Method for automatically adjusting background by using blank microspheres and flow cytometer

Abstract

The invention relates to the technical field of flow cytometers, in particular to a method for automatically adjusting a background by using blank microspheres and a flow cytometer, and the method comprises the following steps: collecting the blank microspheres, monitoring fluorescence signals of the blank microspheres in real time, recording background signal data, denoising and normalizing the collected signals, and statistically analyzing the fluorescence signals of the blank microspheres. The method comprises the steps of determining a median and a standard deviation of background signals, calculating a signal difference value according to statistical characteristics of the background signals, comparing collected signals with a target background signal value, classifying effective signals and background signals, judging the applicability of a background signal threshold value according to a real-time analysis result, and dynamically adjusting the background threshold value. And the processed data and the analysis result are displayed on the user interface and stored in the computer for subsequent analysis, so that the inconvenience and inaccuracy caused by the traditional manual background adjustment method are solved, and the accuracy and repeatability of measurement are improved.

Description

Technical Field

[0001] The invention relates to the technical field of flow cytometers, and in particular to a method for automatically adjusting background using blank microspheres and a flow cytometer. Background Art

[0002] Flow cytometers are widely used in biomedicine, drug development, clinical diagnosis and other fields. They use laser light sources to stimulate fluorescent markers in cells and then detect cell characteristics. However, flow cytometers are often affected by ambient light and instrument noise during use, resulting in changes in background signals (background signals). Background signals affect the fluorescence signals of cells, resulting in reduced accuracy and repeatability of measurement results, thereby affecting the accuracy and repeatability of measurements. Therefore, setting an appropriate background signal threshold is crucial to distinguish between real signals and background noise.

[0003] Blank microspheres are tiny spheres without any markers, usually made of polymers or other biocompatible materials. Blank microspheres are often used in flow cytometers and other biotechnology experiments. The main purpose is to calibrate and evaluate the performance of the instrument, especially in measuring background signals and fluorescence intensity. In the absence of cell samples, by detecting the signal of blank microspheres, the background level of the instrument can be evaluated to ensure that the signal and noise can be accurately distinguished when analyzing real samples.

[0004] At present, traditional flow cytometers usually adjust the background signal by manually setting the background signal threshold. During the acquisition process, it is necessary to manually gate the data distribution of each flow graph, manually set the height signal and area signal of each fluorescent channel according to the background signal data position, and then manually re-acquire until the measured background signal can reach the set target range before stopping the debugging process. The traditional background debugging process is cumbersome, takes a long time to debug, has low debugging efficiency, and the debugging effect depends on the experience of the debugger. The results are easily affected by human subjective factors. Different debuggers or the same debugger in different debugging processes will have different results, which leads to the accuracy and repeatability of the instrument measurement cannot be guaranteed. Summary of the invention

[0005] The purpose of the present invention is to provide a method and a flow cytometer for automatically adjusting the background using blank microspheres, which solves the technical problem that the existing flow cytometer usually adjusts the background signal by manually setting the background signal threshold, the debugging process is cumbersome, the debugging time is long, the debugging efficiency is low, and the debugging effect depends on the experience of the debugger. The result is easily affected by human subjective factors and there are result differences, thereby causing the accuracy and repeatability of the instrument measurement to be unable to be guaranteed.

[0006] To achieve the above-mentioned object, in a first aspect, the present invention provides a flow cytometer, comprising a laser light source module, an optical detection module, a signal processing unit module and a computer data processing and user interface, wherein the laser light source module, the optical detection module, the signal processing unit module and the computer data processing and user interface are connected in sequence;

[0007] The laser light source module is used to excite the sample;

[0008] The optical detection module is used to detect the fluorescent signal emitted by the sample after excitation;

[0009] The signal processing unit module is used to process and analyze the collected signals and transmit the analysis results to the computer system;

[0010] The computer data processing and user interface is used to perform data processing on the received analysis results and display the processed measurement results.

[0011] In a second aspect, the present invention also provides a method for automatically adjusting the background using blank microspheres, comprising the following steps:

[0012] Blank microsphere configuration, selecting appropriate blank microspheres and configuring reagents for the blank microspheres;

[0013] Background signal collection: starting the flow cytometer, using a laser light source to excite the blank microspheres, monitoring their fluorescence signals in real time, and recording background signal data;

[0014] Data preprocessing: denoising and normalizing the collected signals to improve data quality;

[0015] Background signal analysis: statistically analyzing the fluorescence signal of the blank microspheres to determine the median and standard deviation of the background signal;

[0016] Background threshold calculation, calculating the signal threshold according to the statistical characteristics of the background signal;

[0017] Signal classification and analysis: compare the collected signals with the threshold, classify the effective signals and background signals, and perform aggregate analysis;

[0018] Feedback and adjustment: Based on the real-time analysis results, the applicability of the signal threshold is judged and dynamically adjusted;

[0019] Result output: The processed data and analysis results are displayed on the user interface and stored in the database for subsequent analysis.

[0020] The specific steps of background signal collection are as follows:

[0021] Starting the flow cytometer, allowing the flow cytometer to generate a laser light source to excite the blank microspheres, and after the excitation, placing the blank microsphere test tube on the injector;

[0022] Open the software automatic background adjustment interface, click the "Automatic Background Adjustment" button, and start automatically collecting the fluorescence signal emitted by the blank microspheres;

[0023] After the fluorescent signal emitted by the blank microspheres is collected in real time, the collected fluorescent signal data is recorded in real time to achieve the recording of background signal data.

[0024] The specific steps of data preprocessing are as follows:

[0025] De-noising, using a median filter to filter the collected background signal data to smooth the signal and remove high-frequency noise in the signal;

[0026] Signal normalization, after noise removal, normalizes the signal intensity of the background signal data to eliminate the influence of sample concentration or laser intensity changes.

[0027] Wherein, the specific steps of the background signal analysis are:

[0028] The blank microsphere population is obtained by using the MeanShift mean shift clustering algorithm based on the data distribution of the FSC-H and SSC-H scatter plots, and the rectangular gate is automatically calculated so that all the blank microspheres are contained in the rectangular gate;

[0029] The signal peak of each fluorescence channel is automatically identified on the histogram by the AMPD peak detection algorithm, and the blank microspheres in the histogram of each fluorescence channel are included in the linear gate by automatically calculating the linear gate position, and the median and standard deviation of the signal of the blank microspheres are calculated;

[0030] Based on this, the characteristics of all background signals are evaluated and the normal range is determined.

[0031] The specific steps of calculating the background threshold are as follows:

[0032] The median of the background signal measurements for each fluorescent channel was calculated;

[0033] Then calculate the difference between the measured value and the target median position;

[0034] A background threshold is set, and the difference is set as the background threshold.

[0035] The specific steps of feedback and adjustment are as follows:

[0036] Real-time feedback, comparing the analysis results with the preset target value to determine whether the current signal threshold needs to be adjusted;

[0037] If the signal median is within the target median range, no further adjustment is required. If the signal median is outside the target median range, repeated acquisition and adjustment of the background threshold are required automatically.

[0038] Dynamic update: based on real-time collected data, the background signal estimation and threshold calculation are continuously updated, and the calculated background threshold is automatically sent to the instrument for automatic re-collection;

[0039] When all channels meet the target value conditions or reach the set number of iterations, the acquisition will stop automatically;

[0040] After stopping the acquisition, the median of the acquired data and the background threshold are automatically used to determine whether they meet the set target value conditions. If yes, the debugging is successful and passed; if not, the debugging fails. The automatic judgment process ensures the accuracy of the measurement.

[0041] The present invention discloses a method and flow cytometer for automatically adjusting background using blank microspheres, which automatically adjusts the background signal to reduce the influence of background noise, so that the target signal is clearer, the measurement result is more accurate, and the measurement accuracy can be improved. The real-time monitoring and adjustment mechanism ensures that the instrument always maintains the best performance under different environmental conditions, reduces the measurement instability caused by environmental changes, and enhances the stability of the instrument. The simplified operation interface and automatic adjustment function enable even novice users to quickly get started, reduce training costs, and increase user-friendliness. The use of blank microspheres for real-time monitoring and automatic adjustment enhances the convenience of the instrument's operation, adapts to different experimental conditions, and ensures that the flow cytometer can automatically adjust the background signal before leaving the factory, thereby providing consistent and reliable performance, reducing debugging time, and significantly improving debugging efficiency and the accuracy of instrument testing. The automated process reduces the need for users to manually debug the background, allowing experimenters to focus on data analysis and experimental design, improve work efficiency, and save time and labor costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0043] Figure 1 4 is a system module diagram of a flow cytometer according to a first embodiment of the present invention.

[0044] Figure 2 1 is a flowchart of a method for automatically adjusting background using blank microspheres according to a second embodiment of the present invention.

[0045] Figure 3It is a diagram of specific steps of background signal collection in the second embodiment of the present invention.

[0046] Figure 4 It is a diagram of specific steps of data preprocessing according to the second embodiment of the present invention.

