JPH10253624A - Particle measuring device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a ratio in size between a cell and a cell nucleus with high efficiency and accuracy by calculating each size from the images of the cell and the fluorescence signals of the nucleus. SOLUTION: The computing part of an analytical part calculates the diameter N of a nucleus through the use of a conversion factor from a side fluorescence intensity signal S2 among the signals S1 and S2 outputted from the first and second photodetectors 7 and 11, respectively. A pulse light source 2 only instantly emits light by a light emission trigger signal. The flow of a sample is irradiated with pulse light L2 via an optical fiber 2a, a collimating lens 12, a cold mirror 13, and a condensing lens 14 to pick up the image of a cell with good contract. The pulse light L2 transmitted through the flow of a sample forms an image on the light receiving plane of a video camera 17 via a collimating lens 8, a dichroic mirror 9, a BPF 16, a condensing lens 15, and the transmitted light image of the whole cell is picked up. A cell image is cut out at an image processing part from an image signal VS from the video camera 17 and binarized, the diameter C of the cell is calculated from the area of the binarized cell image, and a N/C ratio is computed through the use of the diameter N of the nucleus.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a particle measuring apparatus, and more particularly to a particle measuring apparatus for measuring a ratio of a nucleus size to a blood cell or cell size useful for identifying abnormal blood cells or cancer cells.

[0002]

2. Description of the Related Art Conventionally, in order to identify abnormal blood cells and cancer cells, the ratio of the diameter N of the nucleus to the diameter C of blood cells or cells (N / C)
Is a useful indicator. The following method is known as a method for discriminating between a nuclear portion and a cytoplasmic portion of a cell and analyzing the same (for example, see JP-A-6-58926).

[0003] That is, after the cells are fixed on a slide and washed, the cells are stained with a special dye, and further washed to remove excess dye. Irradiating the created specimen with infrared rays, imaging cells, digitizing the cell image, and analyzing it by computer.

[0004]

However, in such a conventional analysis method, not only is it necessary to prepare a cell sample, but also the cells are flattened on a slide.
Since it is not possible to capture an original image of a cell having a three-dimensional shape, there is a problem that information such as the diameters and profiles of cells and nuclei cannot be obtained correctly.
In addition, there is a problem that the analysis accuracy is significantly reduced when the number of cells is small. Further, the reference does not describe calculating the N / C ratio. On the other hand, with respect to obtaining the N / C ratio, there is known a detection method called slit scanning in which flow particles are irradiated with slit-like light to detect light from the particles (for example, Leon.
"Slit scanning" by El Wheeles Jr., flow cytometry and sorting, 1990
2nd edition, pp. 109-125). In this method, the fluorescence signal shape of cells stained with acridine orange or the like is detected, and the ratio between the signal pulse width of the weakly stained cell chamber and the signal pulse width of the strongly stained nucleus is defined as the N / C ratio. Acridine orange and the like are dyes for staining nucleic acids, and the degree of staining of cell membrane components is low. In addition, there is a problem that nucleic acids and inclusion bodies present in the extranuclear cytoplasm are strongly stained, and both the nucleus and the cytoplasm may not be correctly recognized as electric signal pulse shapes. In addition, this method has a problem that stable data cannot be obtained because the method largely depends on how to take a threshold value of an electric signal indicating a cytoplasm or a nucleus.

The present invention has been made in view of such circumstances, and it is possible to obtain an original three-dimensional image of a cell and fluorescence from a nucleus by using an imaging flow cytometer. It is possible to calculate the cell diameter C and the nucleus N with high accuracy from the obtained image and the fluorescence intensity.
An object of the present invention is to provide a device capable of calculating the / C ratio. More specifically, the present invention enables the N / C ratio to be favorably obtained even when the captured image has an unclear nucleus.

