JPS63231244A - Cell analysis instrument - Google Patents

Abstract

PURPOSE:To reduce the size of an instrument without lowering the efficiency of a continuous optical analysis by detecting the time intervals at which individual cells pass two points on the optical focus of an optical system for projection and measurement, emitting a pulse after lapse of the time of specified times said time intervals and making measurement. CONSTITUTION:A cell suspension marked by a fluorescent label is fed by a pressure through a sample liquid inlet 1 into a flow cell 3 and a physiological salt soln. is fed by a pressure through a sheath liquid inlet 2 into said cell, respectively. The light from a continuous light projection system 5 converges at the central axis of a capillary 4 and is scattered by the cells passing the same. The scattered light is detected by an optical system 6 for detecting the scattered light. The light signals which two photodetecting elements of a photodetector 7 receive are the pulses parted by the time interval T. The pulse of a light emission signal is generated and the pulse light is emitted by a projection system 9 for the pulse light upon lapse of the time nT after the light signals enter the photodetector 7 when the light signals are discriminated as the desired cell in a signal processing system 8 from the height of the pulses.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention involves pouring a cell suspension into a capillary channel or ejecting it from a nozzle, irradiating each cell with light, and detecting the scattered light or fluorescence from the cells. The present invention relates to a cell analysis device, and particularly to a cell analysis device suitable for multi-item cell analysis.

[Conventional technology]

Traditionally, so-called flow cytometers, devices that optically measure and analyze cells while flowing cells, require a laminar flow state in order to flow the cell suspension (sample solution) stably into the measurement section without clogging. A method is adopted that utilizes rapid contractions to flow the sample liquid so that it is surrounded by physiological saline. This method is called a sheath-flow one-way method, and is an effective means for flow systems in cell analyzers. In conventional technology, the sample liquid is sufficiently squeezed using a sheath flow, and in order to analyze the shape, size, and type of cells floating inside, the cells are irradiated with continuous light such as a laser beam, and the scattering emitted from the cells is analyzed. The light intensity and the fluorescence intensity emitted from specific cells that have been labeled with fluorescent labels are measured.

A similar product is disclosed in U.S. Patent No. 4,395,39.
No. 7 specification.

[Problem that the invention seeks to solve]

The above conventional technology uses continuous light as a light source, so a powerful light source is required to obtain detectable scattered light and fluorescence, which has the disadvantage of making the device large and expensive. .

In particular, when detecting the fluorescence intensity of cells marked with a fluorescent label, depending on the type of fluorescent label, the fluorescence is extremely weak, and even with a high-output laser as the light source, the fluorescence may not be detected. In addition, marking with many types of fluorescent labels,
In order to analyze multiple items at the same time, it is necessary to use multiple light sources with wavelength bands suitable for each fluorescent label, which results in increasingly larger equipment.

Some pulsed light sources have a stronger instantaneous light intensity than continuous light sources, but simply replacing a continuous light source with a pulsed light source does not make it possible to match the timing of the pulsed light emission with the moment when the cell passes over the optical focus. 1 analysis becomes inefficient.

An object of the present invention is to miniaturize the apparatus without reducing analysis efficiency.

[Means for solving problems]

The above purpose is to provide a first irradiation measurement optical system that uses continuous light as a light source and a second irradiation measurement optical system that uses pulsed light as a light source, so that individual cells are placed on the optical focus of the first irradiation measurement optical system. This is achieved by detecting the time interval between two adjacent points of .

[Effect]

Since the first irradiation measurement optical system uses a continuous light source, it can detect all flowing cells. Since the first irradiation measuring optical system measures the time interval at which a cell crosses two adjacent points on the optical focus, it is possible to detect the speed of each individual cell. Since there is a second irradiation measurement optical system downstream that is a certain number of times the distance between the two points monitored by the first irradiation measurement optical system, after the cells pass through the first irradiation measurement optical system, By emitting pulsed light after a certain number of times the time interval at which the cell crosses between these two points, the focus of the second irradiation measuring optical system can be measured at the moment when the cell crosses. With this configuration, measurements that do not require a high-power light source are performed using the first irradiation measurement optical system that uses a small, low-power continuous light source, and measurements that require a high-power light source are performed using instantaneous light. Since this can be carried out using the second irradiation measurement optical system that uses a high-intensity pulsed light source, the entire apparatus can be miniaturized.

