JP6489167B2 - Data display method and fine particle analyzer - Google Patents

Description

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

  Generally, when analyzing biologically relevant microparticles such as cells, microorganisms, and liposomes, flow cytometry (flow cytometer) is used (for example, see Non-Patent Document 1). Flow cytometry irradiates laser light (excitation light) of a specific wavelength to micro particles that flow in a line in a flow path, and detects fluorescence and scattered light emitted from each micro particle. In this method, a plurality of fine particles are analyzed one by one. In this flow cytometry, the type, size, and structure of individual microparticles can be determined by converting the light detected by the photodetector into an electrical signal, digitizing it, and performing statistical analysis.

  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 usually include a plurality of light sources having different wavelengths and a plurality of detectors for detecting light emitted from each dye. On the other hand, since the fluorescent dye has a spectrum, if a plurality of fluorescent dyes are used in one measurement as in multi-color analysis, light from fluorescent dyes other than the target leaks into the detector, and the analysis accuracy 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 electrical signal and converted into a numerical value, mathematical correction, that is, Fluorescence correction is performed.

JP 2006-230333 A Special table 2008-500558

Supervised by Hiromitsu Nakauchi, "Cell Engineering Separate Volume, Experimental Protocol Series, Flow Cytometry," Second Edition, Shujunsha Co., Ltd., August 31, 2006

  As described above, each fluorescent dye has a unique spectrum, and the spectral information is important data representing the characteristics of the fluorescent dye itself. However, a conventional flow cytometer detects light from a target fluorescent dye with a sensor that receives light having a specific bandwidth, for example, and treats the detected data as corresponding data. There is a problem that it is not possible to present the spectrum information.

  Therefore, the main object of the present invention is to provide a technique capable of visually understanding spectrum information of fluorescence emitted from fine particles.

In the present invention, there is provided a data display method for spectrally displaying detection data obtained by simultaneously detecting fluorescence emitted from fine particles flowing through a flow path in a plurality of wavelength regions using wavelength information indicating the wavelength region as a parameter. provide.
In the present invention, the detection may be performed by a multi-channel photomultiplier tube. In this case, the wavelength information may be a channel number or a wavelength of the multichannel photomultiplier tube. In this case, the detection data may be obtained by continuously detecting a plurality of fine particles. In this case, the plurality of microparticles may be microparticles modified with two or more dyes or two or more kinds of microparticles modified with different dyes. In this case, the number of channels of the multichannel photomultiplier tube may be equal to or greater than the number of the dyes.
Furthermore, in the present invention, the detection data may be further processed and the result displayed.
In addition, in the present invention, the spectrum may display the fluorescence intensity based on the detection data as a vertical axis.
In the present invention, the spectrum may be displayed with the wavelength information as a horizontal axis.
Furthermore, in the present invention, data of the area subjected to the gating process may be displayed in accordance with the gating process for the spectrum. In this case, the data of the area subjected to the gating process may be displayed as a two-dimensional plot.

In the present invention, a light irradiation unit that irradiates microparticles flowing through a flow path with excitation light, a detection unit that simultaneously detects fluorescence emitted from the microparticles in a plurality of wavelength regions, and detection based on the detection unit There is also provided a microparticle analyzer having at least a display unit for displaying data with spectrum information using the wavelength information indicating the plurality of wavelength regions as a parameter .
In the present invention, the detection unit may include a multichannel photomultiplier tube. In this case, the wavelength information may be a channel number or a wavelength of the multichannel photomultiplier tube.
Furthermore, in this invention, you may further have an analysis part which acquires the detection data corresponding to the light for every said wavelength range acquired with the said detection part.

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

It is a figure which shows the structure of the microparticle analyzer based 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 example of a display shown in FIG. It is a figure which shows the example of a display after a gate.

DESCRIPTION OF EMBODIMENTS Hereinafter, embodiments for carrying out the present invention will be described in detail with reference to the accompanying drawings. In addition, this invention is not limited to each embodiment shown below. The description will be given in the following order.

