JPH08178826A - Flow cytometer - Google Patents

Abstract

PURPOSE: To display a desired image easily by picking up a particle image when the coordinates representative of a detected particle are present in a designated coordinate region. CONSTITUTION: A data processor 100 obtains various feature parameters and an image pickup control circuit 200 judges the kind of each cell in real time using these parameters and controls so that the image only of a previously designated kind of cell can be selectively picked up. When a designated kind of cell is judged to be so, an emission trigger signal for picking up the image of that cell is outputted to a pulse light source 2. A pulse light is then introduced through an optical fiber to a sheath flow cell and a sample flow is irradiated therewith. A pulse light transmitted through the sample flow is focused through a projection lens onto the light receiving face of a video camera and the optical transmission image of the cell is produced. A video signal VS is outputted from the camera 15 to a data processor 300 and a digital image is stored therein.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a flow cytometer having a function of imaging cells and particles flowing in a sheath flow cell.

[0002]

2. Description of the Related Art A cell suspension treated with a reagent such as a fluorescent dye is introduced into a thin glass tube, and the sample stream is irradiated with laser light.
A flow cytometer is known that images cells and particles of a desired size flowing in a thin glass tube and stores the image (for example, Japanese Patent Laid-Open No. 63-941).
No. 56).

[0003]

However, such a conventional flow cytometer has a problem that it is not easy to select and display a desired image from a large number of stored images. The present invention has been made in consideration of such circumstances, and provides a flow cytometer capable of easily displaying an image desired to be displayed on a distribution map.

[0004]

The present invention relates to a sheath flow cell for forming a sample flow containing particles, a detecting means for detecting a signal from particles in a first flow region of the sample flow, and a second flow region of the sample flow. An image pickup means for picking up an image of particles,
Display means, calculating means for calculating a plurality of characteristic parameters representing the characteristics of each particle based on the output of the detecting means, distribution map creating means for creating a distribution map of the characteristic parameters and displaying it on the display means, And a coordinate designating means for designating an arbitrary coordinate area, and when the coordinate area is designated by the coordinate designating means and the coordinates representing the particles detected in the first basin are present in the designated coordinate area, the particle image is captured. Image pickup control means for making the means take an image, image storage means for storing the imaged particle image in association with the coordinates, and coordinates representing the imaged particles in the distribution map when the coordinate designating means designates the coordinates. The present invention provides a flow cytometer including a reading means for reading the corresponding particle image from the image storage means and displaying it on the display means.

The sheath flow cell of the present invention is a flow cell capable of forming a thin flow of a sample liquid by a hydrodynamic effect by wrapping a sample liquid containing particles in the sheath liquid and flowing the sample liquid. Can be used.

The particles targeted by the flow cytometer of the present invention are mainly blood cells and cells contained in blood and urine, but microorganisms such as yeast and lactic acid bacteria, or industrial powders can also be measured. Good. In order to classify the types of blood cells and cells, intracellular types, nucleic acids, etc. may be reacted with a specific fluorescent reagent and the fluorescence intensity thereof may be measured.

As the detection means for detecting the particle signal, it is possible to use a means by electrical detection, a means by optical detection or the like. For example, a light source that irradiates the first flow region of the sample flow with light (for example, a continuous light source that irradiates light continuously such as a laser, a halogen lamp, or a tungsten lamp),
A photodetector that outputs the intensity of light such as scattered light or fluorescence from particles illuminated by this light source (eg, photodiode, phototransistor or photomultiplier)
It can be composed of and.

A light source is preferably provided for illuminating the second stream of the sample stream for imaging, which includes a laser,
Continuous light source that continuously emits light such as a halogen lamp or a tungsten lamp, or a pulsed laser (for example,
An intermittent light source that emits light intermittently, such as Spectra-Physics, 7000 series) or multi-strobe (for example, Sugawara Laboratory Ltd., DSX series) can be used. It is usually preferable to use an optical shutter in combination with the continuous light source as an intermittent light source. As the optical shutter, a known acousto-optic effect element (acounsto-opt) is used.
ic modulator 9 or electro-optic effect element
modulator) etc. can be used.

