Flow cytometer laser delay calibration method and device

Abstract

The present invention discloses a method and device for calibrating the laser delay of a flow cytometer. The method includes: removing the filter in front of the forward detector; controlling the laser system to emit N lasers; allowing a sample without fluorescent particles to flow to a waste liquid barrel after passing through an injection device and a flow chamber; collecting N continuous pulse signals as N laser signals; calculating the time difference between two adjacent pulse peaks as a laser delay correction value; using a certain laser as a reference, and performing laser delay on the N laser signals according to the laser delay correction value to achieve alignment. The present invention can perform laser delay calibration simply and quickly, and has low sample requirements. Any particle without fluorescence is feasible. It can greatly reduce the cost of laser delay calibration, increase the calibration frequency, and thus ensure the accuracy of the delay. The device is simple and can be implemented by only installing a movable device on the filter of the forward detector of a traditional flow cytometer. The calculation method is simple and has high accuracy and reliability, and wide adaptability.

Description

Technical Field

[0001] The present invention belongs to the technical field of cell analysis instruments, and in particular to a flow cytometer data processing method and device. Background Art

[0002] Flow cytometry (FCM) is a commonly used single-cell, multi-parameter, high-throughput instrument. Sheath fluid compresses and focuses the sample onto a streamline, forcing cells to line up and pass through a laser detection area. Detectors receive scattered light and fluorescence signals emitted by the cells to analyze their physical, chemical, and biological properties.

[0003] In a spatially excited multi-laser flow cytometer, the distance between two adjacent laser spots is approximately 100 to 200 microns. Figure 1 As shown in the figure, the particles pass through 488nm, 405nm, and 638nm lasers in sequence. There will be a certain delay between the light signals obtained by each laser. Therefore, how to correctly splice the data detected by multiple lasers is an important step to ensure the accuracy of the detection results.

[0004] Laser delay is typically set to a default value at the factory. However, changes in external conditions such as temperature and humidity can cause fluctuations in the liquid flow and drift in the laser spot, leading to changes in the laser delay. Therefore, laser delay calibration requires regular calibration. Currently, only theoretical research on delay calibration exists, but no specific technical solutions are available. Summary of the Invention

[0005] The purpose of the present invention is to provide a method and device for real-time automatic calibration of the laser delay of a flow cytometer, thereby ensuring the accuracy of the laser delay in real time and thus ensuring the accuracy of the flow detection results.

[0006] The technical solution for achieving the purpose of the present invention is as follows: on the one hand, a flow cytometer laser delay calibration device is provided, which includes a sample injection device, a flow chamber, a waste liquid bucket, a laser system, a detector and a spectroscopic system, an electronic system, a computer and a movable device; the detector and spectroscopic system includes a forward detector and a filter located in front of the forward detector, the filter being arranged on the movable device, and the filter can be moved to the front of the forward detector by the movable device and the two are coaxial, and can also be moved away from the forward detector;

[0007] The sample passes through the injection device and the flow chamber and flows into the waste liquid barrel. The N lasers emitted by the laser system pass through the flow chamber and are incident on the detector and spectrometer system. The laser signal is transmitted to the computer through the electronic system. The computer processes the laser signal to obtain the laser delay correction value, and uses this laser delay correction value to align the signals generated by each laser.

[0008] Furthermore, the receiving aperture of the forward detector can cover N laser spot ranges.

[0009] In another aspect, a method for calibrating laser delay of a flow cytometer is provided, the method comprising the following steps:

[0010] Step 1, removing the filter in front of the forward detector by a movable device;

[0011] Step 2: Control the laser system to emit N laser beams;

[0012] Step 3, the sample without fluorescent particles passes through the sampling device and the flow chamber and then flows into the waste liquid bucket;

[0013] Step 4: collect N continuous pulse signals as signals corresponding to N lasers;

[0014] Step 5, calculating the time difference between two adjacent pulse peaks as the laser delay correction value;

[0015] Step 6: Using a certain laser as a reference, perform laser delay on the N laser signals according to the laser delay correction value obtained in step 5 to achieve alignment.

[0016] Furthermore, before step 3 is executed, the method further includes:

[0017] The sample without fluorescent particles is diluted so that there is a signal in only one laser area in the same time period, that is, two adjacent groups of N continuous pulse signals do not overlap.

[0018] Furthermore, the laser pulse signal is collected in step 4 when the signal value received by the electronic system is greater than a set threshold.

[0019] Furthermore, before executing step 6, the following steps may be further included:

[0020] Repeat steps 4 and 5 several times;

[0021] Calculate the mean or median of the multiple groups of laser delay correction values ​​to obtain a group of laser delay correction values ​​as the final laser delay correction values.

[0022] Furthermore, the calculation of the mean of the multiple groups of laser delay correction values ​​may be replaced by: making a histogram of the multiple groups of laser delay correction values ​​to find the peak value.

[0023] Furthermore, the method further comprises:

[0024] Step 7: Move the filter back to the front of the forward detector by the movable device to detect the sample to be detected.

[0025] Compared with the prior art, the present invention has the following significant advantages:

[0026] 1) The device and method for detecting laser delay proposed in the present invention can perform laser delay calibration simply and quickly.