1. First Embodiment (Example of spectrum display method)
2. Second Embodiment (Example of Fine Particle Analyzer for Spectrum Display)

<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 this 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 format]
In the data display method of this embodiment, the detection data for each wavelength region obtained by the above-described method is spectrally displayed. The display method is not particularly limited, and examples thereof include density plots, dot plots, and contour lines. It is also possible to extract only data in a specific area from the displayed spectrum and display the spectrum chart in an enlarged manner, 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 microparticles and display the result. Examples of the calculation process include integrating detection data and calculating an average value of detection data of all fine particles. Furthermore, in the data display method of the present embodiment, the display can be performed while being detected, but the detected data may be temporarily stored, and the stored data may be read and displayed. Also in this case, the detection data may be processed and the result displayed.

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

  The data display method of the present embodiment is not limited to analysis devices such as microparticles and cells, but can be applied to, for example, manufacturing apparatuses, inspection apparatuses, and medical apparatuses.

<2. Second Embodiment>
[Overall configuration of the device]
Next, a microparticle analyzer according to a second embodiment of the present invention will be described. FIG. 1 is a diagram showing a configuration of a microparticle analysis apparatus according to the present embodiment, and FIG. As shown in FIG. 1, the microparticle analysis apparatus 1 according to the present embodiment includes 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 part 2 should just be the structure which can detect simultaneously the fluorescence emitted from the microparticle of analysis object in a several wavelength area | region. Specifically, a detector capable of detecting a plurality of lights simultaneously such as a configuration in which a sensor capable of detecting the wavelength region is arranged for each wavelength region, or a multi-channel photomultiplier tube (PMT). It can be set as the structure provided with. The number of wavelength regions detected by the detection unit 2, that is, the number of channels or sensors of the detectors disposed in the detection unit 2 may be equal to or greater than the number of dyes to be used. Then, since 6 to 8 colors are common and 10 to 12 colors at the maximum, it is preferably 12 or more.

  Further, in the microparticle analysis apparatus 1 of the present embodiment, a spectroscope is provided in the detection unit 2, and the fluorescence emitted from the microparticles is split by the spectroscope and then enters a detector such as a multi-channel PMT. It is good also as a structure. Furthermore, the detection unit 2 may be provided with an objective lens, a condenser lens, a pinhole, a band cut filter, a dichroic mirror, and the like as necessary.

[Configuration of Conversion Unit 3]
The conversion unit 3 converts light in each wavelength region detected by the detection unit 2 into voltage pulses (electrical signals). For example, an AD converter or the like can be used.

[Configuration of Analysis Unit 4]
In the analysis part 4, the voltage pulse converted by the conversion part 3 is processed and quantified using an electronic computer etc., and it is set as detection data. Specifically, the height (peak), width (width), or area (integral) of the voltage pulse is obtained, and these are used as detection data. 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 microparticle analysis apparatus 1 of the present embodiment. Specifically, for example, as shown in FIG. 2, the display unit 5 has a “density plot” in which the horizontal axis represents the channel number of the detection unit and the vertical axis represents the intensity, indicating the fluorescence intensity for each channel. Is displayed. At this time, if the detection wavelength is increased as the channel number increases, spectrum information of the detection light (fluorescence) can be obtained.

[Operation of Fine Particle Analyzer 1]
Next, operation | movement of the microparticle analyzer 1 of this embodiment is demonstrated. The fine particles to be analyzed by the fine particle analyzer 1 of the present embodiment are not particularly limited, and examples thereof include cells and micro beads. The kind and number of fluorescent dyes that modify these fine particles are not particularly limited, and FITC (fluorescein isothiocyanete: C ₂₁ H ₁₁ NO ₅ S), PE (phycoerythrin), PerCP (periidinin chlorophyll protein), PE Known dyes such as -Cy5 and PE-Cy7 can be appropriately selected and used as necessary. Furthermore, each microparticle may be modified with a plurality of fluorescent dyes.

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

  Thereafter, the data of each wavelength region acquired by the detection unit 2 is converted into a voltage pulse by the conversion unit 3, and further quantified and stored by the analysis unit 4. 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 a result of measuring a mixture of a sample modified only with FITC, a sample modified only with PE, and a negative sample using a 32-channel PMT.