As the image pickup means for picking up the image of the particles, a general video camera for picking up a two-dimensional image may be used. In that case, an image intensifier for enhancing a weak light image is used. It is preferable to use the provided one.
Further, the image intensifier may be provided with shutter means. The second watershed may be downstream of the first watershed or in a similar position.

A CRT, a liquid crystal display or the like can be used as the display means. Distribution map creating means for creating a distribution map of the characteristic parameters and displaying it on the display means, imaging control means for causing the imaging means to capture the particle image, image storage means for storing the captured particle image in association with its coordinates, and stored. The reading means for reading the particle image from the image recording means and displaying it on the display means is a CPU, a ROM and a RAM.
It consists of a microcomputer. The distribution diagram is a general term for representing the distribution state of characteristic parameters, and a suitable example is a two-dimensional scattergram.

The coordinate designating means for designating an arbitrary coordinate area in the distribution chart can designate the coordinates of an arbitrary dot in the distribution chart and the coordinate area of an arbitrary shape, such as a keyboard or a mouse. It is preferable to use the input means of.

[0012]

In the present invention, the sheath flow cell forms a sample flow containing particles, the light detecting means detects a signal from the particles in the first flow region of the sample flow, and the imaging means uses the second flow area.
Take an image of the particles in the watershed. The distribution chart creating means creates a distribution chart of characteristic parameters representing the characteristics of each particle based on the output of the detecting means and displays it on the displaying means.

The coordinate designating means designates arbitrary coordinates and coordinate areas in the displayed distribution map. The imaging control means,
When the coordinate area is designated by the designating means and the coordinates representing the particles detected in the first watershed are present in the designated area, the image capturing means captures the particle image, and the image storing means
The captured particle image is stored in association with its coordinates. Then, when the coordinates designating the imaged particles in the distribution map are designated by the coordinate designating means, the reading means reads the particle image corresponding to the coordinates from the image storage means and causes the display means to display the particle image.

[0014]

DESCRIPTION OF THE PREFERRED EMBODIMENTS An optical system according to an embodiment of the present invention is shown in FIGS. In this embodiment, two light sources, that is, a continuous light emitting laser light source 1 for detecting scattered light and fluorescence and a pulse light source 2 for capturing a cell image are provided. This 2
Lights L1 and L2 from the two light sources 1 and 2 are 90 ° so as to be orthogonal to each other with respect to the rectangular sheath flow cell 3 (the sample flow flows in a direction perpendicular to the paper surface in FIG. 1) as shown in FIG.
Irradiate by crossing. Further, in this embodiment, the pulsed light for capturing the cell image is applied to the sample flow in the sheath flow cell 3 downstream (for example, about 0.5 mm) from the irradiation position of the continuous emission laser light source 1. I have to. By shifting the irradiation position in this way, a clearer cell image that is not affected by scattered light or fluorescence from cells can be captured.

The cell suspension treated with an appropriate reagent is guided to the sheath flow cell 3 to form a sample flow which is narrowed down by the sheath liquid. The continuous emission laser light is narrowed down by the condenser lens 4 and applied to the sample flow. When cells P1, P2, P3 ... Pn flow into this irradiation area, scattered light or fluorescence from each cell is collected by the condenser lens 5.
The scattered light collected by is reflected by the dichroic mirror 6 and received by the photodiode 7. The green and red fluorescences are the dichroic mirror 8 and
The light is reflected by the mirror 9 and received and multiplied by the photomultiplier tubes 10 and 11.

Photodiode 7, photomultiplier tube 10,
The scattered light intensity signal S1 and the fluorescence intensity signals S2 and S3 detected by 11 are the signal processing device 100 shown in FIG.
The information such as the height, area and width of each detection signal pulse is A / D converted and various characteristic parameters are calculated.

