Signal synchronization system of flow cytometer

Abstract

The present invention relates to the technical field of cell analysis, and discloses a flow cytometer signal synchronization system, which comprises: a liquid flow module, which is used for generating single to-be-detected sample liquid flow in uniform arrangement; the light source module is used for generating laser beams to enter the liquid flow module; the photoelectric conversion module is used for collecting a pulse light signal generated by the liquid flow module in response to the laser beam and converting the pulse light signal into a pulse analog signal; and the signal synchronization module is connected with the photoelectric conversion module and is used for generating an external trigger synchronization signal according to the magnitude of the pulse analog signal, so that a spectrograph performs exposure according to the external trigger synchronization signal, and a single to-be-detected sample liquid flow generates a fluorescence signal. According to the scheme, the signal analysis flexibility and the signal acquisition accuracy are improved.

Description

Technical Field

[0001] This invention relates to the field of cell analysis technology, and more specifically to a flow cytometer signal synchronization system. Background Technology

[0002] Flow cytometry, as a powerful single-cell analysis technique, enables rapid, multi-parameter quantitative analysis of various cellular parameters, playing a crucial role in biomedical research. Fluorescent probes, as important tools for labeling cells or biomolecules, are constantly increasing in variety and quantity. Novel fluorescent probes possess unique spectral characteristics, providing richer biological information. Traditional full-spectrum flow cytometers, due to their relatively fixed spectroscopic system design and fluorescence acquisition system, struggle to flexibly adapt to the diverse excitation and emission spectra of novel fluorescent probes, and also suffer from low resolution.

[0003] In related technologies, a spectrometer is used as the fluorescence detection system in flow cytometry. The spectrometer can perform high-resolution continuous spectral detection of fluorescence signals, transforming the discontinuous fluorescence acquired by traditional full-spectrum flow cytometry into a continuous fluorescence spectrum. However, during flow cytometry operation, cells pass through the detection area at high speed, requiring the fluorescence detection system to quickly and accurately capture the fluorescence signal of each cell. Ensuring precise time synchronization between the spectrometer and other parts of the flow cytometer (such as the cell sample introduction system and laser excitation system) has become a pressing problem to be solved. Summary of the Invention

[0004] This invention provides a flow cytometer signal synchronization system to solve the signal synchronization problem between a spectrometer and a flow cytometer.

[0005] This invention provides a flow cytometer signal synchronization system, comprising: The flow module is used to generate a uniformly distributed flow of liquid from a single sample to be tested. A light source module is used to generate a laser beam that is incident on the liquid flow module; A photoelectric conversion module is used to collect the pulsed light signal generated by the fluid flow module in response to the laser beam, and convert the pulsed light signal into a pulsed analog signal; The signal synchronization module, connected to the photoelectric conversion module, is used to generate an external trigger synchronization signal based on the magnitude of the pulsed analog signal, so that the spectrometer can perform exposure based on the external trigger synchronization signal, thereby causing the single sample liquid flow to generate a fluorescence signal.

[0006] In one optional implementation, the signal synchronization module includes a programmable logic device; the programmable logic device is used to sequentially filter, shape, and level-convert the pulse analog signal to generate the external trigger synchronization signal.

[0007] In one optional implementation, the signal synchronization module includes a voltage comparator; the voltage comparator is used to compare a pulsed analog signal with a reference voltage and generate the external trigger synchronization signal based on the comparison result.

[0008] In an optional implementation, the voltage comparator is further configured to output a high level when the amplitude of the pulse analog signal is less than the reference voltage, and otherwise output a low level, so as to convert the pulse analog signal into a square wave signal, and use the rising edge of the square wave signal as the start signal of the external trigger synchronization signal.

[0009] In one optional embodiment, the flow cytometer signal synchronization system further includes a laser beam-shrinking module; the laser beam-shrinking module is disposed between the light source module and the fluid flow module, and the laser beam-shrinking module is used to shrink the laser beam to a size scale corresponding to a single sample to be tested.

[0010] In one alternative embodiment, the laser beam-shrinking module includes beam expanders arranged in sequence and a first microscope objective.

[0011] In one optional embodiment, the flow cytometer signal synchronization system further includes a fluorescence detection module; the fluorescence detection module is connected to the signal synchronization module; the fluorescence detection module is also connected to the spectrometer; the fluorescence detection module is used to receive the external trigger synchronization signal and control the spectrometer to enter the exposure state according to the external trigger synchronization signal; the fluorescence detection module is also used to collect the fluorescence signal.

[0012] In one optional embodiment, the flow cytometer signal synchronization system further includes a second microscope objective and a third microscope objective disposed between the flow module and the fluorescence detection module; the second microscope objective and the third microscope objective are coaxial and arranged in opposite directions.

[0013] In one optional implementation, the flow cytometer signal synchronization system further includes a host computer; the host computer is connected to the flow module and is used to control the flow rate of the individual sample to be tested; the host computer is also connected to the signal synchronization module and is used to send adjustment commands to the signal synchronization module according to the flow rate of the individual sample to be tested, so as to calibrate the generation time of the external trigger synchronization signal.

[0014] In one optional implementation, the host computer is also connected to the fluorescence detection module to receive the fluorescence signal and analyze the liquid flow of the single sample to be tested based on the fluorescence signal.