[0047] Figure 5 It is a diagram of specific steps of background signal analysis according to the second embodiment of the present invention.

[0048] Figure 6 4 is a diagram showing the specific steps of background threshold calculation according to the second embodiment of the present invention.

[0049] Figure 7 It is a diagram of specific steps of feedback and adjustment of the second embodiment of the present invention.

[0050] Figure 8 It is a specific step diagram of the result output of the second embodiment of the present invention.

[0051] Fig. 9 This is a diagram of the automatic background adjustment interface of the second embodiment of the present invention.

[0052] Fig.10 4 is a diagram of the “automatic background adjustment” button of the second embodiment of the present invention.

[0053] Fig.11 4 is a signal status diagram before background threshold adjustment according to the second embodiment of the present invention.

[0054] Fig.12 4 is a signal status diagram after the background threshold is adjusted according to the second embodiment of the present invention.

[0055] Fig.13 This is a measurement diagram of the initial value before background adjustment according to the second embodiment of the present invention.

[0056] Fig.14 4 is a measurement diagram of the background-adjusted value of the second embodiment of the present invention.

[0057] Fig.15 It is a debugging report diagram of the second embodiment of the present invention.

[0058] Fig.16 This is a report list diagram according to the second embodiment of the present invention.

[0059] Fig.17 This is a diagram of the automatic background debugging result of the second embodiment of the present invention.

[0060] In the figure: 101 - laser light source module, 102 - optical detection module, 103 - signal processing unit module, 104 - computer data processing and user interface. DETAILED DESCRIPTION

[0061] Embodiments of the present invention are described in detail below. Examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and are intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0062] First embodiment:

[0063] See also Figure 1 The present invention provides a flow cytometer, including a laser light source module, an optical detection module, a signal processing unit module and a computer data processing and user interface.

[0064] In this embodiment, the characteristics of the sample to be tested can be detected by using the laser light source module, the optical detection module, the signal processing unit module and the computer data processing and user interface in coordination.

[0065] Among them, the laser light source module, the optical detection module, the signal processing unit module and the computer data processing and user interface are connected in sequence; the laser light source module is used to excite the sample; the optical detection module is used to detect the fluorescence signal emitted by the excited sample; the signal processing unit module is used to process and analyze the collected signal and transmit the analysis result to the computer system; the computer data processing and user interface is used to perform data processing on the received analysis result and display the processed measurement result.

[0066] Secondly, the samples excited by the laser light source module include cell samples to be analyzed and blank microsphere samples.

[0067] When using a flow cytometer of the present embodiment, the laser light source module is used to excite samples, wherein the samples include cell samples to be analyzed and blank microsphere samples. After excitation, the optical detection module is used to detect the fluorescent signal emitted by the excited sample. After detection, the signal processing unit module is used to process and analyze the collected signal, and the analysis result is transmitted to the computer system, so that the computer data processing and user interface can perform data processing on the received analysis results and display the processed measurement results, thereby completing the detection of the characteristics of the sample.

[0068] Second embodiment:

[0069] Based on the first embodiment, please refer to Figures 2 to 17 The present invention also provides a method for automatically adjusting the background using blank microspheres, comprising the following steps:

[0070] S1. Blank microsphere configuration: select appropriate blank microspheres and configure reagents for the blank microspheres.

[0071] Specifically, appropriate blank microspheres are selected, and the fluorescence characteristics of the selected blank microspheres are ensured to be within the experimental range, and then the blank microspheres are used to prepare blank microsphere reagents.

[0072] S2, background signal collection, start the flow cytometer, use the laser light source to excite the blank microspheres, monitor their fluorescence signals in real time, and record the background signal data.

[0073] Specifically, the specific steps of background signal collection are:

[0074] S21, start the flow cytometer, let the flow cytometer generate a laser light source to excite the blank microspheres, and after the excitation, place the blank microsphere test tube on the injector;

[0075] S22, opening the software automatic background adjustment interface, clicking the "automatic background adjustment" button, and starting to automatically collect the fluorescence signal emitted by the blank microspheres;

[0076] S23, after real-time collection of the fluorescent signal emitted by the blank microspheres, the collected fluorescent signal data is recorded in real time to achieve the recording of background signal data.

[0077] S3. Data preprocessing: denoising and normalizing the collected signals to improve data quality.

[0078] Specifically, the specific steps of the data preprocessing are:

[0079] S31, denoising processing, using a median filter to filter the collected background signal data to smooth the signal and remove high-frequency noise in the signal;

[0080] S32, signal normalization, after the noise removal process, the signal intensity of the background signal data is normalized to eliminate the influence of the sample concentration or the laser intensity change.

