Automatic system of quantitative fluorescence flow cytometer

Abstract

The invention discloses an automatic system of a quantitative fluorescence flow cytometer, and belongs to the technical field of cell detection, the automatic system comprises a sample injection module, a fluorescence detection module and a data processing module, the sample injection module controls operation of an injection pump through an upper computer program, a fluid system is flexibly controlled, and the flow velocity and volume of a sample are adjusted according to needs; the fluorescence detection module receives a signal of the data acquisition card in real time through a signal acquisition program and adjusts the gain of a photomultiplier at any time so as to monitor and adjust the signal quality, and the data processing module demodulates and filters an original signal through virtual lock-in amplifier software and can restore the original waveform of the signal; fitting the signal into a trapezoid by the feature extraction software, and extracting cell size and protein quantity information of each channel contained in the signal; and the channel matching software matches the signals among different channels according to the trapezoidal signals and the time delay relationship among the channels, and finally complete single cell feature information is generated.

Description

Technical Field

[0001] The invention belongs to the technical field of cell detection, and in particular relates to an automated system of 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 one or more protein molecules in a large number of single cells for certain proteins with lower content in a short period of time. The results of this protein quantitative analysis provide key parameters for cell heterogeneity, which is 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. It uses cells stained with fluorescently labeled antibodies to be flushed through capillaries, and the fluorescence intensity is quantified by a photomultiplier tube. Subsequently, based on chemically modified calibration beads, fluorescence flow cytometry can accurately quantify single-cell surface proteins, but this method cannot accurately quantify the number of single-cell intracellular protein molecules due to the lack of corresponding intracellular protein calibration beads.

[0004] In the face of the demand for rapid and accurate typing of abnormal white blood cells, a quantitative fluorescence flow cytometer system based on uniform light field is developed to address the key bottlenecks of data acquisition in current fluorescence flow cytometers (uneven energy distribution of the excitation spot, difficulty in converting fluorescence intensity into the number of specific proteins, and easy clogging of small-sized channels). Its core channel is an integrated three-dimensional focusing channel with a microscale optical baffle made by quartz dry etching. When working, three syringe pumps with individually controllable flow rates are used to access the sample flow and the horizontal / vertical sheath flow inlet, and the 3D focusing effect of different geometric sizes is achieved by adjusting the flow rate ratio of the sample flow and the two sheath flows. When cells are injected into the channel with the sample flow, the cells are focused on the uniform light field detection area located at the bottom center of the channel under the action of the fluid. In this area, the edge of the excitation light spot with severely uneven light intensity is blocked by the optical baffle, thus forming a uniform excitation light field with consistent light intensity in the detection area; and for detecting 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 entering the objective lens. Therefore, fluorescence calibration can be achieved based on spatial equivalence, and the fluorescence data of the cell can be converted into the number of membrane proteins, cytoplasmic proteins, and nuclear proteins expressed by using the calibration curve of fluorescence value and molecular number established by gradient concentration fluorescent solution.

[0005] As a multi-channel, quantitative cell protein detection method, quantitative fluorescence flow cytometry has great application potential. Through the quantitative fluorescence flow cytometer system, high-sensitivity abnormal white blood cell fluorescence data sets can be obtained at high throughput and an identification system can be established to promote the rapid and accurate typing of acute leukemia cells and provide a reliable basis for the initial screening of acute leukemia. Summary of the invention

[0006] The quantitative fluorescence flow cytometer system mentioned in the background technology still has the following problems: 1. The automation level of sample injection is low, and more manual operations are required during operation; 2. There is a lack of effective quality control means; 3. For the collected raw data, there is a lack of 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 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 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;

[0010] 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 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 the computer, and 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 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.

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

[0014] In the above technical solution, the signal acquisition program stores the original signal in a 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 peak generated when a single cell passes by from the fluorescence intensity signal, fit the signal peak into a trapezoidal peak, and extract the time 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 of the rising edge and the falling edge of the trapezoidal signal (corresponding to the time difference between the upper and lower vertices on the left and right sides) and the peak duration of the trapezoidal signal (corresponding to the time difference between the upper and lower vertices on the left and right sides).

[0019] In the above technical solution, the channel matching program is implemented as follows: the trapezoidal peak signals of each channel are arranged in time, the earliest trapezoidal peak is selected from each channel to form a set A, the earliest trapezoidal peak a1 is selected from the set A, and the delay time is estimated by the width of the trapezoidal peak; according to the delay time between different channels, other trapezoidal peaks matching the trapezoidal peak are selected from the set, and empty values ​​are filled in for channels that have not been successfully matched, and together they 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 add the next peak of the channel corresponding to this peak in time to the set. If there is no next peak, fill in the empty value; continue the above matching process until all peaks are matched.

[0020] In the above technical solution, in the data processing module, after the data is processed, a data table of the content and size of each channel protein in a series of single cells is 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 as needed. Only by adjusting the direction of the reversing valve can the automatic injection and sample operation process be completed, solving the problem of a large number of manual operations during the sample operation process.

[0023] 2. The fluorescence detection module receives the signal 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, thereby 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 can demodulate and filter the original signal through the virtual phase-locked amplifier software, and restore the original waveform of the signal; the feature extraction software can fit the signal into a trapezoid and extract the cell size and protein amount information of each channel contained in the signal; the channel matching software can match the signals between different channels according to the trapezoidal signal and the time delay relationship between channels, and finally generate complete single-cell feature information. Therefore, the data processing module solves the problem of lack of perfect processing methods for the collected raw data. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Schematic diagram of the automated system composition of quantitative fluorescence flow cytometer.

