An automated system for a quantitative fluorescence flow cytometer

Abstract

The present invention discloses an automated system for a quantitative fluorescence flow cytometer, belonging to the technical field of cell detection. The system includes a sample injection module, a fluorescence detection module, and a data processing module. The sample injection module controls the operation of an injection pump through a host computer program, flexibly controls the fluid system, and adjusts the sample flow rate and volume as needed. The fluorescence detection module receives the signals of a data acquisition card in real time through a signal acquisition program, and adjusts the gain of a photomultiplier tube at any time, realizing the monitoring and adjustment of signal quality. The data processing module demodulates and filters the original signals through virtual lock-in amplifier software, and can restore the original waveform of the signals. The feature extraction software fits the signals into trapezoids and extracts the cell size and the information of the protein amount in each channel contained in the signals. The channel matching software matches the signals between different channels according to the trapezoidal signals and the time delay relationship between channels, and finally generates complete single-cell feature information.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell detection, and particularly relates to an automated system for a quantitative fluorescence flow cytometer. Background Art

[0002] High-resolution quantitative detection of single-cell proteins refers to the quantitative detection of the number of a certain or multiple protein molecules in a large number of single cells for some proteins with relatively low content within a short period of time. The results of this protein quantitative analysis provide key parameters for cell heterogeneity, and are helpful for the mechanism research and clinical diagnosis and treatment of tumors.

[0003] Currently, fluorescence flow cytometry is an important technology in the field of single-cell protein quantification. In fluorescence flow cytometry, cells stained with fluorescently labeled antibodies are flushed through a capillary tube, and the fluorescence intensity is quantified by a photomultiplier tube. Subsequently, based on calibration beads after chemical modification, fluorescence flow cytometry can accurately quantify the surface proteins of single cells. However, due to the lack of corresponding intracellular protein calibration beads, this method cannot accurately quantify the number of intracellular proteins in single cells.

[0004] In the face of the need for rapid and accurate typing of abnormal white blood cells, aiming at the key bottlenecks in current fluorescence flow cytometers in data acquisition (uneven energy distribution of the excitation spot, difficult conversion of fluorescence intensity into the number of specific proteins, and easy blockage of small-size channels), a quantitative fluorescence flow cytometer system based on a uniform light field is developed. Its core channel is an integrated three-dimensional focusing channel with microscale optical baffles fabricated by dry etching of quartz. During operation, three injection pumps with individually controllable flow rates are respectively connected to the sample flow and the horizontal / vertical sheath flow inlets, and different geometric 3D focusing effects are achieved by adjusting the flow rate ratios of the sample flow and the two sheath flows. When cells are injected into the channel along with the sample flow, the cells are focused on the uniform light field detection area located at the center of the bottom of the channel under the action of the fluid. In this area, the edge part with severely uneven light intensity in the excitation spot is blocked by the optical baffle, thus forming a uniform excitation light field with consistent light intensity in the detection area; for the detected fluorescence, since the height of the microchannel is much smaller than the distance between the microchannel and the objective lens and the area of the detection window formed by the optical baffle is much smaller than the target surface of the photomultiplier tube (PMT), the fluorescence at different positions in the microchannel has the same proportion to enter the objective lens. Therefore, fluorescence calibration can be achieved based on spatial equivalence. With the calibration curve of fluorescence value and molecule number established by a gradient concentration fluorescent solution, the fluorescence data of cells can be converted into the expression numbers of membrane proteins, cytoplasmic proteins, and nuclear proteins.

[0005] As a multi-channel and quantitative method for detecting cellular proteins, quantitative fluorescence flow cytometry has high application potential. Through a quantitative fluorescence flow cytometer system, highly sensitive abnormal white blood cell fluorescence datasets can be obtained in a high-throughput manner and an identification system can be established, promoting the rapid and accurate typing application of acute leukemia cells and providing a reliable basis for the primary screening of acute leukemia. Summary of the Invention

[0006] Regarding the following problems still existing in the quantitative fluorescence flow cytometer system mentioned in the background art: 1. The automation level of sample injection is relatively low, and a lot of manual operations are required during the operation process; 2. Lack of effective quality control means; 3. For the collected raw data, there is a lack of a perfect processing means to extract the protein content characteristics and size information of each detected cell for analysis. The present invention proposes an automated system for a quantitative fluorescence flow cytometer.