[0006]

SUMMARY OF THE INVENTION The present invention provides a sheath flow cell for wrapping a sample solution containing stained cells in a sheath solution and converting it into a sample flow, a first light source for irradiating the sample flow with light, and a first light source. An imager for capturing an image of cells irradiated with light from a light source to obtain an image; a second light source for irradiating the sample stream with light; and a light detector for detecting a fluorescence signal generated from a nucleus of the cells exposed to the light from the second light source Instrument, comprising an analysis unit for analyzing the image and the fluorescence signal,
The analysis unit calculates a cell size C from the image, a nucleus size N from the fluorescence signal, and calculates an N / C ratio, and an output for outputting the calculated N / C. The present invention provides a particle measuring device comprising a part.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The cells in the present invention include all cells containing cytoplasm and nucleus, and include, for example, peripheral blood cells and urinary cells. Further, the staining treatment in the present invention is a treatment for staining at least the nucleus with a second dye capable of fluorescence excitation, and preferably, in addition to this, the cells are placed in a near-infrared region which does not overlap with the excitation wavelength of the second dye. This is a process of further dyeing with a first dye having an absorption wavelength.

[0008] As the first dye, for example, HITC
Cyanine dyes such as iodide and IR-125
As the second dye, propidium iodide (P
Dyes such as I) and ethidium bromide (EB).

Further, the sheath flow cell of the present invention is a flow cell capable of forming a thin sample flow by a hydrodynamic effect by wrapping a sample containing cells with a sheath liquid and flowing the same. Can be used.

In imaging a cell, if the wavelength of the light illuminating the cell is short, the light is greatly reduced by scattering on the cell surface, making it difficult to image the cell. Further, the fluorescence excitation wavelength overlaps with the fluorescence excitation wavelength of the second dye, and the fluorescence excitation light is not correctly detected.

On the other hand, when the wavelength of light is long, the dimming due to cells is reduced (the transmittance is increased). The absorbance of water increases. Image resolution decreases. These factors make it difficult to obtain a good image.

If the wavelength of the illumination light for imaging the cell overlaps with the wavelength of the fluorescence excitation light or the fluorescence, it is difficult to detect the signal from the cell. Has a wavelength of 720 to 1500 nm
It is preferable to irradiate the sample stream with near-infrared light in the range of (1). Specifically, a near-infrared pulsed semiconductor laser (720 to 980 nm), a color center laser (60
0 to 900 nm), dye laser (600 to 1200 n)
m), a YAG laser (1.064 μm), a YLF laser (1.048 μ), or a Ti sapphire laser (mode-lock type with a cavity damper) or the like.

When the cells are previously stained with a dye having an absorption wavelength in the near infrared region, it is naturally preferable that the first light source is a light source in the near infrared wavelength range.

A video camera that captures a general two-dimensional image may be used as an imager that captures cells. In this case, a video camera that has an image intensifier that amplifies weak light is used. Preferably, it is used. Further, the image intensifier may be provided with shutter means.

The second light source has, for example, 400 to 700 n.
a visible light source that emits light having a wavelength of m,
A photodiode or a photomultiplier tube can be used for the photodetector.

The analyzing section can be constituted by a microcomputer comprising a CPU, a ROM and a RAM or a personal computer.

[0017]

FIG. 1 shows an optical system according to an embodiment of the present invention. In this embodiment, a cell-containing sample solution is supplied to a square sheath flow cell 3. The cell-containing sample solution is previously stained with a first dye whose cytoplasm has an absorption wavelength in the near-infrared light region and a second dye whose nucleus can be fluorescently excited as follows.

A sample for measurement was prepared as follows. Propidium iodide as a first staining solution the first dye 250μg /
1 liter of an aqueous solution (pH 8.5) containing methanol solution diluent Tris-HCl 20 mM NaHCO ₃ 60 mM NaCl 60 mM containing 4 mg / ml of HITC iodide as the second dye in PBS solution second staining solution containing 2 ml of 1 ml of the diluent was placed in a test tube, into which 10 μl of blood, 15 μl of the second stain, and 5 μl of the first stain
Add 0 μl and stain for 30 minutes at room temperature protected from light.

A sample for measurement may be prepared as follows. First staining solution Auramine O 2.6% Ethylene glycol 95.9% Second staining solution Methanol solution containing 10 mg / ml HITC iodide Third staining solution First staining solution and second staining solution were mixed at a ratio of 5: 1. 3
Use as a staining solution. Then, 1950 μl of the diluent and 10 μl of blood
1, 40 μl of the third staining solution is mixed and allowed to react for 30 seconds to 5 minutes, and then guided to a flow cell for measurement.