[0015] The flow cytometer signal synchronization system provided by this invention has the following beneficial effects: The flow cytometer signal synchronization system provided by this invention can directly obtain complete spectral information of fluorescence signals through a spectrometer, overcoming the problem of poor compatibility of flow cytometers with fluorescent probes in related technologies and improving the flexibility of signal analysis. It provides a uniformly distributed liquid flow of a single sample through a liquid flow module, a continuous and stable laser beam through a light source module, and an external trigger synchronization signal for the spectrometer through a photoelectric conversion module and a signal synchronization module. This ensures strict synchronization between the exposure process and the time window of the sample passing through the laser spot area, thereby achieving accurate acquisition of fluorescence signals and guaranteeing the subsequent acquisition of high-quality spectral data based on the fluorescence signals. Attached Figure Description

[0016] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0017] Figure 1 This is a schematic diagram of the structure of a flow cytometer signal synchronization system according to an embodiment of the present invention; Figure 2 This is a schematic diagram of another flow cytometer signal synchronization system according to an embodiment of the present invention; Figure 3 This is a schematic diagram of the pulse conversion signal according to an embodiment of the present invention; Figure 4 This is a schematic diagram of another pulse conversion signal according to an embodiment of the present invention; Figure 5 This is a schematic diagram of the signal flow corresponding to the programmable logic device in an embodiment of the present invention; Figure 6 This is a schematic diagram of signal synchronization corresponding to the programmable logic device in an embodiment of the present invention; Figure 7 This is a schematic diagram of the signal flow corresponding to the voltage comparator in an embodiment of the present invention; Figure 8 This is a schematic diagram of signal synchronization corresponding to the voltage comparator in an embodiment of the present invention.

[0018] Explanation of reference numerals in the attached figures: 1. Light source module; 2. Beam expander; 3. First microscope objective; 4. Fluid flow module; 5. Second microscope objective; 6. Third microscope objective; 7. Photoelectric conversion module; 8. Signal synchronization module; 9. Fluorescence detection module; 10. Host computer. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, 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 embodiments of the present invention, not all embodiments. 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.

[0020] In the description of this invention, it should be noted that the terms "center," "upper," "lower," "left," "right," "vertical," "horizontal," "inner," and "outer," etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are used only for the convenience of describing the invention and for simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on the invention. Furthermore, the terms "first," "second," and "third" are used for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0021] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "linking" should be interpreted broadly. For example, they can refer to a fixed connection, a detachable connection, or an integral connection; they can refer to a mechanical connection or an electrical connection; they can refer to a direct connection or an indirect connection through an intermediate medium; and they can refer to the internal connection of two components. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0022] Furthermore, the technical features involved in the different embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0023] In the biomedical field, with the deepening research into the molecular mechanisms of life processes, the demand for precise analysis of cells and biomolecules is increasing. Flow cytometry, as a powerful single-cell analysis technique, can perform rapid, multi-parameter quantitative analysis of various cellular parameters, playing a crucial role in many biomedical research areas such as immunology, oncology, and hematology. In this process, fluorescent probes, as important tools for labeling cells or biomolecules, are constantly increasing in type and quantity. Novel fluorescent probes possess unique spectral characteristics, providing richer biological information to explore complex biological processes such as cell function, phenotype, and cell-cell interactions. Traditional full-spectrum flow cytometers, due to their relatively fixed spectroscopic system design and fluorescence acquisition system, struggle to flexibly adapt to the diverse excitation and emission spectra of novel fluorescent probes. When the spectral characteristics of a novel fluorescent probe do not match the preset detection band of a traditional flow cytometer, signal detection efficiency significantly decreases, or even becomes ineffective. Furthermore, since the excitation and emission spectra of fluorescent dyes often have a certain width, when the excitation bands of two dyes are close together, their emission spectra easily overlap. In the traditional flow cytometry process, this spectral overlap can cause different fluorescence signals to interfere with each other, making it difficult for the detection system to accurately distinguish and differentiate signals from different fluorescent dyes. This results in poor spectral resolution of traditional full-spectrum flow cytometers, thus affecting the accuracy and reliability of experimental results.

[0024] In related technologies, a spectrometer is used as the fluorescence detection system in flow cytometry. The spectrometer enables high-resolution continuous spectral detection of fluorescence signals, transforming the discontinuous fluorescence acquired by traditional full-spectrum flow cytometry into a continuous fluorescence spectrum. This significantly improves spectral resolution, allowing for clearer differentiation of dye signals with similar excitation bands, reducing signal overlap interference, and demonstrating good compatibility with novel fluorescent dyes. The spectrometer's continuous detection capability enables the complete acquisition and analysis of fluorescence signals from various novel fluorescent probes, providing strong support for the widespread application of novel fluorescent probes in biomedical research. Furthermore, compared to the complex filter system of traditional flow cytometers, the application of a spectrometer simplifies the instrument's optical structure, reduces structural complexity, and lowers instrument costs and maintenance difficulty.

[0025] However, when applying spectrometers to flow cytometry fluorescence detection systems, signal synchronization becomes a critical challenge that urgently needs to be addressed. During flow cytometry operation, cells pass through the detection area at high speed, requiring the fluorescence detection system to quickly and accurately capture the fluorescence signal of each cell. The working principle and data acquisition method of spectrometers differ from those of traditional flow cytometry. Ensuring precise time synchronization between the spectrometer and other parts of the flow cytometer (such as the cell sample introduction system and laser excitation system) to guarantee accurate and timely acquisition and recording of the fluorescence signal of each cell is a pressing issue. Failure to effectively solve the signal synchronization problem will lead to data loss, signal mismatch, and other issues, severely impacting the accuracy and completeness of the detection results.