[0007] The technical solution of the present invention is as follows:

[0008] An automated system for a quantitative fluorescence flow cytometer, comprising a sample injection module, a fluorescence detection module, and a data processing module;

[0009] The sample injection module includes an injection pump, a sample injection flow channel, a host computer, a serial communication bus between the host computer and the injection pump, and an injection pump control program in the host computer; the sample injection flow channel includes a liquid guiding hose, a connector, and a reversing valve connected in sequence, the host computer is connected to the injection pump through the serial communication bus, and the injection pump control program communicates with the injection pump through the serial communication bus;

[0010] The fluorescence detection module includes a photomultiplier tube, a data acquisition card, and a signal acquisition program in the host computer. There are multiple photomultiplier tubes, each photomultiplier tube corresponds to a fluorescence channel, the photomultiplier tube is connected to the data acquisition card through a data line, the data acquisition card is connected to the host computer through a data line, and the signal acquisition program receives the signal of the data acquisition card and can control the photomultiplier tube;

[0011] The data processing module includes a virtual lock-in amplifier program, a cell feature extraction program, and a channel matching program in a computer. The original signal passes through the virtual lock-in amplifier program, the feature extraction program, and the channel matching program in sequence.

[0012] In the above technical solution, the injection pump control program controls the operation or stop of the injection pump, controls the injection pump to be in the suction or injection mode, and monitors and controls the time and volume of suction or injection of the injection pump.

[0013] In the above technical solution, the signal acquisition program monitors the real-time signals of all channels and controls the gain size of the photomultiplier tube.

[0014] In the above technical solution, the signal acquisition program stores the original signal in the computer.

[0015] In the above technical solution, the virtual lock-in amplifier program is used to demodulate and filter the original signal to generate a fluorescence intensity signal.

[0016] In the above technical solution, the feature extraction program is used to identify the peaks generated when a single cell passes through the fluorescence intensity signal, fit the signal peaks into a trapezoidal peak, and extract the times of the four vertices of the trapezoidal signal.

[0017] In the above technical solution, the feature extraction program calculates the fluorescence intensity of the corresponding channel through the height of the trapezoidal signal, and then calculates the protein content.

[0018] In the above technical solution, the feature extraction program calculates the cell size information through the time taken by the rising edge and falling edge of the trapezoidal signal (corresponding to the time difference between the left and right upper base vertices and the lower base vertices) and the peak duration of the trapezoidal signal (corresponding to the time difference between the left and right upper base vertices).

[0019] In the above technical solution, the implementation method of the channel matching program is as follows: Arrange the trapezoidal peak signals of each channel in time order, select the trapezoidal peak with the earliest time from each channel to form a set A, select the trapezoidal peak a1 with the earliest time in set A, and estimate the delay time with the width of this trapezoidal peak; According to the delay time between different channels, select other trapezoidal peaks that match this trapezoidal peak from the set, and fill in null values for the channels that do not match successfully, and jointly form a cell signal C x (x = 1, 2, 3,...); Remove the trapezoidal peaks that have participated in the matching from the set in turn, and at the same time add the next peak in time of the channel corresponding to this peak to the set. If there is no next peak, fill in null values; Continuously perform the above matching process until all peak matches are completed.

[0020] In the above technical solution, in the data processing module, after the data is processed, a series of data tables of the protein content and size of each single cell in each channel are generated.

[0021] Beneficial effects:

[0022] 1. The injection module can control the operation of the injection pump through the host computer program, can flexibly control the fluid system, and adjust the sample flow rate and volume according to needs. Only by adjusting the direction of the switching valve, the automatic sample injection and sample running process can be completed, solving the problem of a large number of manual operations during the sample running process.