〔Example〕

An embodiment of the present invention will be described below with reference to FIG.

In the figure, a cell suspension marked with a fluorescent label is injected into a flow cell 3 through a sample liquid inlet 1, and physiological saline is injected into a flow cell 3 through a sheath fluid inlet 2, respectively. Within the flow cell 3, the cell suspension forms a sheath flow surrounded by physiological saline, forms a cell row 11, and flows on the central axis of the capillary tube 4 in a laminar flow state. ``The capillary tube 4 is made of a transparent material and has a rectangular cross section.The light emitted from the continuous light irradiation system 5 is focused in a narrow area on the central axis of the capillary tube 4, and is scattered by the cells passing through it. The scattered light is detected by the scattered light detection optical system 6. The photodetector 7 of the scattered light detection optical system 6 consists of two photodetecting elements, and the center of the capillary tube 11 is located on each photodetecting element. The light scattered when a cell passes two points close to each other on the axis forms an image.
The optical signals received by the two photodetecting elements of the photodetector 7 when one cell passes are pulses spaced apart by a time interval T, as shown in a) and b) of FIG. The height of this pulse is related to the size of the cell, and the signal processing system 8 determines whether the passed cell is the target cell based on the height of the pulse. When the signal processing system 8 determines that the cell is the target cell, the signal is processed by nT, which is n times the time interval T between the optical signal pulses received by the two photodetecting elements after the optical signal enters the photodetector 7. After the elapse of time, a light emission signal pulse is generated as shown in FIG. 2C), and the pulsed light irradiation system 9 emits pulsed light. The light emitted from the pulsed light irradiation system 9 converges on the central axis of the capillary tube 4 at a point downstream from the convergence point of the continuous light irradiation system S. The interval between the convergence point of the continuous light irradiation system 5 and the convergence point of the pulsed light irradiation system 9 is n times the interval between the two points that form images on the two photodetector elements of the photodetector 7, and the pulse At the moment when light is emitted, cells are flowing through the convergence point.

Since the cells are marked with fluorescent labels, they are excited by the strong pulsed light from the pulsed light irradiation system 9 and emit fluorescence. Fluorescence emitted from cells is detected by a fluorescence detection optical system 10, and the information is statistically processed by a signal processing system 8.

In this embodiment, since only the measurement of scattered light that can be measured with a relatively weak light source is performed using the irradiation measurement optical system using continuous light as the light source, a small and inexpensive continuous light irradiation system 5 can be used. In addition, for fluorescence measurements that require a strong light source, a small pulsed light source with high instantaneous output is used, so the device can be made smaller. Furthermore, since each cell is measured at the moment when it passes through the convergence point of the pulsed light irradiation system 9, each cell can be measured under the same conditions, and measurement accuracy can be improved. In addition, since the convergence point of the continuous light irradiation system 5 and the convergence point of the pulsed light irradiation system 9 are located apart, the lenses, etc. do not become cluttered, and the light from another light irradiation system is prevented. This prevents interference from occurring and impairing measurement accuracy. In addition, since each cell is measured, it is possible to measure even if the flow rate fluctuates to some extent, and the cell flow system does not need to be highly accurate.
The cost of the device can be reduced.

Furthermore, the discrimination is made based on the intensity of the scattered light.

Even if the cell suspension contains cells other than the target cells or dirt, it is possible to remove them from the measurement.

FIG. 3 shows another embodiment of the present invention, in which an imager 12 is used instead of the fluorescence detection optical system. Pulsed light irradiation system 9
emits pulsed light with a very short emission time and can obtain still images of flowing cells. From the obtained image information, each cell is analyzed by single image processing or the like.

