JP6741100B2 - Microparticle analyzer and microparticle analysis method - Google Patents

Description

The present invention relates to a display method and a fine particle analyzer. More specifically, it relates to a technique for analyzing the type of fluorescence emitted from microparticles.

In general, flow cytometry (flow cytometer) is used to analyze biologically relevant microparticles such as cells, microorganisms, and liposomes (see, for example, Non-Patent Document 1). Flow cytometry is a method of irradiating laser light (excitation light) of a specific wavelength to microparticles that flow in a row in a channel and detect fluorescence and scattered light emitted from each microparticle. , A method of analyzing a plurality of fine particles one by one. In this flow cytometry, the light detected by the photodetector is converted into an electric signal, converted into a numerical value, and statistically analyzed to determine the type, size, and structure of each individual microparticle.

Further, in recent years, multi-color analysis using a plurality of fluorescent dyes has become widespread in flow cytometry (see, for example, Patent Documents 1 and 2). These conventional flow cytometers are usually provided with a plurality of light sources having different wavelengths and a plurality of detectors for detecting the light emitted from each dye. On the other hand, since a fluorescent dye has a spectrum, when a plurality of fluorescent dyes are used in one measurement like multicolor analysis, light from fluorescent dyes other than the target leaks into the detector, and the accuracy of analysis decreases. Therefore, in the conventional flow cytometer, in order to extract only the light information from the target fluorescent dye, when the light detected by the photodetector is converted into an electric signal and numerically converted, a mathematical correction, that is, Fluorescent correction is performed.

JP, 2006-230333, A Japanese Patent Publication No. 2008-500558

Supervision by Keiko Nakauchi, "Cell Engineering Separate Volume, Experimental Protocol Series, Free Flow Cytometry", 2nd Edition, Shujunsha Co., Ltd., published August 31, 2006

As described above, each fluorescent dye has a unique spectrum, and its spectral information is important data as a characteristic of the fluorescent dye itself. However, the conventional flow cytometer detects the light from the target fluorescent dye by, for example, a sensor that receives light having a specific bandwidth and treats the detected data as corresponding data. However, there is a problem in that the spectrum information of can not be presented.

Therefore, it is a main object of the present invention to provide a technique capable of visually understanding spectral information of fluorescence emitted from microparticles.

In the present invention, a plurality of microparticles modified with a dye flowing through the channel, a light irradiation unit for irradiating one excitation light, and the fluorescence emitted from the microparticles by irradiation of the excitation light, a plurality of A detection unit having a plurality of detectors that simultaneously detect in the wavelength region, and detecting fluorescence in the wavelength region of the number of dyes for each of the microparticles in the detection unit, based on the detected fluorescence data, the microparticles Provided is a microparticle analysis device having an analysis unit for analyzing information regarding a dye modified in the above.
The present invention may include a conversion unit that converts the light of each wavelength region detected by the detection unit into a voltage pulse.
Further, in the present invention, the plurality of microparticles modified with the dye are undyed microparticles, microparticles modified with one dye, microparticles modified with two or more dyes, or any of these. Alternatively, a mixture of two or more kinds may be used.
Further, in the present invention, the light irradiation unit may include a plurality of light sources that emit excitation light having different wavelengths.
In addition, in the present invention, the detector may be a sensor or a channel of a multichannel photomultiplier tube, in which case the number of channels of the sensor or the multichannel photomultiplier tube may be six or more. ..
Further, in the present invention, the detection unit may have detectors equal to or more than the number of dyes modified in the fine particles.
Further, in the present invention, the analysis unit may analyze the information regarding the dye modified to the microparticles based on the detected fluorescence data to specify the group of microparticles.
In addition, in the present invention, the detected fluorescence data may have a display unit that displays a spectrum using wavelength information indicating the wavelength region as a parameter, and in this case, the display unit is a gate for the spectrum. The data of the area subjected to the gating processing may be displayed according to the processing. Further, in this case, the data of the area subjected to the gating processing may be displayed as a two-dimensional plot.