[0081] Specifically, the signal normalization adopts the minimum-maximum normalization method. In order to better display the low-value signal on the flow chart, the channel histogram of the background needs to be adjusted, and all are displayed using Logical coordinates.

[0082] S4. Background signal analysis: statistically analyzing the fluorescence signal of the blank microspheres to determine the median and standard deviation of the background signal.

[0083] Specifically, the specific steps of the background signal analysis are:

[0084] S41, using the MeanShift mean shift clustering algorithm based on the data distribution of the FSC-H and SSC-H scatter plots to obtain a blank microsphere population, and automatically calculating a rectangular gate so that all the blank microspheres are contained within the rectangular gate;

[0085] S42, using the AMPD peak detection algorithm, automatically identifying the signal peak of each fluorescence channel on the histogram, automatically calculating the linear gate position, allowing the blank microspheres in the histogram of each fluorescence channel to be included in the linear gate, and calculating the median and standard deviation of the signal of the blank microspheres;

[0086] S43. Based on this, evaluate the characteristics of all background signals and determine the normal range.

[0087] S5. Background threshold calculation: calculating the signal threshold according to the statistical characteristics of the background signal.

[0088] Specifically, the specific steps of calculating the background threshold are:

[0089] S51, calculating the measured median value of the background signal of each fluorescent channel;

[0090] S52, recalculating the difference between the measured value and the target median position;

[0091] S53: Setting a background threshold, and setting the difference as the background threshold.

[0092] S6. Signal classification and analysis: compare the collected signals with the threshold, classify the valid signals and background signals, and perform aggregate analysis.

[0093] Specifically, the collected signal is compared with the calculated threshold, and the effective signal and the background signal are distinguished according to the comparison result, and after classification, an aggregation analysis is performed.

[0094] S7, feedback and adjustment, based on the real-time analysis results, determine the applicability of the signal threshold and make dynamic adjustments.

[0095] Specifically, the specific steps of the feedback and adjustment are:

[0096] S71, real-time feedback, comparing the analysis result with the preset target value to determine whether the current signal threshold needs to be adjusted;

[0097] S72, if the signal median is within the target median range, no further adjustment is required; if the signal median is outside the target median range, repeated acquisition is required automatically and the background threshold is adjusted;

[0098] S73, dynamic update, based on the real-time collected data, continuously update the background signal estimation and threshold calculation, and automatically send the calculated background threshold to the instrument to automatically re-collect;

[0099] S74, when all channels meet the target value conditions or reach the set number of iterations, the acquisition is automatically stopped;

[0100] S75. After stopping the acquisition, the median of the acquired data and the background threshold are automatically used to determine whether the set target value condition is met. If yes, the debugging is successful and passed; if not, the debugging fails. The accuracy of the measurement is ensured through the automatic determination process.

[0101] S8. Result output: the processed data and analysis results are displayed on the user interface and stored in the database for subsequent analysis.

[0102] Specifically, the specific steps of the result output are:

[0103] S81. Displaying the result in the form of a report on the user interface, and whether the result of the automatic background adjustment is successful;

[0104] S82, data storage, storing the processed data and analysis results in a software database for subsequent analysis and comparison.

[0105] Specifically, the results displayed in the form of a report on the user interface include the median background signal of the blank microspheres of each channel and the adjusted background threshold, as well as the target value range.

[0106] When using a method of automatically adjusting the background using blank microspheres of the present embodiment, the background signal is automatically adjusted to reduce the influence of background noise, so that the target signal is clearer, the measurement result is more accurate, and the measurement accuracy can be improved. The real-time monitoring and adjustment mechanism ensures that the instrument always maintains optimal performance under different environmental conditions, reduces the measurement instability caused by environmental changes, and enhances the stability of the instrument. The simplified operating interface and automatic adjustment function enable even novice users to quickly get started, reducing training costs and increasing user-friendliness. Using blank microspheres for real-time monitoring and automatic adjustment enhances the convenience of the instrument's operation, adapts to different experimental conditions, and ensures that the flow cytometer can automatically adjust the background signal before leaving the factory, thereby providing consistent and reliable performance, reducing debugging time, and significantly improving debugging efficiency and the accuracy of instrument testing. The automated process reduces the need for users to manually debug the background, allowing experimenters to focus on data analysis and experimental design, improving work efficiency, and saving time and labor costs.