FIG. 3 shows the configuration of the signal processing system of this embodiment. In this signal processing system, the data processing device 100
Four characteristic parameters of scattered light intensity a, signal pulse width b, green fluorescence intensity c, and red fluorescence intensity d are obtained.
The imaging control circuit 200 determines the type of each cell in real time using these parameters, and controls so that only cells of the type designated in advance as an imaging target can be selected and imaged.

That is, the characteristic parameter of a cell is
Whether or not the characteristic parameters of the cell of the type to be imaged are matched is compared in real time, and if it is determined that the cell is the image to be imaged, the light emission trigger signal Ts for imaging the cell is set. Supply to the pulse light source 2.

The pulse light source 2 is a type of light source that emits light for a moment (about several tens of nanoseconds) in response to the light emission trigger signal Ts. It can be imaged without. As shown in FIG. 1, the pulsed light is guided to the flow cell 3 by the optical fiber 12, narrowed down by the condenser lens 13, and irradiated on the sample flow.

By irradiating through the optical fiber 12, the coherency of the pulsed light can be lowered and a cell image with less diffraction fringes can be picked up. The pulsed light transmitted through the sample flow is projected onto the video camera 1 by the projection lens 14.
An image is formed on the light receiving surface of No. 5, and a transmitted light image of the cell is taken.
The video signal Vs from the video camera 15 is passed to the data processing device 300 shown in FIG. 3, and is stored and saved as a digital image.

Characteristic parameters a to d such as scattered light intensity and fluorescence intensity are passed to the data processing device 300, and a scattergram (two-dimensional frequency distribution) is analyzed by a combination of these parameters. The input device 400 is composed of a keyboard and a mouse, and specifies a set of characteristic parameters for forming a scattergram, specifies coordinates and coordinate areas in the scattergram, and sets the number of times of imaging. A display device 500, for example, a CRT, displays a scattergram, a cell image, and the like. In addition, the signal processing device 100, the imaging control circuit 200, and the data processing device 300 include a CPU, a ROM, and an R.
It is composed of a microcomputer composed of AM.

The operation in such a configuration will be described with reference to the flowchart shown in FIG. First, the user uses the input device 400 to set the X-axis and Y-axis of the scattergram.
Characteristic parameters to be assigned to the axes are a and b, respectively.
Is specified, the scattergram SG is displayed as shown in FIG.
Is displayed on the display device 500, and when a desired coordinate area is designated by the input device 400, the designated coordinate area A is displayed in the scattergram SG as shown in FIG. 5 (step S1). , S2).

Next, as shown in FIGS. 1 and 2, cells P1, P2, P3, ...
n sequentially flows, and the photodiode 7 and the photomultiplier tubes 10 and 11 first detect the light from the cell P1 (step S3), and the detection signals S1 to S3 are A / D converted in the signal processing device 100. Then, the characteristic parameters a1 to d1 are calculated (steps S4 and S5).

Next, it is judged whether or not the coordinates (a1, b1) of the cell P1 are within the area A (step S6), and FIG.
As described above, when the coordinates (a1, b1) are within the area A, the imaging control circuit 200 immediately determines whether or not the cell P1 can be imaged by the video camera 15 (step S7).

When it is determined that the image pickup is possible, the image pickup control circuit 200 outputs a signal Ts to the pulse light source 2, the pulse light source 2 emits light, and the cell P1 is imaged by the video camera 15 (step S8).

The data (each 8 bits) representing the characteristic parameters a, b, c, d created in step S5 is stored in the memory of the data processing device 300 as a data set as shown in FIG. Is stored. When the image pickup of the cell P1 is completed in step S8, as shown in FIG. 8, the flag bit at the end of the data of the cell P1 becomes 1 (ON), and it is recorded that the cell P1 has been imaged (step S9). .

The cells P2 following the cell P1 are also subjected to the processing of steps S3 to S5, and in step S6, the coordinates (a2, b2) of the characteristic parameters of the cell P2.
However, if it is determined that the flag exists outside the designated coordinate area A as shown in FIG. 5 or if the image capturing is not possible in step S7, the flag bit of the characteristic parameter is
As shown in (3), it becomes 0 (OFF), and it is recorded that the cell P2 was not imaged (step S10).