[0023] 2. The fluorescence detection module receives the signals from the data acquisition card in real time through the signal acquisition program, and can adjust the gain of the photomultiplier tube at any time, realizing the monitoring and adjustment of the signal quality, and further realizing the quality control of the quantitative fluorescence flow cytometer.

[0024] 3. The data processing module realizes the demodulation and filtering of the original signals through the virtual lock-in amplifier software, and can restore the original waveform of the signals; the feature extraction software can fit the signals into trapezoids and extract the cell size and the information of the protein amount in each channel contained in the signals; the channel matching software can match the signals between different channels according to the trapezoidal signals and the time delay relationship between channels, and finally generate the complete single-cell feature information. Therefore, the data processing module solves the problem of lacking perfect processing means for the collected original data. Brief Description of the Drawings

[0025] Figure 1 It is a schematic diagram of the composition of the automated system of the quantitative fluorescence flow cytometer.

[0026] Figure 2 It is a schematic diagram of the working process of the sample injection module.

[0027] Figure 3 It is a structural diagram of the signal acquisition module.

[0028] Figure 4 It is a flow chart of the data processing module.

[0029] Figure 5 It is the trapezoidal fitting of the cell signals.

[0030] Figure 6 It is a flow chart of the channel matching program.

[0031] 1 is the sample injection module, 1-1 is the syringe pump, 1-2 is the sample injection flow channel, 2 is the fluorescence detection module, 2-1 is the photomultiplier tube, 2-2 is the data acquisition card, 3 is the data processing module, 4 is the quantitative fluorescence flow cytometer, and 5 is the host computer. Detailed Embodiments

[0032] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. However, the following embodiments are only for explaining the present invention, and the protection scope of the present invention should include all the contents of the claims. Moreover, through the description of the following embodiments, those skilled in the art can fully implement all the contents of the claims of the present invention.

[0033] Embodiment

[0034] As Figure 1As shown in the figure, this embodiment discloses an automated system for a quantitative fluorescence flow cytometer, which includes a sample injection module 1, a fluorescence detection module 2, and a data processing module 3. The outlet of the sample injection module 1 is connected to the sample channel of the quantitative fluorescence flow cytometer, and is used to introduce the sample into the detection area. The fluorescence detection module 2 is used to convert the fluorescence collected by the fluorescence collection optical path of the quantitative fluorescence flow cytometer 4 into an electrical signal through a photomultiplier tube 2-1 (PMT) and save it. The data processing module 3 is used to process the signals saved by the fluorescence detection module 2 into a series of characteristic data of the detected cells.

[0035] The sample injection module 1 consists of a sample injection flow path 1-2 composed of a hose, a hose connector, and a reversing valve, an injection pump 1-1, and an injection pump control program in the host computer 5. The sample injection flow path 1-2 has three interfaces, which are respectively connected to the injection pump 1-1, the sample injection port of the quantitative fluorescence flow cytometer 4, and used as a sample injection port. The sample injection process is as Figure 2 shown. First, the flow path is filled with buffer solution by the injection pump, then the sample is drawn into the flow path from the sample injection port by the injection pump and temporarily stored, and finally the sample in the flow path is pushed into the detection channel of the quantitative fluorescence flow cytometer 4 by the injection pump. The injection pump control program is connected to the injection pump 1-1 through a USB (Universal Serial Bus) interface and controls the injection pump 1-1. The injection pump control program is written in LabVIEW and has functions of controlling the operation or stop of the injection pump 1-1, controlling the injection pump 1-1 to be in the suction or injection mode, and monitoring and controlling the time and volume of the suction or injection of the injection pump 1-1, and can realize flexible control of the sample injection process.

[0036] The fluorescence detection module consists of a photomultiplier tube 2-1 (PMT), a data acquisition card 2-2, and a signal acquisition program in the host computer 5, as Figure 3 shown. The photomultiplier tube 2-1 is used to convert the fluorescence collected by the quantitative fluorescence flow cytometer 4 into an electrical signal and transmit it to the data acquisition card 2-2 through a data line. The data acquisition card 2-2 acquires data at a high frequency and sends the data in a packet to the host computer 5 through a data line. The signal acquisition program in the host computer 5 is written in LabVIEW, can monitor the fluorescence signal uploaded from the data acquisition card 2-2 in real time, and can also save the signal to the host computer 5. According to the intensity of the fluorescence signal monitored in real time, the gain of the photomultiplier tube 2-1 can be adjusted in the signal acquisition program to make the sensitivity and signal-to-noise ratio of the quantitative fluorescence flow cytometer 4 optimal, and realize the quality control of the quantitative fluorescence flow cytometer 4.