  In this “density plot”, the PMT channel number (wavelength-dependent number) is taken on the horizontal axis, the fluorescence intensity is taken on the vertical axis, the accumulated number 0 is blue, and the accumulated number increases, that is, as the density increases. The color is red. At this time, if the number of the channel with the smallest detection wavelength is 1, and the detection wavelength is increased as the number increases, information corresponding to the fluorescence spectrum can be obtained. From this spectrum information, the user can recognize what fluorescence the sample is labeled.

  Furthermore, in the microparticle analyzer 1 of the present embodiment, it is possible to perform gating that selects only a specific sample group from the data shown in FIG. 2 and extracts and displays only the data of the sample (microparticle). The display form at that time is not particularly limited, and can be displayed 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 a conventional flow cytometer, since a gating process is usually performed on a two-dimensional plot, only information for two channels is gated.

  On the other hand, as shown in FIG. 3, in the microparticle analyzer 1 of this embodiment, gates are collectively applied for, for example, 10 channels for the fluorescence spectrum. Then, as shown in FIG. 4, it is possible to set the data of the gated sample to the dot plot mode or change the color by the spectrum plot. As a result, more explicit gating can be realized, and the user can clearly check the data. It is also possible to display the data of the gated sample as a two-dimensional plot.

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

  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”, and the like may be displayed. Is possible. It is also possible to continuously measure a plurality of microparticles, integrate the data detected by the detection unit 2 as needed, and reflect it in real time on the fluorescence spectrum displayed on the display unit 5. Furthermore, the stored data can be read out and used, or the results of continuous measurement of a plurality of microparticles can be temporarily stored and displayed using the average value of all samples.

DESCRIPTION OF SYMBOLS 1 Fine particle analyzer 2 Detection part 3 Conversion part 4 Analysis part 5 Display part

Claims (15)

For each of a plurality of fine particles flowing through the flow path, detection data obtained by simultaneously detecting fluorescence emitted by light irradiation in a plurality of wavelength regions is spectrum-displayed using the wavelength information indicating the wavelength region as a parameter. ,
A data display method for displaying different colors according to the integrated number of detection data of the plurality of microparticles .   The data display method according to claim 1, wherein the detection is performed by a multi-channel photomultiplier tube.   The data display method according to claim 2, wherein the wavelength information is a channel number or a wavelength of the multichannel photomultiplier tube.   The data display method according to claim 2 or 3, wherein the detection data is acquired by continuously detecting a plurality of fine particles.   The data display method according to claim 4, wherein the plurality of microparticles are microparticles modified with two or more dyes or two or more kinds of microparticles modified with different dyes.   The data display method according to claim 5, wherein the number of channels of the multichannel photomultiplier tube is equal to or greater than the number of the dyes.   Furthermore, the data display method as described in any one of Claim 4 to 6 which calculates the said detection data, and displays the result.   The data display method according to any one of claims 1 to 7, wherein the spectrum displays a fluorescence intensity based on the detection data as a vertical axis.   The data display method according to claim 1, wherein the spectrum displays the wavelength information as a horizontal axis.   The data display method according to any one of claims 1 to 9, further comprising displaying data of the area subjected to the gating process in accordance with a gating process for the spectrum.   The data display method according to claim 10, wherein the data of the gated region is displayed as a two-dimensional plot. A light irradiation unit for irradiating excitation light to a plurality of microparticles flowing through the flow path;
A detection unit that simultaneously detects fluorescence emitted by irradiation of excitation light in a plurality of wavelength regions for each of the plurality of microparticles;
The display unit displays the detection data based on the detection unit as a parameter with wavelength information indicating the plurality of wavelength regions as a parameter, and displays the color according to the integrated number of detection data of the plurality of microparticles ;
A microparticle analyzer having at least.   The microparticle analyzer according to claim 12, wherein the detection unit includes a multichannel photomultiplier tube.   The fine particle analyzer according to claim 12 or 13, wherein the wavelength information is a channel number or a wavelength of the multichannel photomultiplier tube. An analysis unit for acquiring detection data corresponding to the light for each wavelength region acquired by the detection unit;
The fine particle analyzer according to any one of claims 12 to 14, further comprising: