing and control capabilities. The memory includes non-volatile and / or volatile storage media and internal memory. The non-volatile storage media stores an operating system, computer programs, and a database. The internal memory provides an environment for the operation of the operating system and computer programs in the non-volatile storage media. The network interface of the computer device is used to communicate with external clients via a network connection. When the computer program is executed by the processor, it implements the functions or steps of a modulation-demodulation-based fluorescence signal separation method on the server side.
[0048] In one embodiment, a computer device is provided, which can be a client. The computer device includes a processor, memory, a network interface, a display screen, and an input device connected via a system bus. The processor of the computer device provides computing and control capabilities. The memory of the computer device includes a non-volatile storage medium and internal memory. The non-volatile storage medium stores an operating system and computer programs. The internal memory provides an environment for the operation of the operating system and computer programs in the non-volatile storage medium. The network interface of the computer device is used to communicate with an external server via a network connection. When the computer program is executed by the processor, it implements the functions or steps of a modulation-demodulation-based fluorescence signal separation method on the client side.
[0049] In one embodiment, a computer device is provided, including a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein the processor, when executing the computer program, performs the following steps: Acquire mixed fluorescence signals, wherein the mixed fluorescence signals are generated by simultaneously focusing multiple pulsed laser beams of different frequencies onto the sample; Based on the demodulation device, the reference signal, and the mixed fluorescence signal, signal separation is performed on the mixed fluorescence signal.
[0050] This invention enables the separation of mixed fluorescence signals by using a demodulation device and a reference signal, thereby achieving multi-target spatiotemporal synchronous detection of fluorescence signals.
[0051] In one embodiment, a computer-readable storage medium is provided that stores a computer program, which, when executed by a processor, performs the following steps: Acquire mixed fluorescence signals, wherein the mixed fluorescence signals are generated by simultaneously focusing multiple pulsed laser beams of different frequencies onto the sample; Based on the demodulation device, the reference signal, and the mixed fluorescence signal, signal separation is performed on the mixed fluorescence signal.
[0052] This invention enables the separation of mixed fluorescence signals by using a demodulation device and a reference signal, thereby achieving multi-target spatiotemporal synchronous detection of fluorescence signals.
[0053] It should be noted that the functions or steps that can be implemented by the computer-readable storage medium or computer device described above can be referred to the relevant descriptions on the server side and client side in the foregoing method embodiments. To avoid repetition, they will not be described one by one here.
[0054] Those skilled in the art will understand that all or part of the processes in the methods of the above embodiments can be implemented by a computer program instructing related hardware. The computer program can be stored in a non-volatile computer-readable storage medium. When executed, the computer program can include the processes of the embodiments of the above methods. Any references to memory, storage, databases, or other media used in the embodiments provided in this application can include non-volatile and / or volatile memory. Non-volatile memory may include read-only memory (ROM), programmable ROM (PROM), electrically programmable ROM (EPROM), electrically erasable programmable ROM (EEPROM), or flash memory. Volatile memory may include random access memory (RAM) or external cache memory. By way of illustration and not limitation, RAM is available in a variety of forms, such as static RAM (SRAM), dynamic RAM (DRAM), synchronous DRAM (SDRAM), dual data rate SDRAM (DDRSDRAM), enhanced SDRAM (ESDRAM), synchronous link DRAM (SLDRAM), RAMbus direct RAM (RDRAM), direct memory bus dynamic RAM (DRDRAM), and memory bus dynamic RAM (RDRAM), etc.
[0055] Those skilled in the art will clearly understand that, for the sake of convenience and brevity, the above-described division of functional units and modules is used as an example. In practical applications, the above functions can be assigned to different functional units and modules as needed, that is, the internal structure of the device can be divided into different functional units or modules to complete all or part of the functions described above.
[0056] The above-described embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention, and should all be included within the protection scope of the present invention.
Claims
1. A modulation demodulation based fluorescence signal separation method, characterized in that, The modulation-based fluorescence signal separation method comprises: acquiring a mixed fluorescence signal, wherein the mixed fluorescence signal is generated by simultaneously focusing multiple beams of pulsed laser with different frequencies on a sample; performing signal separation on the mixed fluorescence signal based on a demodulation device, a reference signal, and the mixed fluorescence signal.
2. The modulation-based fluorescence signal separation method according to claim 1, wherein, The demodulation device is a lock-in, and the step of performing signal separation on the mixed fluorescence signal based on the demodulation device and the reference signal comprises: determining an input signal based on the mixed fluorescence signal; obtaining amplitude and phase information of an output signal based on the input signal, the reference signal, and the lock-in, wherein the lock-in is used to perform mixing processing on the input signal and the reference signal to obtain in-phase and quadrature components, and perform low-pass filtering processing on the in-phase and quadrature components to obtain target in-phase and quadrature components, so as to calculate amplitude and phase of the output signal based on the target in-phase and quadrature components.
3. The modulation-based fluorescence signal separation method according to claim 2, wherein, The mixed fluorescent signal comprises a fluorescent signal a with a frequency of and a fluorescent signal b with a frequency of The reference signal has the same frequency as the fluorescent signal a, and the step of obtaining the amplitude and phase information of the output signal based on the input signal, the reference signal and the lock-in amplifier lock-in comprises: by lock-in, using a reference signal of the same frequency as the fluorescent signal a separating the mixed fluorescent signal to obtain the amplitude and phase of the output signal corresponding to the fluorescent signal a; The input signal is represented by the following formula: wherein , are the amplitudes of the fluorescence signal a, fluorescence signal b, respectively, , are the phases of the fluorescence signal a, fluorescence signal b, respectively. The reference signal As represented by the following formula: wherein is the phase of the reference signal; The mixing signal is obtained by multiplying the input signal and the reference signal, and is represented by the following formula: 。 4. The modulation-based fluorescence signal separation method according to claim 3, wherein, The in-phase component is a result of directly multiplying the input signal and the reference signal, and is represented by the following formula: wherein is the in-phase component; The quadrature component is a result of multiplying a signal with a phase difference of 90 degrees from the reference signal and the input signal, and the signal with a phase difference of 90 degrees from the reference signal is represented by the following formula: i.e. ; The quadrature component is represented by the following formula: 。 5. The modulation-based fluorescence signal separation method according to claim 4, wherein, The in-phase component The signal after low-pass filtering is a target in-phase component The target in-phase component Is expressed as follows: 。 6. The modulation-based fluorescence signal separation method according to claim 5, wherein, The quadrature component The signal after low-pass filtering is a target quadrature component , the target quadrature component is expressed as follows: 。 7. The modulation-based fluorescence signal separation method of claim 5, wherein, amplitude of the output signal as shown by the following equation: phase of the output signal as shown by the following equation: 。 8. The modulation-based fluorescence signal separation method according to claim 3, wherein, The mixed fluorescent signal comprises a fluorescent signal a with a frequency of and a fluorescent signal b with a frequency of The reference signal has the same frequency as the fluorescent signal b, and the step of obtaining the amplitude and phase information of the output signal based on the input signal, the reference signal and the lock-in amplifier lock-in comprises: by lock-in, using a reference signal of the same frequency as the fluorescent signal b the mixed fluorescent signal is separated to obtain the amplitude and phase of the output signal corresponding to the fluorescent signal b.
9. A computer device comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, characterized in that, The processor implements the steps of the data processing method according to any one of claims 1 to 8 when executing the computer program.
10. A computer-readable storage medium storing a computer program, the computer program comprising instructions that, when executed by a computer, cause the computer to perform the method of any one of claims 1-9. The computer program implements the steps of the data processing method according to any one of claims 1 to 8 when executed by the processor.