[0037] The data processing module 3 includes a virtual lock-in amplifier program, a cell feature extraction program, and a channel matching program in the host computer 5. All three programs are written in Matlab, and their working process is as Figure 4As shown in the figure. The original signal collected from the photomultiplier tube 2-1 first passes through the virtual lock-in amplifier program. According to the fluorescence channel of the photomultiplier tube 2-1, the modulation frequency of the original signal can be known. The virtual lock-in amplifier program can demodulate and filter the original signal according to the corresponding modulation frequency to restore the waveform of the fluorescence signal. Then, through the feature extraction program, the baseline and the signal peaks representing cells in the fluorescence signal are identified, and the signal peaks are fitted into trapezoids. The schematic diagram of the trapezoidal signal is as shown in Figure 5 As shown. The peak height and the time of the four vertices (lower left base, upper left base, upper right base, lower right base) can be extracted from the trapezoidal signal. The cell size information is calculated by the time passed by the rising edge and falling edge of the trapezoidal signal (corresponding to the time difference between the upper base vertex and the lower base vertex on the left and right sides) and the peak duration of the trapezoidal signal (corresponding to the time difference between the upper base vertices on the left and right sides). The protein content, size, and the time of the four vertices are used as the features of the trapezoidal signal and recorded in the trapezoidal peak information of each channel. The flowchart of the channel matching program is as shown in Figure 6 As shown. The trapezoidal peak information of each channel is arranged in time. The trapezoidal peak with the earliest time is selected from each channel to form a set A. The trapezoidal peak a1 with the earliest time is selected from the set A, and the delay time is estimated by the width of this trapezoidal peak; according to the delay time between different channels, other trapezoidal peaks that match this trapezoidal peak are selected from the set, and null values are filled in for the channels that do not match successfully, jointly forming a cell signal C x (x = 1, 2, 3,...); the trapezoidal peaks that have participated in the matching are removed from the set in turn, and at the same time, the next peak in time of the channel corresponding to this peak is added to the set. If there is no next peak, a null value is filled in; the above matching process is continuously carried out until all peak matches are completed. The method for judging the delay time is:

[0038] ,

[0039] ,

[0040] Among them, is the time corresponding to the upper right base vertex of the reference peak, is the time corresponding to the upper left base vertex of the reference peak, is the time corresponding to the lower left base vertex of the peak to be matched, is the time corresponding to the lower right base vertex of the peak to be matched, is the delay time between the reference peak and the peak to be matched.

[0041] In this embodiment, as a preferred implementation method, the reversing valve still remains manually adjustable, and other reversing valves that can be automatically adjusted can also be used to form the liquid path.

[0042] In this embodiment, as a preferred implementation method, the serial communication bus uses a USB cable, and other serial communication cables compatible with the syringe pump do not affect the realization of the basic functions. The syringe pump control program is written in LabVIEW, and other serial communication control programs with similar functions do not affect the realization of the basic functions.

[0043] In this embodiment, as a preferred implementation method, the hardware for signal acquisition is implemented by a number of photomultiplier tubes 2-1, and the use of other photoelectric conversion elements such as photodiodes and avalanche photodiodes does not affect the realization of the basic functions.

[0044] In this embodiment, as a preferred implementation method, the signal acquisition program of the fluorescence detection module 2 is written in LabVIEW, and other control programs with similar functions do not affect the realization of the basic functions.

[0045] In this embodiment, as a preferred implementation method, the transmission of signals to the host computer 5 is realized by a data acquisition card 2-2, and the use of other data acquisition hardware such as single-chip microcomputers and FPGAs (field programmable gate arrays) does not affect the realization of the basic functions.