Hereinafter, the cells P3 to Pn are also sequentially
Similar processing is performed to create a data set (FIG. 8) of characteristic parameters of the cells P1 to Pn (step S1).
1).

Next, the flag bits of the cells P1 to Pn of FIG. 8 are searched (step S12), and the coordinates of the characteristic parameter of the cell having the flag bit of 1 are black square dots as shown in FIG. The coordinates of the characteristic parameter of the particle whose flag bit is 0 are displayed on the scattergram SG (step S13), and are displayed on the scattergram SG by small black dots as shown in FIG. 6 (step S13).
14).

Next, when the user uses the input device 400 to specify the coordinates of the cell Pk among the coordinates displayed by the black square dots (step S15), the coordinates of the cell Pk of the scattergram SG are specified. The dot representing the color changes to a large black circle as shown in FIG. 7 (step S16),
At the same time, the cell image of the cell Pk is transferred to the data processing device 30.
0 is read from the memory and the display screen D of the display device 500 is displayed.
It is displayed on P (step S17).

At this time, the values of the characteristic parameters ak to dk of the cell Pk are displayed on the display device 500. Regarding the dots on the scattergram, in this embodiment, the coordinates of the imaged cells are displayed as black square dots, the coordinates of the cells in the cell image are displayed as large black circles, and the Although the identification is facilitated, these may be displayed by dots of other shapes or dots of different colors.

[0032]

According to the present invention, the characteristic parameter and the image are stored in association with the particles (cells), and the distribution position of the distribution map and the particle image can be displayed so that the correspondence between the two is clear. Therefore, the morphology of the particles can be observed. Furthermore, it is convenient because the particle image can be seen immediately every time the distribution points of the distribution map are specified. For example, if there are particles distributed in a region where normal cells are not distributed, it is possible to confirm what kind of particles they are, and this is useful for finding abnormal particles. In addition, since the judgment of the user can be added to the image for particles whose classification is unknown only by the data of the characteristic parameter, the analysis accuracy is improved, and the particle image can be easily used to support the particle classification result. it can.

[Brief description of drawings]

FIG. 1 is an explanatory diagram showing a configuration of an optical system according to an embodiment of the present invention.

FIG. 2 is a perspective view of an essential part of FIG.

FIG. 3 is a block diagram showing the configuration of the control system of the embodiment.

FIG. 4 is a flowchart showing the operation of the embodiment.

FIG. 5 is an explanatory diagram showing a display example of the embodiment.

FIG. 6 is an explanatory diagram showing a display example of the embodiment.

FIG. 7 is an explanatory diagram showing a display example of the embodiment.

FIG. 8 is an explanatory diagram showing a data set of characteristic parameters according to the embodiment.

[Explanation of symbols]

 1. Continuous emission laser 2. Pulse light source 3. Sheath flow cell 4. Condenser lens 5. Condensing lens 6. Dichroic mirror 7. Photodiode 8. Dichroic mirror 9. Mirror 10. Photomultiplier tube 11. Photomultiplier tube 12. Optical fiber 13. Condenser lens 14. Projection lens 15. Video camera

Claims (1)

[Claims] 1. A sheath flow cell for forming a sample flow containing particles, a detection unit for detecting a signal from the particles in a first flow region of the sample flow, and an imaging unit for capturing an image of the particles in a second flow region of the sample flow. A display means, a calculating means for calculating a plurality of characteristic parameters representing the characteristics of each particle based on the output of the detecting means, a distribution map creating means for creating a distribution map of the characteristic parameters and displaying it on the display means, Coordinate designating means for designating an arbitrary coordinate area in the figure and a particle image when the coordinate area is designated by the coordinate designating means and the coordinates representing the particles detected in the first basin are present in the designated coordinate area Imaging control means for causing the imaging means to image
When the coordinate designating unit designates the image storing unit that stores the imaged particle image in association with the coordinate and the coordinate representing the imaged particle in the distribution map, the particle image corresponding to the coordinate is output from the image storing unit. A flow cytometer comprising reading means for reading and displaying on a display means.