[0026] According to an embodiment of the present invention, in one aspect, a flow cytometer signal synchronization system is provided. Figure 1 This is a schematic diagram of the structure of a flow cytometer signal synchronization system according to an embodiment of the present invention, as shown below. Figure 1 As shown, the flow cytometer signal synchronization system includes a fluid flow module, a light source module, a photoelectric conversion module, and a signal synchronization module.

[0027] This flow module is used to generate a uniformly distributed flow of liquid from a single sample to be tested.

[0028] This light source module is used to generate a laser beam that is incident on the liquid flow module.

[0029] The photoelectric conversion module is used to collect the pulsed light signal generated by the liquid flow module in response to the laser beam, and convert the pulsed light signal into a pulsed analog signal.

[0030] The signal synchronization module is connected to the photoelectric conversion module. The signal synchronization module is used to generate an external trigger synchronization signal based on the magnitude of the pulse analog signal, so that the spectrometer can perform exposure based on the external trigger synchronization signal, thereby causing the single sample liquid flow to generate a fluorescence signal.

[0031] Figure 1 The working principle of the flow cytometer signal synchronization system shown is as follows: This fluid flow module, based on fluid dynamics, primarily utilizes sheath flow focusing technology to generate single sample droplets. During operation, the module organizes randomly dispersed individual test samples (such as cells or microparticles) into a spatially ordered particle stream through specific hydrodynamic forces—a uniformly distributed stream of individual test samples. Once formed, each particle sequentially passes through a laser beam emitted by the light source module. The particles then block the laser beam, generating a pulsed light signal. This fluid flow module organizes cells and converts them into detectable light signals, providing the foundation for subsequent photoelectric conversion based on these pulsed light signals and is crucial for signal synchronization.

[0032] In one embodiment, the flow module includes a flow system conduit for containing the flow of the single sample to be tested.

[0033] This light source module provides a stable optical signal source for the system. Its core principle revolves around the emission of continuous laser light, the generation of pulsed light signals, and photoelectric conversion. The module can use a continuous laser as the core light source, outputting continuous and stable energy to provide constant laser irradiation for the entire optical detection process. When the continuous laser beam is focused on the fluid flow module, a single-column stream of particles, ordered by the fluid flow module, sequentially passes through the laser irradiation area. Each particle passing through blocks the laser beam, causing periodic interruptions to the originally continuous laser irradiation, thus forming a pulsed light signal synchronized with the particle flow. The generated pulsed light signal is then transmitted to the photoelectric conversion module, which uses photoelectric conversion characteristics to accurately convert the pulsed light signal into a corresponding electrical signal (pulse analog signal) to generate an external trigger synchronization signal.

[0034] During flow cytometry, based on the principle of optical scattering, a laser beam is focused on the detection area. As individual test samples encased in sheath fluid pass through sequentially, the samples scatter and block the laser light, causing changes in light intensity distribution and generating uniformly spaced pulsed light signals. This photoelectric conversion module uses an internal photomultiplier tube (PMT) or avalanche photodiode (APD) as a detector to convert the received pulsed light signal into a pulsed analog signal using the photoelectric effect. This pulsed analog signal carries the timestamp information of a single test sample passing through the detection area. The amplitude of this pulsed analog signal is directly related to the degree of laser blockage by the test sample; the larger the sample volume, the greater the blockage, the stronger the scattered light intensity, and the larger the amplitude of the converted pulsed analog signal. The waveform characteristics of this pulsed analog signal are modulated by factors such as cell morphology and surface structure. Irregular test samples can complicate laser blockage, causing corresponding fluctuations in the pulsed analog signal waveform. The pulsed analog signal output from this photoelectric conversion module is input to the back-end signal synchronization module.

[0035] This signal synchronization module accurately converts the pulsed analog signal output from the photoelectric conversion module into a control signal adapted to the external triggering requirements of the spectrometer, i.e., an external trigger synchronization signal. The performance of this signal synchronization module directly affects the accuracy and timeliness of spectral data acquisition. The signal synchronization module includes two implementation schemes: one based on programmable logic devices and the other based on voltage comparators. These two schemes achieve signal conversion and trigger control through different technical approaches.

[0036] In one embodiment, the signal synchronization module includes a programmable logic device; the programmable logic device is used to sequentially filter, shape, and level-convert the pulse analog signal to generate the external trigger synchronization signal.

[0037] In one embodiment, the signal synchronization module further includes a data acquisition card for converting the pulse analog signal into a pulse digital signal and sending the pulse digital signal to the programmable logic device. The programmable logic device filters the received pulse digital signal and performs edge detection to obtain the edge signal of the pulse digital signal and outputs an external trigger synchronization signal. The data acquisition card can be an analog-to-digital converter.