[0027] 2) The sample requirements are low, and any non-fluorescent particles are feasible, which can greatly reduce the cost of laser delay calibration and increase the calibration frequency, thereby ensuring the accuracy of the delay.

[0028] 3) The device is simple and can be realized by simply installing a movable device on the filter of the forward detector of a traditional flow cytometer.

[0029] 4) The calculation method is simple with high accuracy and reliability, can adapt to various situations, and has wide applicability.

[0030] The present invention is further described in detail below with reference to the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the delay between the optical signals obtained by each laser in a multi-laser flow cytometer with spatial excitation.

[0032] Figure 2 Schematic diagram of a laser delay calibration device for a flow cytometer in one embodiment.

[0033] Figure 3 FIG. 4 is a pulse signal diagram of three laser beams in one embodiment.

[0034] Figure 4 FIG. 1 is a pulse signal diagram with a clear dividing line in one embodiment.

[0035] Figure 5 Flowchart of a method for calibrating laser delay of a flow cytometer in one embodiment. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of this application more clear, the following further describes this application in detail with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain this application and are not intended to limit this application.

[0037] It should be noted that if the embodiments of the present invention involve directional indications (such as up, down, left, right, front, back, etc.), the directional indications are only used to explain the relative position relationship, movement status, etc. between the various components under a certain specific posture (as shown in the accompanying drawings). If the specific posture changes, the directional indications will also change accordingly.

[0038] In one embodiment, combined Figure 2, provides a flow cytometer laser delay calibration device, the device comprising a sample introduction device 101, a flow chamber 102, a waste liquid bucket 103, a laser system 201, a detector and spectroscopic system 202, an electronic system 301, a computer 401 and a movable device; the detector and spectroscopic system comprises a forward detector and a filter located in front of the forward detector, the filter being disposed on the movable device, the filter being movable to the front of the forward detector via the movable device and being coaxial with the forward detector, and also being movable away from the forward detector;

[0039] The filter can be removed by a movable device to deviate from the forward detector. The sample without fluorescent particles passes through the sampling device and the flow chamber and flows into the waste liquid barrel. The N lasers emitted by the laser system pass through the flow chamber and are incident on the detector and the spectroscopic system. The laser signal is transmitted to the computer through the electronic system. The computer processes the laser signal to obtain the laser delay correction value, and uses the laser delay correction value to align the signals generated by each laser.

[0040] During normal testing, the filter in front of the forward detector is active, allowing only light of a specific wavelength to pass through. When performing laser delay calibration, the filter is removed, allowing the forward detector to receive light signals of various wavelengths.

[0041] In one embodiment, combined Figure 5 , provides a flow cytometer laser delay calibration method, the method comprising the following steps:

[0042] Step 1, removing the filter in front of the forward detector by a movable device;

[0043] Step 2: Control the laser system to emit N laser beams;

[0044] Step 3: The non-fluorescent particle sample (blank pellet or other non-fluorescent particles) passes through the injection device and the flow chamber and flows into the waste liquid bucket;

[0045] Step 4: collect N continuous pulse signals as signals corresponding to N lasers;

[0046] Step 5, calculating the time difference between two adjacent pulse peaks as the laser delay correction value;

[0047] Step 6: Using a certain laser as a reference, perform laser delay on N laser signals according to the laser delay correction value obtained in step 5 to achieve alignment;

[0048] Step 7: Move the filter back to the front of the forward detector by the movable device to detect the sample to be detected.

[0049] Furthermore, in one embodiment, before executing step 3, the method further includes:

[0050] The sample without fluorescent particles is diluted so that there is a signal in only one laser area in the same time period, that is, two adjacent groups of N continuous pulse signals do not overlap.

[0051] Furthermore, in one embodiment, the laser pulse signal is collected in step 4 when the received signal value is greater than a set threshold.

[0052] Furthermore, in one embodiment, before executing step 6, the following steps are further included:

[0053] Repeat steps 4 and 5 several times;

[0054] The average of the multiple groups of laser delay correction values ​​is calculated to obtain a group of laser delay correction values ​​as the final laser delay correction value.

[0055] Here, the calculation of the mean of the multiple groups of laser delay correction values ​​may be replaced by: making a histogram of the multiple groups of laser delay correction values ​​to find the peak value.

[0056] As a specific example, in one of the embodiments, the present invention is further verified and explained in detail.

[0057] In the embodiment of the present invention, the flow cytometer includes three lasers: a blue laser, a purple-red laser, and a red laser. The blue laser is selected as the trigger laser and the reference laser (delay is 0). It should be noted that in actual work, any laser can be selected as the trigger laser and the reference laser.

[0058] The specific steps of the flow cytometer laser delay calibration method are as follows:

[0059] Step 1, remove the filter in front of the forward detector;

[0060] Step 2: Load 10 μm white non-fluorescent beads;

[0061] Step 3, select 488 blue laser as the trigger signal. When the signal value is greater than a certain value, start sampling at a fixed time, and get three consecutive pulse signals, which are the signals corresponding to the three lasers. You can also mine and collect signals at any time, intercept one group of pulses as the signal corresponding to the three lasers. Figure 3 shown.