[0026] Figure 2 Schematic diagram of the injection module workflow.

[0027] Figure 3 This is the structure diagram of the signal acquisition module.

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

[0029] Figure 5 is a trapezoidal fit to the cell signal.

[0030] Figure 6 Flowchart of the channel matching procedure.

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

[0032] The present invention is described in detail below in conjunction with the accompanying drawings and specific embodiments. However, the following embodiments are limited to explaining the present invention, and the protection scope of the present invention should include the entire contents of the claims, and through the description of the following embodiments, those skilled in the art can fully implement the entire contents of the claims of the present invention.

[0033] Example

[0034] like Figure 1As shown, this embodiment discloses an automated system for a quantitative fluorescence flow cytometer, including a sample injection module 1, a fluorescence detection module 2 and a data processing module 3, wherein the outlet of the sample injection module 1 is connected to the sample channel of the quantitative fluorescence flow cytometer, and is used to pass 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, and the data processing module 3 is used to process the signal saved by the fluorescence detection module 2 into a series of characteristic data of the detected cells.

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

[0036] The fluorescence detection module is composed of a photomultiplier tube 2-1 (PMT), a data acquisition card 2-2 and a signal acquisition program in a host computer 5, such as Figure 3 As 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 the data line. The data acquisition card 2-2 collects data at a high frequency and sends it to the host computer 5 through the data line. The signal acquisition program in the host computer 5 is written by LabVIEW, which 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 optimize the sensitivity and signal-to-noise ratio of the quantitative fluorescence flow cytometer 4, thereby achieving quality control of the quantitative fluorescence flow cytometer 4.

[0037] The data processing module 3 includes the virtual phase-locked amplifier program, the cell feature extraction program and the channel matching program in the host computer 5. The three programs are all written in Matlab. The workflow is as follows: Figure 4As shown. The original signal collected from the photomultiplier tube 2-1 first passes through the virtual phase-locked 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 phase-locked 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 peak representing the cell in the fluorescence signal are identified, and the signal peak is fitted into a trapezoid. The schematic diagram of the trapezoidal signal is 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 taken for the rising and falling edges of the trapezoidal signal (corresponding to the time difference between the upper and lower vertices on the left and right sides) and the peak duration of the trapezoidal signal (corresponding to the time difference between the upper and lower vertices on the left and right sides). The protein content, size, and the time of the four vertices are recorded in the trapezoidal peak information of each channel as features of the trapezoidal signal. The flow chart of the channel matching program is shown in Figure 6 As shown, the trapezoidal peak information of each channel is arranged by time, and the earliest trapezoidal peak is selected from each channel to form a set A. The earliest trapezoidal peak a1 is selected from the set A, and the width of the trapezoidal peak is used to estimate the delay time; according to the delay time between different channels, other trapezoidal peaks matching the trapezoidal peak are selected from the set, and empty values ​​are filled in for channels that have not been successfully matched, and together they form a cell signal C x (x=1,2,3,...); remove the matched trapezoidal peaks from the set in turn, and add the next peak of the corresponding channel of this peak to the set. If there is no next peak, fill in a null value; continue the above matching process until all peaks are matched. The delay time judgment method is:

[0038] ,

[0039] ,

[0040] in, is the time corresponding to the top vertex on the right side of the reference peak, is the time corresponding to the top vertex on the left side of the reference peak, is the time corresponding to the left bottom vertex of the peak to be matched, is the time corresponding to the right bottom 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 is still manually adjusted, and other reversing valves that can be automatically adjusted can also be used to form a liquid circuit.

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

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

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

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

[0046] In this embodiment, as a preferred implementation method, signal demodulation is implemented by a virtual phase-locked amplifier program. Using a physical phase-locked amplifier to replace the combination of a data acquisition card and a virtual phase-locked amplifier program does not affect the basic function implementation. Various filtering methods and combinations can be used in the virtual phase-locked amplifier program.

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

[0048] The above description is only a specific implementation of the present application, so that those skilled in the art can understand or implement the present application. Various modifications to these embodiments will be apparent to those skilled in the art, and 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 the embodiments shown herein, but will conform to the widest range consistent with the principles and novel features applied for 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, and the original signal passes through the virtual lock-in amplifier program, the feature extraction program and the channel matching program in sequence.

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 virtual lock-in amplifier program is used to demodulate and filter the original signal to generate a fluorescence intensity signal.

6. The automated system for quantitative fluorescence flow cytometry according to claim 1, characterized in that: The feature extraction program is used to identify the peak generated when a single cell passes by from the fluorescence intensity signal, fit the signal peak into a trapezoidal peak signal, and extract the time of the four vertices of the trapezoidal peak signal.

7. 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.

8. 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.

9. The automated system for quantitative fluorescence flow cytometry according to claim 1, characterized in that: The implementation method of the channel matching program is as follows: the trapezoidal peak signals of each channel are arranged in time, the earliest trapezoidal peak is selected from each channel to form a set A, the earliest trapezoidal peak a1 is selected from the set A, and the delay time is estimated by the width of the trapezoidal peak; according to the delay time between different channels, other trapezoidal peaks matching the trapezoidal peak are selected from the set, and the channels that have not been successfully matched are filled with null values, and together 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 add the next peak of the channel corresponding to this peak in time to the set. If there is no next peak, fill in the empty value; continue the above matching process until all peaks are matched.

10. 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.

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