[0038] This signal synchronization module, incorporating programmable logic devices, utilizes phase-locked loop (PLL) synchronization technology and the signal processing capabilities of programmable logic devices (such as FPGAs) to filter, shape, and level-convert pulsed analog signals. It converts these signals into TTL (Transistor-Transistor Logic) level signals that meet the external trigger control requirements of the spectrometer, serving as the external trigger synchronization signal. This external trigger synchronization signal acts as the spectrometer's exposure start trigger signal, precisely controlling the exposure sequence and ensuring accurate capture of the fluorescence signal generated by laser irradiation of the sample. Combined with the spectral characteristics of the fluorescent marker, it enables qualitative component analysis and quantitative concentration determination of the sample. Specifically, the FPGA-based signal synchronization module's core advantages lie in its high-speed parallel processing and flexible programming capabilities. The module first uses a high-precision analog-to-digital converter (ADC) to sample and quantize the pulsed analog signal output from the photoelectric conversion module, converting the continuous analog signal into a discrete digital signal sequence (pulse digital signal). This process involves the rigorous application of the Nyquist sampling theorem, ensuring that the sampling frequency is at least twice the highest frequency of the pulsed analog signal to avoid spectral aliasing. The converted pulse digital signal is input into the FPGA. Leveraging the FPGA's abundant logic resources and high-speed clock management unit, an edge detection algorithm precisely captures the falling edge of the pulse digital signal. The falling edge of the pulse digital signal corresponds to the critical time point information of the sample entering the laser detection area. Through a preset timing logic circuit, the FPGA converts this time point information into an external trigger synchronization signal that conforms to the spectrometer's external trigger control protocol (such as TTL level standards, pulse width requirements, etc.). The FPGA's programmability allows for flexible adjustment of parameters such as trigger delay and pulse width to adapt to the triggering requirements of different spectrometer models. Furthermore, the FPGA's parallel processing capabilities enable synchronous processing and trigger control of multi-channel signals.

[0039] In one embodiment, the signal synchronization module includes a voltage comparator; the voltage comparator is used to compare a pulsed analog signal with a reference voltage and generate the external trigger synchronization signal based on the comparison result. The signal synchronization module can also use a voltage comparator to compare the pulsed analog signal as input with a reference voltage and convert the pulsed analog signal into a TTL level signal that meets the external trigger control requirements of the spectrometer.

[0040] In one embodiment, the voltage comparator is further configured to output a high level when the amplitude of the pulse analog signal is less than the reference voltage, and output a low level otherwise, so as to convert the pulse analog signal into a square wave signal, and use the rising edge of the square wave signal as the start signal of the external trigger synchronization signal.

[0041] This signal synchronization module, which includes a voltage comparator, achieves signal conversion based on the fast response characteristics of analog circuits. The module uses the pulsed analog signal output from the photoelectric conversion module as one input to the comparator, while simultaneously introducing an adjustable reference voltage as the other input. According to the basic working principle of a voltage comparator, when the amplitude of the pulsed analog signal is lower than the reference voltage, the comparator quickly outputs a high-level signal; conversely, it outputs a low-level signal. By appropriately setting the reference voltage value, the pulsed analog signal generated by the sample passing through the detection area can be converted into a square wave signal with steep edges. The rising edge of this square wave signal can then serve as the start signal for external trigger control of the spectrometer.

[0042] In one embodiment, the flow cytometer signal synchronization system further includes a laser beam-shrinking module. This module is positioned between the light source module and the flow module, and is used to shrink the laser beam to a size scale corresponding to a single sample. The laser beam-shrinking module is a key adjustment unit in the optical path. Its core function is to shrink the spot size of the original laser beam output from the light source module, ensuring that the laser irradiation range matches the scale of a single sample. Specifically, the laser beam-shrinking module includes a beam-shrinking system composed of precision lenses. Utilizing the principles of beam focusing and shrinking in geometric optics, it shrinks the divergence or initial spot size of the laser beam output from the light source module. After the original laser beam is shrunken by the lens group, the spot diameter is precisely reduced to an order of magnitude comparable to the size of the sample (typically on the micrometer scale), forming a highly focused micro-spot. When this highly focused micro-spot acts on the sample channel, its spatial coverage is strictly limited. At any given time, only a single sample arranged in an orderly fashion by the flow module can be irradiated, while adjacent samples are outside the spot and not irradiated by the laser. When a single test sample passes through the light spot area, the light signal generated by blocking or scattering the laser corresponds only to that single test sample. This avoids signal superposition or mixing caused by adjacent test samples being simultaneously irradiated by the laser, providing a spatial basis for the subsequent photoelectric conversion and signal synchronization module to acquire the pulse analog signal and external trigger synchronization signal corresponding to a single test sample.

[0043] In one embodiment, the laser beam-shrinking module includes a beam expander and a first microscope objective arranged in sequence. The beam expander and the first microscope objective work together to form a laser beam-shrinking system, which compresses the laser spot to a specific size required by the system through coordinated control of the optical path.

[0044] In one embodiment, the flow cytometer signal synchronization system further includes a fluorescence detection module; the fluorescence detection module is connected to the signal synchronization module; the fluorescence detection module is also connected to the spectrometer; the fluorescence detection module is used to receive the external trigger synchronization signal and control the spectrometer to enter the exposure state according to the external trigger synchronization signal; the fluorescence detection module is also used to acquire the fluorescence signal. The fluorescence detection module is the core unit in the flow cytometer for realizing fluorescence signal capture and conversion. The working mechanism of the fluorescence detection module closely depends on the linkage with the signal synchronization module, and the specific process revolves around receiving the external trigger synchronization signal, detection triggering, and fluorescence signal acquisition. After the signal synchronization module completes the timing calibration of the flow of a single sample and the light source module, it sends a precise external trigger synchronization signal to the fluorescence detection module. This external trigger synchronization signal serves as a start command, directly controlling the exposure timing of the spectrometer. The fluorescence detection module only responds when the signal synchronization module generates the external trigger synchronization signal, driving the spectrometer to enter the exposure state. At this point, the specific fluorescence generated by the laser excitation of the sample (such as the fluorescence emitted by fluorescently labeled cellular components) is focused onto the photosensitive surface of the spectrometer through an optical collection system (such as lenses and filters). The spectrometer utilizes its highly sensitive photoelectric detection capability to convert the fluorescence signal into a corresponding digital image signal or intensity data, completing real-time acquisition and temporary storage. Throughout the process, the fluorescence detection module remains in a passively controlled state. The activation of the fluorescence detection module's detection action and the setting of the exposure time are strictly regulated by the signal synchronization module, ensuring that the acquisition of the fluorescence signal and the time window of the sample passing through the laser irradiation area completely coincide, achieving end-to-end timing matching of "particle-excitation-fluorescence-acquisition". It should be noted that the spectrometer can be housed within the fluorescence detection module; that is, the fluorescence detection module can include the spectrometer.

[0045] In one embodiment, the flow cytometer signal synchronization system further includes a second and a third microscope objective disposed between the flow module and the fluorescence detection module; the second and third microscope objectives are coaxial and arranged in opposite directions. The second microscope objective can collect fluorescence signals diverging throughout the space at a wide angle and convert them into parallel light output, ensuring a perfect match with the entrance aperture of the third microscope objective; the third microscope objective further focuses the parallel light into a point light source, enabling the point light source to precisely match the entrance slit of the fluorescence detection module. The dual-objective collaborative design formed by the second and third microscope objectives significantly improves the capture efficiency of weak fluorescence signals, providing a guarantee for subsequent high-sensitivity detection.

[0046] In one embodiment, the flow cytometer signal synchronization system further includes a host computer; the host computer is connected to the flow module and is used to control the flow rate of the single sample to be tested; the host computer is also connected to the signal synchronization module and is used to send adjustment instructions to the signal synchronization module according to the flow rate of the single sample to be tested, so as to calibrate the generation time of the external trigger synchronization signal.

[0047] As the core control and data processing hub of the flow cytometer signal synchronization system, the host computer operates within a closed-loop process of "system regulation - signal coordination - data processing," achieving centralized management of multiple modules through hardware and software collaboration. Specifically, at the system control level, the host computer first precisely regulates the flow rate of the fluidization module through preset parameters or real-time commands. Based on experimental requirements (such as sample type and detection accuracy), it sends control signals to the fluidization drive devices (such as peristaltic pumps and pressure controllers) within the fluidization module, dynamically adjusting the movement speed of individual test samples within the fluidization system's pipeline pathway. This ensures that each test sample passes through the laser irradiation area in a stable sequence that meets detection requirements. For the signal synchronization module, the host computer plays a dynamic adaptation role, acquiring the flow rate data of the fluidization module in real time. It calculates the time interval between the test samples passing through the laser detection area using a built-in algorithm and sends adjustment commands to the signal synchronization module accordingly, precisely calibrating the generation time of the external trigger synchronization signal. For example, when the flow rate increases, the sample passes through faster, and the host computer will shorten the interval of the external trigger synchronization signal to ensure that actions such as laser irradiation and fluorescence detection are synchronized with the movement of the sample, thus avoiding signal misalignment caused by flow rate fluctuations.

[0048] In one embodiment, the host computer is also connected to the fluorescence detection module to receive the fluorescence signal and analyze the flow of a single sample based on the fluorescence signal. In the data processing stage, the host computer receives raw fluorescence signal data from the spectrometer via a data interface. After preprocessing such as noise reduction, filtering, and feature extraction, it combines the time-series information of the sample to complete data association, binding each fluorescence signal to the corresponding sample attribute (such as transit time and flow rate parameters). This ultimately generates structured data that can be used for analysis (such as fluorescence intensity distribution maps and particle count statistics), while also supporting data storage, visualization, and export.

[0049] In one embodiment, the light source module includes multiple lasers; wherein different lasers correspond to different laser bands, laser powers, and operating times.

[0050] It should be noted that, on the one hand, this invention improves the detector used in spectral data acquisition scenarios. Traditional flow cytometers generally use point detectors to receive fluorescence signals, while more advanced full-spectrum flow cytometers are upgraded to a 32-channel photomultiplier tube (PMT) array receiving architecture. These detectors have rise times in nanoseconds (ns), offering not only fast detection speeds but also excellent sensitivity to weak light signals, enabling efficient capture of low-intensity fluorescence signals. However, this technology still has significant shortcomings, such as poor compatibility with fluorescent probes, limitations in the spectral response range of fixed channels, difficulty in adapting to diverse fluorescent labeling needs, and inability to flexibly handle multi-target labeling scenarios for complex samples. The flow cytometer signal synchronization system proposed in this invention directly obtains the complete spectral information of the fluorescence signal through a spectrometer, breaking through the inherent limitations of traditional detectors in terms of technical principles and providing a new approach to solving the fluorescent probe compatibility problem and improving signal resolution flexibility. On the other hand, this invention proposes a corresponding signal synchronization system based on the improved detector. Spectrometers typically use linear CCD or linear CMOS arrays as their photoelectric detection cores. The inherent photoelectric characteristics of these devices dictate that a certain duration of photoelectric accumulation of the fluorescence signal is required to ensure effective detection and output of high-quality spectral data. This characteristic places stringent demands on timing accuracy; the exposure start-up time of the linear array detector must be strictly aligned with the moment the fluorescence signal generated by the analyte enters the spectrometer's field of view. Otherwise, problems such as missed signal detection and incomplete acquisition can easily occur, directly affecting detection accuracy. To solve this timing synchronization problem and ensure that the fluorescence signal can be completely captured by the linear array detector, this invention specifically proposes the aforementioned flow cytometer signal synchronization system.