In the present invention, one excitation light is irradiated to a plurality of microparticles modified with a dye flowing through the flow path, and the fluorescence emitted from the microparticles by the irradiation of the excitation light is simultaneously emitted in a plurality of wavelength regions. In a detection unit having a plurality of detectors for detecting, fluorescence is detected in a wavelength region equal to or more than the number of the dyes for each of the microparticles, and based on the detected fluorescence data, information about the dye modified to the microparticles is analyzed. Also provided is a method for analyzing microparticles.

According to the present invention, since it is possible to present the spectrum information of the fluorescence emitted from the fine particles, the user can visually understand the measurement data.

It is a figure which shows the structure of the microparticle analysis apparatus which concerns on the 2nd Embodiment of this invention. It is a figure which shows the example of a display of a measurement result. It is a figure which shows the method of applying a gate about the display example shown in FIG. It is a figure which shows the example of a display after a gate.

Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the accompanying drawings. The present invention is not limited to the embodiments described below. The description will be given in the following order.

1. First embodiment (example of spectrum display method)
2. Second embodiment (an example of a fine particle analyzer for displaying a spectrum)

<1. First Embodiment>
[overall structure]
First, a data display method according to the first embodiment of the present invention will be described. In the data display method of the present embodiment, fluorescence emitted from microparticles such as cells and microbeads is simultaneously detected in a plurality of wavelength regions, and the detection data for each wavelength region is spectrally displayed.

[Detection data]
In the data display method of the present embodiment, the light detected by the photodetector is converted into voltage pulses (electrical signals) for each wavelength region and quantified to obtain detection data. Specifically, the height, width or area of the voltage pulse is obtained and used as detection data.

[Display form]
In the data display method according to this embodiment, the detection data for each wavelength region obtained by the above-described method is displayed as a spectrum. The display method is not particularly limited, and examples thereof include a density plot, a dot plot, and a contour line. Further, it is also possible to extract only the data of a specific region from the displayed spectrum and display the spectrum chart on an enlarged scale, or display a histogram or a two-dimensional plot.

Further, in this data display method, it is also possible to perform arithmetic processing on the detection data acquired by continuously detecting a plurality of fine particles and display the result. The arithmetic processing includes, for example, integrating detection data and calculating an average value of detection data of all particles. Furthermore, in the data display method of the present embodiment, it is possible to display while detecting, but it is also possible to temporarily store the detected data and read out and display this stored data. Also in that case, the detection data may be arithmetically processed and the result may be displayed.

In the conventional two-dimensional plot, it was difficult for the user to visually judge the data even if the fluorescence correction was performed. On the other hand, in the data display method of the present embodiment, since the spectrum information of the fluorescence emitted from the microparticles can be presented, the user can display the measurement data regardless of the presence or absence of the fluorescence correction and the result of the fluorescence correction. Can be visually understood. Further, in the data display method of the present embodiment, it is possible to obtain various kinds of information regarding the fine particles by extracting the data in the specific area.

The data display method according to the present embodiment is applicable not only to analysis devices for microparticles and cells, but also to manufacturing devices, inspection devices, medical devices, and the like.

<2. Second Embodiment>
[Overall configuration of device]
Next, a fine particle analyzer according to the second embodiment of the present invention will be described. FIG. 1 is a diagram showing the configuration of the fine particle analyzer of the present embodiment, and FIG. 2 is a diagram showing a display example of the measurement results. As shown in FIG. 1, the microparticle analysis apparatus 1 of the present embodiment is provided with at least a detection unit 2, a conversion unit 3, an analysis unit 4, and a display unit 5.

[Configuration of Detection Unit 2]
The detection unit 2 may have a configuration capable of simultaneously detecting the fluorescence emitted from the fine particles to be analyzed in a plurality of wavelength regions. Specifically, a configuration in which a sensor capable of detecting each wavelength region is arranged, and a detector capable of simultaneously detecting a plurality of lights such as a multi-channel photo-multiplier tube (PMT). Can be provided. The number of wavelength regions detected by the detection unit 2, that is, the number of detector channels or sensors provided in the detection unit 2 may be equal to or greater than the number of dyes used. In general, 6 to 8 colors are generally used, and the maximum is 10 to 12 colors. Therefore, 12 or more colors are preferable.