[0062] Step 4, calculate Figure 3 The t1 and t2 shown are the laser delays of the violet laser and the red laser relative to the blue laser, respectively. At this point, by observing or calculating the magnitude pattern of the pulse peaks, it is possible to determine whether three consecutive pulse peaks are signals generated by the same particle.

[0063] Step 5: Move the filter of the forward detector and detect other samples normally.

[0064] Step 6: Using t1 and t2 as laser delays, align the pulse signals generated by the blue laser, purple laser, and red laser channels to obtain the signal values ​​of a particle in all detection channels.

[0065] To ensure that there is a signal in only one laser area within the same time period, the sample in step 2 needs to be diluted to a concentration below 105 cells / ml, as shown below. Figure 4 The pulse signals shown have clear dividing lines. Numbers 1 and 2 in the figure are the signals formed by two particles on three lasers.

[0066] The above step 4 can be repeated multiple times, and the peak value can be obtained by taking the average value or making a histogram to serve as the final laser delay, thereby improving the anti-interference ability and thus improving the accuracy of the calculation.

[0067] This method is also applicable to 2 or more lasers.

[0068] The pulse signals provided by this method can also be grouped based on their peak values, ensuring that several pulse signals are generated from the same particle. For example, if lasers are arranged in the order of 488nm, 405nm, and 638nm, the signal sizes obtained at each laser will vary due to the different laser powers. The peak value of the pulse at 488nm is significantly larger than that at the other lasers, so this can be used as the starting point of a group of signals.

[0069] In summary, the method for real-time automatic calibration of the laser delay of a flow cytometer proposed in the present invention can ensure the accuracy of the laser delay in real time, thereby ensuring the accuracy of the flow cytometer detection results.

[0070] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only illustrative of the principles of the present invention. Without departing from the spirit and scope of the present invention, any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

Claims

1. A flow cytometer laser delay calibration device, characterized in that: The device includes a sample injection device, a flow chamber, a waste liquid bucket, a laser system, a detector and a spectroscopic system, an electronic system, a computer, and a movable device; the detector and spectroscopic system include a forward detector and a filter located in front of the forward detector, the filter being arranged on the movable device. The filter can be moved to the front of the forward detector via the movable device, and the two can be coaxial, and can also be moved away from the forward detector. The filter can be removed by a movable device to deviate from the forward detector. The sample without fluorescent particles passes through the sampling device and the flow chamber and flows into the waste liquid barrel. The N lasers emitted by the laser system pass through the flow chamber and are incident on the detector and the spectroscopic system. The laser signal is transmitted to the computer through the electronic system. The computer processes the laser signal to obtain the laser delay correction value, and uses the laser delay correction value to align the signals generated by each laser.

2. The flow cytometer laser delay calibration device according to claim 1, characterized in that: The receiving aperture of the forward detector can cover the range of N laser spots.

3. A method for calibrating laser delay of a flow cytometer based on the device according to any one of claims 1 to 2, characterized in that: The method comprises the following steps: Step 1, removing the filter in front of the forward detector by a movable device; Step 2: Control the laser system to emit N laser beams; Step 3, the sample without fluorescent particles passes through the sampling device and the flow chamber and then flows into the waste liquid bucket; Step 4: collect N continuous pulse signals as signals corresponding to N lasers; Step 5, calculating the time difference between two adjacent pulse peaks as the laser delay correction value; Step 6: Using a certain laser as a reference, perform laser delay on the N laser signals according to the laser delay correction value obtained in step 5 to achieve alignment.

4. The flow cytometer laser delay calibration method according to claim 3, characterized in that: Before step 3 is executed, the following steps are also included: The sample without fluorescent particles is diluted so that there is a signal in only one laser area in the same time period, that is, two adjacent groups of N continuous pulse signals do not overlap.

5. The flow cytometer laser delay calibration method according to claim 3, characterized in that: The laser pulse signal is collected in step 4 when the received signal value is greater than a set threshold.

6. The flow cytometer laser delay calibration method according to claim 3, characterized in that: Before executing step 6, the following steps are also included: Repeat steps 4 and 5 several times; Calculate the mean or median of the multiple groups of laser delay correction values ​​to obtain a group of laser delay correction values ​​as the final laser delay correction values.

7. The flow cytometer laser delay calibration method according to claim 6, characterized in that: The method of calculating the mean of the plurality of groups of laser delay correction values ​​may be replaced by: making a histogram of the plurality of groups of laser delay correction values ​​to find the peak value.

8. The flow cytometer laser delay calibration method according to claim 3, characterized in that: The method further includes: Step 7: Move the filter back to the front of the forward detector by the movable device to detect the sample to be detected.

9. A computer device comprising a memory, a processor, and a computer program stored in the memory and executable on the processor, wherein: When the processor executes the computer program, the steps of the method according to any one of claims 3 to 8 are implemented.

10. A computer-readable storage medium having a computer program stored thereon, characterized in that: When the computer program is executed by a processor, the steps of the method according to any one of claims 3 to 8 are implemented.

Images