[0051] The aforementioned flow cytometer signal synchronization system can directly obtain complete spectral information of fluorescence signals through the spectrometer, overcoming the problem of poor compatibility of flow cytometers with fluorescent probes in related technologies and improving the flexibility of signal analysis. It provides a uniformly distributed liquid flow of a single sample to be tested through the liquid flow module, a continuous and stable laser beam through the light source module, and an external trigger synchronization signal for the spectrometer through the photoelectric conversion module and the signal synchronization module. This ensures that the exposure process and the time window of the sample passing through the laser spot area are strictly synchronized, thereby achieving accurate acquisition of the sample fluorescence signal and ensuring that high-quality spectral data can be obtained from the fluorescence signal in the future.

[0052] According to an embodiment of the present invention, another aspect provides a flow cytometer signal synchronization system. Figure 2 This is a schematic diagram of another flow cytometer signal synchronization system according to an embodiment of the present invention, as shown below. Figure 2As shown, the system includes a light source module 1, a beam expander 2, a first microscope objective 3, a fluid flow module 4, a second microscope objective 5, a third microscope objective 6, a photoelectric conversion module 7, a signal synchronization module 8, a fluorescence detection module 9, and a host computer 10.

[0053] The core function of the first microscope objective 3 is to participate in the precise beam reduction of the laser spot. Since the samples to be tested are at the cellular level (typically 10µm-100µm in size), while the original laser spot size is usually in the range of hundreds of micrometers to millimeters, much larger than the sample size, directly using the original spot would easily lead to multiple samples being irradiated simultaneously, causing signal interference and making accurate detection of a single sample impossible. Therefore, the first microscope objective 3 and the beam expander 2 work together to form a laser beam reduction system. Through coordinated control of the optical path, the laser spot is compressed to the specific size required by the system (typically an elliptical spot of 8µm × 80µm), ultimately ensuring that the laser is focused on only a single sample at any given time, laying the foundation for accurate signal acquisition.

[0054] The core function of the second and third microscope objectives 5 and 6 is to synergistically improve the collection efficiency of fluorescence signals. When a laser irradiates a sample, it emits a fluorescence signal, but this signal is usually weak and disperses 360° into space as a point source. Traditional flow cytometers use a single lens to collect fluorescence, which is limited by the optical angle range and has low collection efficiency. This embodiment optimizes this process by combining two objectives. The second microscope objective 5 first collects the fluorescence signal dispersed throughout the space at a wide angle and converts it into parallel light output, ensuring a perfect match with the entrance aperture of the third microscope objective 6. The third microscope objective 6 then further focuses the parallel light into a point source, precisely matching the entrance slit of the fluorescence detection module 9. This synergistic design of the two objectives significantly improves the capture efficiency of weak fluorescence signals, providing a guarantee for subsequent high-sensitivity detection.

[0055] It should be noted that the incident end of the fluorescence detection module 9 is typically equipped with a slit structure, with a typical slit size of approximately 60µm. This slit is a key adapting component for the fluorescence signal to enter the detection system, directly determining the range and accuracy of signal acquisition. The fluorescence signal, previously collected and converted into parallel light by the second microscope objective 5, needs to be further focused by the third microscope objective 6. The third microscope objective 6 precisely compresses the parallel light into a spot size that matches the 60µm slit size, ensuring that the fluorescence signal can enter the downstream fluorescence detection module 9 efficiently and completely, avoiding signal loss or stray light interference caused by spot size mismatch, and laying the foundation for the accuracy of subsequent spectral detection.

[0056] Figure 3This is a schematic diagram of the pulse conversion signal according to an embodiment of the present invention. The continuous laser emitted by the light source module 1 has a stable output power and a continuous energy supply. The laser beam accurately irradiates the liquid flow system pipeline in the liquid flow module 4. When there is no sample flow in the liquid flow system pipeline, the continuous laser can be directly projected onto the photosensitive area of ​​the photoelectric conversion module 7. At this time, the photoelectric conversion module 7 is continuously excited by the laser, and the output voltage is maintained at a high level (this is a typical output characteristic under laser irradiation). In the initial state without laser irradiation, the photoelectric conversion module 7 outputs a low level. This level change pattern is similar to... Figure 3 The waveforms shown correspond perfectly.

[0057] Figure 4 This is a schematic diagram of another pulse conversion signal according to an embodiment of the present invention. When a sample to be tested flows through the pipeline of the liquid flow system, the irradiation state of the laser will dynamically change with the orderly movement of the sample to be tested in the liquid flow. Specifically, when a single sample to be tested passes through the laser spot area, the sample to be tested will physically block the laser beam, causing the laser energy projected onto the photoelectric conversion module 7 to weaken instantaneously, and the output voltage of the photoelectric conversion module 7 will drop rapidly to a low level; while when the gap between two adjacent samples to be tested passes through the laser spot, the laser beam can directly irradiate the photoelectric conversion module 7 without obstruction, and the output voltage of the photoelectric conversion module 7 will quickly recover to a high level. This cycle repeats, and the photoelectric conversion module 7 will output a pulse analog signal that is consistent with the rhythm of the movement of the sample to be tested. The waveform of this pulse analog signal is as follows: Figure 4 As shown. The level signal output from the photoelectric conversion module 7 is used as an input to the signal synchronization module 8 for signal synchronization conversion.

[0058] When an FPGA is used as the core processing element of the signal synchronization module 8, a predictive delay synchronization method is adopted to achieve high-precision signal synchronization, which is suitable for scenarios where the fluorescence signal is weak and all fluorescence signals need to be collected. Figure 5 This is a schematic diagram of the signal flow corresponding to the programmable logic device in an embodiment of the present invention. The specific processing flow of the signal synchronization module 8 is as follows: Figure 5As shown, the pulse analog signal first undergoes a conversion from the analog domain to the digital domain via an analog-to-digital converter (ADC), transforming it into a pulse digital signal which is then input to the FPGA core processing unit. This step is fundamental to digital signal processing, providing operable digital quantities for subsequent FPGA logic operations and signal analysis. After receiving the pulse digital signal, the FPGA first filters out high-frequency noise and spurious interference signals using a preset digital filtering algorithm to purify the signal and prevent misjudgments in subsequent edge detection due to noise. This ensures the accuracy of the external trigger synchronization signal generation and lays a reliable signal foundation for the entire synchronization process. The filtered pulse digital signal then enters the edge detection stage. The FPGA employs a logic processing method combining "sampling," "registration," and comparison. "Sampling" involves delaying and sampling the signal through multiple registers to obtain the signal state at different times. "Registration" temporarily stores these sampled states in registers. Subsequently, by comparing the registered signals at different times, the edge information of the pulse digital signal is accurately extracted, achieving effective detection of the rising and falling edges of the pulse digital signal.

[0059] When the falling edge of the pulsed digital signal is detected, this moment precisely corresponds to the time when the sample under test enters the laser spot blocking area. Using this as a trigger condition, the FPGA outputs an external trigger synchronization signal through its internal logic circuitry. This external trigger synchronization signal is directly used to control the exposure operation of detection equipment such as the spectrometer, ensuring that the exposure process is strictly synchronized with the time window when the sample under test passes through the laser spot area, thereby achieving accurate acquisition of the sample's fluorescence signal.

[0060] The FPGA-based signal synchronization module 8 employs a predictive delay synchronization method to achieve high-precision timing alignment of signals. The process of generating the external trigger synchronization signal by this module 8 is highly dependent on the dynamic stability of the fluid flow system within the fluid flow module; therefore, a closed-loop control mechanism needs to be constructed via a host computer 10. The host computer 10 collects real-time flow velocity feedback data from the fluid flow system, processes it using an algorithm, and outputs adjustment commands to dynamically correct the fluid flow drive parameters to maintain flow velocity stability. Simultaneously, the host computer 10 transmits the real-time flow velocity information to the signal synchronization module 8 in digital signal form, providing the basic parameters for timing calculations on the FPGA.

[0061] Figure 6 This is a schematic diagram of signal synchronization corresponding to the programmable logic device in an embodiment of the present invention. When the fluid flow system enters a stable operating state, the FPGA's signal synchronization strategy based on predicted delay is as follows: Figure 6As shown, the signal synchronization strategy executes the following core logic: After detecting the pulsed simulated signal generated by the laser being blocked by the current sample under test, it combines the flow velocity parameters transmitted by the host computer 10 with the average spacing characteristics of the samples under test in the liquid flow, and calculates the precise time when the next sample under test will arrive at the laser spot area through a built-in time prediction algorithm; then, it outputs an external trigger synchronization signal in advance within a preset time window, driving the spectrometer to enter the exposure preparation state. This advance triggering mechanism ensures that when the sample under test passes through the laser irradiation area and generates a fluorescence signal, the spectrometer is already in a stable exposure state, thereby achieving full-cycle acquisition of the fluorescence signal and avoiding signal truncation due to trigger delay.

[0062] When a voltage comparator is used as the core device of the signal synchronization module 8, a delay synchronization method is adopted to achieve high-precision signal synchronization, which is suitable for detection scenarios with strong fluorescence signals. In such scenarios, it is not necessary to fully acquire the fluorescence signal to meet the detection requirements. Figure 7 This is a schematic diagram of the signal flow corresponding to the voltage comparator in an embodiment of the present invention. The specific processing flow is as follows: Figure 7 As shown, the pulsed analog signal does not require an additional analog-to-digital conversion stage and can be directly input to the voltage comparator. The core function of the voltage comparator is to compare the input pulsed analog signal with a pre-set voltage threshold (reference voltage) in real time. When the voltage value of the pulsed analog signal is lower than the voltage threshold, this state corresponds to the sample under test entering the obstruction area of ​​the laser spot. At this time, the voltage comparator detects the threshold crossover point and outputs an external trigger synchronization signal in a timely manner. This external trigger synchronization signal is directly used to control the detection equipment such as the spectrometer to perform the exposure action, thereby realizing the timely acquisition of fluorescence signals. The signal synchronization module 8 based on the voltage comparator has a simple signal processing link, which can quickly respond to changes in the pulsed analog signal. In scenarios with strong fluorescence signals, it can efficiently complete signal synchronization and exposure control to meet specific detection requirements.