Further, in the microparticle analysis device 1 of the present embodiment, a spectroscope is provided in the detection unit 2, and the fluorescence emitted from the microparticles is dispersed by this spectroscope and then enters the detector such as the multi-channel PMT. It may be configured. Further, the detection unit 2 may be provided with an objective lens, a condenser lens, a pinhole, a band cut filter, a dichroic mirror, etc., if necessary.

[Configuration of conversion unit 3]
The conversion unit 3 converts the light in each wavelength region detected by the detection unit 2 into a voltage pulse (electrical signal), and for example, an AD converter or the like can be used.

[Configuration of analysis unit 4]
In the analysis unit 4, the voltage pulse converted by the conversion unit 3 is processed and quantified by using an electronic computer or the like to obtain detection data. Specifically, the height (peak), the width (width), or the area (integral) of the voltage pulse is obtained, and these are used as detection data. Then, the result is associated with each wavelength region and stored in a storage unit (not shown) or the like.

[Configuration of display unit 5]
The display unit 5 displays the detection data obtained by the processing in the analysis unit 4, that is, the measurement result of the fine particle analyzer 1 of the present embodiment. Specifically, on the display unit 5, for example, as shown in FIG. 2, the channel number of the detection unit is plotted on the horizontal axis and the intensity is plotted on the vertical axis to show the fluorescence intensity for each channel. Is displayed. At this time, if the detection wavelength is set to increase as the channel number increases, the spectrum information of the detection light (fluorescence) can be obtained.

[Operation of Microparticle Analyzer 1]
Next, the operation of the microparticle analyzer 1 of this embodiment will be described. The microparticles to be analyzed by the microparticle analysis device 1 of the present embodiment are not particularly limited, and examples thereof include cells and microbeads. The type and number of fluorescent dyes that modify these microparticles are not particularly limited, and FITC (fluorescein isothiocyanete: C21H11NO5S), PE (phycoerythrin), PerCP (periidinin chlorophyll protein), PE-Cy5, and PE-Cy7. Known dyes such as can be appropriately selected and used as necessary. Furthermore, each microparticle may be modified with a plurality of fluorescent dyes.

When optically analyzing the microparticles using the microparticle analysis apparatus 1, first, excitation light is emitted from the light source of the light irradiation unit and irradiated to the microparticles flowing in the flow path. Next, the fluorescence emitted from the fine particles is detected by the detection unit 2. At that time, for example, the fluorescence emitted from the microparticles is dispersed by a spectroscope such as a prism or a diffraction grating, and light of different wavelength regions is supplied to each sensor or PMT channel provided in the detection unit 2. Make it incident.

After that, the data of each wavelength region acquired by the detection unit 2 is converted into a voltage pulse by the conversion unit 3, further quantified by the analysis unit 4, and stored. Then, the result is displayed on the display unit 5 in a format such as "density plot" shown in FIG. The “density plot” shown in FIG. 2 is the result of measurement of a mixture of a sample modified with FITC alone, a sample modified with PE only, and a Negative sample using a 32-channel PMT.

In this "density plot", the horizontal axis represents the PMT channel number (wavelength-dependent number), and the vertical axis represents the fluorescence intensity. The cumulative number 0 is blue, and the cumulative number increases, that is, as the density increases. I try to make it red. At this time, if the number of the channel having the smallest detection wavelength is set to 1 and the detection wavelength becomes higher as the number becomes larger, information corresponding to the fluorescence spectrum can be obtained. Then, from this spectrum information, the user can recognize what fluorescence the sample is labeled with.

Further, in the microparticle analyzer 1 of the present embodiment, it is possible to perform gating in which only a specific sample group is selected from the data shown in FIG. 2 and the data of only that sample (microparticle) is extracted and displayed. The display form at that time is not particularly limited, and it is possible to display in various forms such as a histogram, a two-dimensional plot, and a spectrum chart. FIG. 3 is a diagram showing a method of applying a gate to the display example shown in FIG. 2, and FIG. 4 is a diagram showing a display example after the gate. In the conventional flow cytometer, since the gating process is usually performed on the two-dimensional plot, only the information for two channels is gated.