In this embodiment, images of target cells can be obtained while flowing a large amount of cells, so abnormal cells can be discovered quickly. Furthermore, since a pulsed light is emitted to obtain an image at the moment a cell passes, the imager 11 only needs to view a narrow range, and therefore can take an image with a high magnification.

FIG. 4 shows another light detection method that can be used in the scattered light detection optical system 6 of FIG. A light shielding plate 13 with a rectangular hole is placed there. Therefore, the scattered light when a cell passes two points in close proximity enters the photodetector 7, and the optical signal received by the photodetector 7 when one cell passes is shown in Figure 5 a). As shown, the waveform has two peaks.

The signal processing system 8 measures the distance T between these two peaks, and causes the pulsed light irradiation system 9 to emit light after n times the distance T, as shown in FIG. 5C). In this embodiment, since a photodetector consisting of one photodetection element is used to detect scattered light, the cost can be reduced.

FIG. 6 shows another embodiment of the present invention, in which a continuous light irradiation system 5
The optical axis of the pulsed light irradiation system 9 and the optical axis of the pulsed light irradiation system 9 are arranged so as to be orthogonal to each other. By arranging them in this way, the lenses and the like can be prevented from colliding with each other, so that the convergence points of the continuous light irradiation system 5 and the convergence points of the pulsed light irradiation system 9 can be set close to each other. Therefore, the accuracy of the time at which pulsed light is emitted can be improved. Furthermore, the length of each capillary tube 4 can be shortened, and pressure loss in the flow system can be reduced.

FIG. 7 shows another flow system to which the present invention can be applied. The flow cell 3 does not have a capillary tube, and the liquid containing cells is ejected into the atmosphere from the ejection port, and the measurement is performed at the liquid column 14. In this case, the liquid column 14 has a cross section because the radius R is gas. This has the advantage that the velocity distribution within the cell is small and the difference in velocity between individual cells is small.

〔Effect of the invention〕

As described above, according to the present invention, it is possible to use a compact continuous light source and a pulsed light source, so that the device can be downsized.

[Brief explanation of the drawing]

1 and 2 are a configuration diagram and an operation explanatory diagram of one embodiment of the present invention, FIG. 3 is a configuration diagram of another embodiment of the present invention, and FIGS. 4 and 5 are diagrams of another embodiment of the present invention. FIG. 6 is a diagram showing another configuration of the present invention, and FIG. 7 is a diagram showing another flow system to which the present invention can be applied. 3...Flow cell, 5...Continuous light irradiation system, 6...
Scattered guess 1 Stone 2 Diagram

Claims (1)

[Claims] 1. A cell flow system that pressurizes a cell suspension and sends it onto an optical focal point, and irradiates cells floating in the flow flowing down from this cell flow system with intense light. A biological cell analyzer consisting of an irradiation measurement optical system that measures the intensity of scattered light or fluorescence intensity, and a signal processing system that analyzes the type, size, shape, etc. of cells based on signals from the irradiation measurement optical system. , a first irradiation measurement optical system having a light source that emits continuous light, and a second irradiation measurement optical system having a light source that emits intermittent pulsed light; The second irradiation optical system is characterized in that it is configured to measure the time interval between cells passing two adjacent points using an optical system, and to emit pulsed light from the second irradiation optical system after a constant multiple of that interval has elapsed. Cell analysis device. 2. In the cell analyzer according to claim 1, when the intensity of scattered light from the cells is measured by the first irradiation measurement optical system and the intensity of the scattered light is within a preset range. A cell analyzer that emits pulsed light corresponding to only the second irradiation measurement optical system. 3. A cell analyzer according to any one of claims 1 and 2, in which the second irradiation measurement optical system measures fluorescence excited by pulsed light. 4. A cell analysis device according to any one of claims 1 and 2, which uses a microscope as the second irradiation measurement optical system to analyze the shape of cells.