[0063] The signal synchronization module 8, with a voltage comparator as its core, achieves high-precision time alignment of signals through a delay synchronization mechanism. It has low requirements for the stability of the fluid flow system and can maintain synchronization accuracy without strict flow rate control. Figure 8 This is a schematic diagram of signal synchronization corresponding to the voltage comparator in an embodiment of the present invention. The signal synchronization strategy adopted by the signal synchronization module 8, which is based on the voltage comparator, is as follows: Figure 8As shown, the timing link contains two key delay levels: Firstly, the time difference between line 1 and line 2 corresponds to the signal rise time delay of the photoelectric conversion module 7, i.e., the time required for the electrical signal to rise from the baseline to a stable level during the conversion of the pulsed light signal into an electrical signal (pulse analog signal). Secondly, the time difference between line 2 and line 3 is the spectrometer's trigger response delay, which refers to the time interval from when the spectrometer receives the external trigger synchronization signal, completes the internal circuit startup and optical path preparation, to when the exposure officially begins. By presetting the above two-level delay parameters, the signal synchronization module 8 can achieve timing compensation. When the system completes the delay adjustment and the spectrometer starts exposure, the sample to be tested has accurately entered the spectrometer's field of view. This timing design ensures spatial matching between the sample to be tested and the exposure window even with slight fluctuations in the liquid flow, making it particularly suitable for scenarios with strong fluorescence signals. In such scenarios, strong fluorescence signals have a higher signal-to-noise ratio redundancy, and the detection requirements can be met without completely covering the entire signal cycle, further reducing the extreme requirements for system synchronization accuracy.

[0064] Although embodiments of the invention have been described in conjunction with the accompanying drawings, those skilled in the art can make various modifications and variations without departing from the spirit and scope of the invention, and such modifications and variations all fall within the scope defined by the invention.

Claims

1. A flow cytometer signal synchronization system, characterized in that, include: The flow module is used to generate a uniformly distributed flow of liquid from a single sample to be tested. A light source module is used to generate a laser beam that is incident on the liquid flow module; A photoelectric conversion module is used to collect the pulsed light signal generated by the fluid flow module in response to the laser beam, and convert the pulsed light signal into a pulsed analog signal; The signal synchronization module, connected to the photoelectric conversion module, is used to generate an external trigger synchronization signal based on the magnitude of the pulsed analog signal, so that the spectrometer can perform exposure based on the external trigger synchronization signal, thereby causing the single sample liquid flow to generate a fluorescence signal.

2. The flow cytometer signal synchronization system according to claim 1, characterized in that, The signal synchronization module includes a programmable logic device; the programmable logic device is used to sequentially filter, shape and level-convert the pulse analog signal to generate the external trigger synchronization signal.

3. The flow cytometer signal synchronization system according to claim 1, characterized in that, The signal synchronization module includes a voltage comparator; the voltage comparator is used to compare the pulse analog signal with a reference voltage and generate the external trigger synchronization signal based on the comparison result.

4. The flow cytometer signal synchronization system according to claim 3, characterized in that, The voltage comparator is also used to output a high level when the amplitude of the pulse analog signal is less than the reference voltage, and output a low level otherwise, so as to convert the pulse analog signal into a square wave signal and use the rising edge of the square wave signal as the start signal of the external trigger synchronization signal.

5. The flow cytometer signal synchronization system according to any one of claims 1 to 4, characterized in that, The flow cytometer signal synchronization system also includes a laser beam shrinking module; the laser beam shrinking module is disposed between the light source module and the fluid flow module, and the laser beam shrinking module is used to shrink the laser beam to a size scale corresponding to a single sample to be tested.

6. The flow cytometer signal synchronization system according to claim 5, characterized in that, The laser beam-shrinking module includes beam expanders arranged in sequence and a first microscope objective.

7. The flow cytometer signal synchronization system according to any one of claims 1 to 4, characterized in that, The flow cytometer signal synchronization system further includes a fluorescence detection module; the fluorescence detection module is connected to the signal synchronization module; the fluorescence detection module is also connected to the spectrometer; the fluorescence detection module is used to receive the external trigger synchronization signal and control the spectrometer to enter the exposure state according to the external trigger synchronization signal; the fluorescence detection module is also used to collect the fluorescence signal.

8. The flow cytometer signal synchronization system according to claim 7, characterized in that, The flow cytometer signal synchronization system further includes a second microscope objective and a third microscope objective disposed between the flow module and the fluorescence detection module; the second microscope objective and the third microscope objective are coaxial and arranged in opposite directions.

9. The flow cytometer signal synchronization system according to claim 7, characterized in that, The flow cytometer signal synchronization system also includes a host computer; the host computer is connected to the flow module and is used to control the flow rate of the single sample to be tested; the host computer is also connected to the signal synchronization module and is used to send adjustment commands to the signal synchronization module according to the flow rate of the single sample to be tested, so as to calibrate the generation time of the external trigger synchronization signal.

10. The flow cytometer signal synchronization system according to claim 9, characterized in that, The host computer is also connected to the fluorescence detection module to receive the fluorescence signal and analyze the liquid flow of the single sample to be tested based on the fluorescence signal.

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