On the other hand, as shown in FIG. 3, in the microparticle analysis apparatus 1 of the present embodiment, the fluorescence spectra are gated together for, for example, 10 channels. Then, as shown in FIG. 4, the data of the gated sample can be put in the dot plot mode or the color can be changed in the spectrum plot. Thereby, more explicit gating can be realized, and the user can clearly confirm the data. It is also possible to display the data of the gated sample in a two-dimensional plot.

Further, since the gated region can be selected based on the fluorescence spectrum, for example, two kinds of fluorescent dyes are immobilized on one microparticle, or two microparticles modified with different fluorescent dyes may be included. It can be easily carved. Furthermore, by gating the part where there is no leakage from other fluorescent dyes, the user can visually recognize various information from the displayed data regardless of the presence or absence of the fluorescence correction and the result of the fluorescence correction. It is possible to obtain.

In addition, in the microparticle analysis apparatus 1 of the present embodiment, not only the “density plot” and “dot plot” described above, but also “contour plot”, “two-dimensional plot”, “two-dimensional histogram”, etc. may be displayed. It is possible. It is also possible to continuously measure a plurality of fine particles, integrate the data detected by the detection unit 2 at any time, and reflect the data in the fluorescence spectrum displayed on the display unit 5 in real time. Furthermore, the stored data can be read and used for display, or the results of continuous measurement of a plurality of fine particles can be temporarily stored and displayed using the average value of all samples.

1 Micro Particle Analyzer 2 Detecting Section 3 Converting Section 4 Analyzing Section 5 Display Section

Claims (11)

A light irradiation unit for irradiating at least one excitation light to a plurality of fine particles modified with a dye flowing through the flow path,
The fluorescence emitted from the fine particles by irradiation of the excitation light, and a detector having a plurality of detectors that detect a plurality of wavelengths,
Detecting fluorescence in the wavelength region of the number of dyes or more for each of the microparticles in the detection unit,
Based on the detected fluorescence data, an analysis unit that analyzes information about the dye modified to the microparticles ,
The detected fluorescence data, the wavelength information indicating the wavelength region as a parameter, a display unit for displaying a spectrum,
Have
The fine particle analysis apparatus, wherein the spectrum display is displayed in different colors according to the integrated number of detection data of the plurality of fine particles . The microparticle analyzer according to claim 1, further comprising a conversion unit that converts the light of each wavelength region detected by the detection unit into a voltage pulse. The plurality of microparticles modified with the dye are undyed microparticles, microparticles modified with one dye, microparticles modified with two or more dyes, or a mixture of any two or more thereof. The microparticle analyzer according to claim 1 or 2, which is obtained by The microparticle analyzer according to claim 1, wherein the light irradiation unit has a plurality of light sources that emit excitation light having different wavelengths. The microparticle analyzer according to any one of claims 1 to 4, wherein the detector is a sensor or a channel of a multichannel photomultiplier tube. The microparticle analyzer according to claim 5, wherein the number of channels of the sensor or the multichannel photomultiplier tube is 6 or more. 7. The microparticle analysis apparatus according to claim 1, wherein the detection unit has a number of detectors equal to or more than the number of dyes modified on the microparticles. The microparticle analysis according to any one of claims 1 to 7, wherein the analysis unit analyzes information about a dye modified on the microparticles based on the detected fluorescence data to identify a group of microparticles. apparatus. The microparticle analyzer according to any one of claims 1 to 8, wherein the display unit displays the data of the region subjected to the gating processing according to the gating processing on the spectrum. The microparticle analyzer according to claim 9 , wherein the data of the region subjected to the gating process is displayed as a two-dimensional plot. Irradiating at least one excitation light to a plurality of dye-modified microparticles flowing through the channel,
The fluorescence emitted from the fine particles by irradiation of the excitation light, the detection unit having a plurality of detectors that detect a plurality of wavelengths, the fluorescence by the number or in the wavelength range of the dye for each of the fine particles Detect and
Based on the detected fluorescence data, analyze information about the dye modified to the microparticles ,
The detected fluorescence data is displayed as a spectrum using wavelength information indicating the wavelength region as a parameter,
The method for analyzing microparticles , wherein the spectrum display is displayed in different colors according to the integrated number of detection data of the plurality of microparticles .