[0046] In this embodiment, as a preferred implementation method, signal demodulation is realized by a virtual lock-in amplifier program. The use of a physical lock-in amplifier to replace the combination of the data acquisition card and the virtual lock-in amplifier program does not affect the realization of the basic functions, and various filtering methods and combinations can be used in the virtual lock-in amplifier program.

[0047] In this embodiment, as a preferred implementation method, the cell feature extraction program and the channel matching program are written in Matlab, and other programs with similar data processing functions do not affect the realization of the basic functions.

[0048] The above are only the specific implementation manners of the present application, enabling those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be obvious to those skilled in the art. The general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application will not be limited to these embodiments shown herein, but rather will conform to the widest scope consistent with the principles and novel features claimed herein.

Claims

1. An automated system for quantitative fluorescence flow cytometry, characterized in that: It includes a sample injection module, a fluorescence detection module and a data processing module; The injection module includes a syringe pump, an injection channel, a host computer, a serial communication bus between the host computer and the syringe pump, and a syringe pump control program in the host computer. The injection channel includes a liquid guide hose, a joint and a reversing valve connected in sequence. The host computer and the syringe pump are connected via a serial communication bus, and the syringe pump control program communicates with the syringe pump via the serial communication bus. The fluorescence detection module includes a photomultiplier tube, a data acquisition card and a signal acquisition program in a host computer. There are multiple photomultiplier tubes, each photomultiplier tube corresponds to a fluorescence channel. The photomultiplier tube is connected to the data acquisition card through a data line, and the data acquisition card is connected to the host computer through a data line. The signal acquisition program receives the signal from the data acquisition card and controls the photomultiplier tube. The data processing module includes a virtual lock-in amplifier program, a cell feature extraction program and a channel matching program in the computer. The original signal passes through the virtual lock-in amplifier program, the feature extraction program and the channel matching program in sequence. The virtual lock-in amplifier program is used to demodulate and filter the original signal to generate a fluorescence intensity signal. The feature extraction program is used to identify the peak generated when a single cell passes through the fluorescence intensity signal, and fit the signal peak into a trapezoidal peak signal, and extract the time of the four vertices of the trapezoidal peak signal; The implementation method of the channel matching program is as follows: arrange the trapezoidal peak signals of each channel in time, select the earliest trapezoidal peak from each channel to form a set A, select the trapezoidal peak a1 with the earliest time in the set A, and use the width of the trapezoidal peak to estimate the delay time; according to the delay time between different channels, select other trapezoidal peaks that match the trapezoidal peak from the set, and fill in null values ​​for channels that have not been successfully matched, together forming a cell signal Cx (x=1,2,3,...); remove the trapezoidal peaks that have participated in the matching from the set in turn, and add the next peak of the channel corresponding to this peak in time to the set, and fill in null values ​​if there is no next peak; continue the above matching process until all peaks are matched.

2. The automated system for quantitative fluorescence flow cytometry according to claim 1, characterized in that: The syringe pump control program controls the syringe pump to run or stop, controls the syringe pump to be in the suction or injection mode, and monitors and controls the time and volume of the syringe pump suction or injection.

3. The automated system for quantitative fluorescence flow cytometry according to claim 1, characterized in that: The signal acquisition program monitors the real-time signals of all channels and controls the gain of the photomultiplier tubes.

4. The automated system for quantitative fluorescence flow cytometry according to claim 1, characterized in that: The signal acquisition program stores the original signal in a computer.

5. The automated system for quantitative fluorescence flow cytometry according to claim 1, characterized in that: The feature extraction program calculates the fluorescence intensity of the corresponding channel by the height of the trapezoidal signal, and then calculates the protein content.

6. The automated system for quantitative fluorescence flow cytometry according to claim 1, characterized in that: The feature extraction program calculates the cell size information through the time of the rising and falling edges of the trapezoidal signal and the duration of the peak value of the trapezoidal signal.

7. The automated system for quantitative fluorescence flow cytometry according to claim 1, characterized in that: In the data processing module, the data is processed to generate a series of data tables of the content and size of each channel protein in a single cell.

Images