[0107] What is disclosed above is only one or more preferred embodiments of the present application, and cannot be used to limit the scope of rights of the present application. Ordinary technicians in this field can understand that all or part of the processes of implementing the above embodiments and equivalent changes made according to the claims of the present application are still within the scope covered by the present application.

Claims

1. A flow cytometer, characterized in that: It includes a laser light source module, an optical detection module, a signal processing unit module and a computer data processing and user interface, wherein the laser light source module, the optical detection module, the signal processing unit module and the computer data processing and user interface are connected in sequence; The laser light source module is used to excite the sample; The optical detection module is used to detect the fluorescent signal emitted by the sample after excitation; The signal processing unit module is used to process and analyze the collected signals and transmit the analysis results to the computer system; The computer data processing and user interface is used to perform data processing on the received analysis results and display the processed measurement results.

2. A method for automatically adjusting background using blank microspheres, applied to the flow cytometer of claim 1, characterized in that: The following steps are involved: Blank microsphere configuration, selecting appropriate blank microspheres and configuring reagents for the blank microspheres; Background signal collection: starting the flow cytometer, using a laser light source to excite the blank microspheres, monitoring their fluorescence signals in real time, and recording background signal data; Data preprocessing: denoising and normalizing the collected signals to improve data quality; Background signal analysis: statistically analyzing the fluorescence signal of the blank microspheres to determine the median and standard deviation of the background signal; Background threshold calculation, calculating the signal threshold according to the statistical characteristics of the background signal; Signal classification and analysis: compare the collected signals with the threshold, classify the effective signals and background signals, and perform aggregate analysis; Feedback and adjustment: Based on the real-time analysis results, the applicability of the signal threshold is judged and dynamically adjusted; Result output: The processed data and analysis results are displayed on the user interface and stored in the database for subsequent analysis.

3. The flow cytometer according to claim 2, wherein: The specific steps of background signal collection are: Starting the flow cytometer, allowing the flow cytometer to generate a laser light source to excite the blank microspheres, and after the excitation, placing the blank microsphere test tube on the injector; Open the software automatic background adjustment interface, click the "Automatic Background Adjustment" button, and start automatically collecting the fluorescence signal emitted by the blank microspheres; After the fluorescent signal emitted by the blank microspheres is collected in real time, the collected fluorescent signal data is recorded in real time to achieve the recording of background signal data.

4. The method for automatically adjusting background using blank microspheres as claimed in claim 2, characterized in that: The specific steps of the data preprocessing are: De-noising, using a median filter to filter the collected background signal data to smooth the signal and remove high-frequency noise in the signal; Signal normalization, after noise removal, normalizes the signal intensity of the background signal data to eliminate the influence of sample concentration or laser intensity changes.

5. The method for automatically adjusting background using blank microspheres as claimed in claim 2, characterized in that: The specific steps of the background signal analysis are: The blank microsphere population is obtained by using the MeanShift mean shift clustering algorithm based on the data distribution of the FSC-H and SSC-H scatter plots, and the rectangular gate is automatically calculated so that all the blank microspheres are contained in the rectangular gate; The signal peak of each fluorescence channel is automatically identified on the histogram by the AMPD peak detection algorithm, and the blank microspheres in the histogram of each fluorescence channel are included in the linear gate by automatically calculating the linear gate position, and the median and standard deviation of the signal of the blank microspheres are calculated; Based on this, the characteristics of all background signals are evaluated and the normal range is determined.

6. The method for automatically adjusting background using blank microspheres as claimed in claim 2, characterized in that: The specific steps of calculating the background threshold are: The median of the background signal measurements for each fluorescent channel was calculated; Then calculate the difference between the measured value and the target median position; A background threshold is set, and the difference is set as the background threshold.

7. The method for automatically adjusting background using blank microspheres as claimed in claim 2, characterized in that: The specific steps of the feedback and adjustment are: Real-time feedback, comparing the analysis results with the preset target value to determine whether the current signal threshold needs to be adjusted; If the signal median is within the target median range, no further adjustment is required. If the signal median is outside the target median range, repeated acquisition and adjustment of the background threshold are required automatically. Dynamic update: based on real-time collected data, the background signal estimation and threshold calculation are continuously updated, and the calculated background threshold is automatically sent to the instrument for automatic re-collection; When all channels meet the target value conditions or reach the set number of iterations, the acquisition will stop automatically; After stopping the acquisition, the median of the acquired data and the background threshold are automatically used to determine whether they meet the set target value conditions. If yes, the debugging is successful and passed; if not, the debugging fails. The automatic judgment process ensures the accuracy of the measurement.

Images