As shown in FIG. 1, the sheath flow cell 3 is provided with two light sources, a continuous light source 1 for detecting scattered light and fluorescence, and a pulse light source 2 for picking up a cell image. I have. The light source 1 is a visible light source, and the light source 2 is a near-infrared light source. An Ar laser emitting visible light having a center wavelength of 488 nm is used for the light source 1, and a pulse laser diode emitting near-infrared light having a center wavelength of 780 nm and a spectral width of 7 nm is used for the light source 2. You are using

Continuous visible light L from the two light sources 1 and 2
1 and the pulsed near-infrared light L2 are supplied to the sheath flow cell 3 (FIG. 1).
In this case, the sample flows in a direction perpendicular to the paper surface).

When cells flow into this irradiation area, forward scattered light from the cells is collected by a condenser lens 5 and received by a first photodetector 7 comprising a photodiode via a band-pass filter 6 to receive a signal. It is output as S1 (the beam stopper is not shown). Lateral fluorescence generated from the fluorescently stained nucleus is received and multiplied by a second photodetector 11 composed of a photomultiplier tube via a collimator lens 8, a dichroic mirror 9, and a condenser lens 10, and is output as a signal S2. .

FIG. 2 shows the configuration of the signal processing system according to this embodiment. Of the signals S1 and S2 output by the first and second photodetectors 7 and 11, respectively, the side fluorescence intensity signal S
2 is input to the particle analyzer 100a of the analyzer 100,
A / D conversion of the height information of the detection signal pulse is performed and the arithmetic unit 10
At 0d, it is converted to the core diameter N using a conversion coefficient.

The imaging control section 100c supplies the pulse light source 2 with a light emission trigger signal Ts for imaging a cell at the same time or after a predetermined time when the signal S1 is input to the analysis section 100 from the first detector.

The pulse light source 2 is a light source of a type that emits light only for a moment (about 25 nanoseconds) by the light emission trigger signal Ts. Images can be taken.

As shown in FIG. 1, the pulsed light L2 is guided to a collimating lens 12 by a kaleidoscope (which can be a multimode optical fiber) 2a, which is an example of a coherence reducing means, passes through a cold mirror 13, and passes through a condenser. The light is narrowed down by the lens 14 and is irradiated on the sample flow.

By irradiating the light via the optical fiber 2a, the coherency of the pulsed light L2 is reduced, and a cell image can be captured with high contrast. The optical fiber 2a has a core diameter of 800 μm and a length of 2 m (MKH-800 manufactured by Sumitomo Electric Industries, Ltd.).

The pulse light transmitted through the sample flow is converted into parallel light by a collimating lens 8 and reflected by a dichroic mirror 9 to form an image on a light receiving surface of a video camera 17 via a band pass filter 16 and a condenser lens 15. Then, a monochrome transmitted light image of the entire cell is captured.

In the obtained monochrome image, cells are relatively well captured, but nuclei are not always well captured. Therefore, the image signal Vs from the video camera 17 is passed to the image processing unit 100b shown in FIG. Image processing unit 1
At 00b, the cell image is cut out, binarized, and stored as a digital image. The calculation unit 100d calculates the equivalent circle diameter as the cell diameter C from the area of the binarized cell image,
The N / C ratio is calculated from the core diameter N already calculated. In the case of the present embodiment, even if the image of the nucleus is unclear, the size of the nucleus can be calculated from the fluorescence signal intensity.

The input unit 200 shown in FIG. 2 includes a keyboard and a mouse, and performs various commands and setting of conditions to the analyzing unit 100. In addition, the output unit 100e has a CR
It outputs a calculation result and the like obtained by the calculation unit 100d, which is composed of T and a printer.

Here, in order to detect side fluorescence excited by light having a wavelength of 488 nm and emitted from the nucleus as light having a wavelength of 500 to 700 nm, light having a wavelength of 500 to 700 nm is transmitted and light having a wavelength of 700 nm or more is transmitted. A reflecting dichroic mirror 9 is used. And the wavelength 780
In order to obtain a near-infrared light image having a wavelength of 780 nm or more, a bandpass filter 16 that transmits light having a wavelength of 780 nm is used.

Therefore, the video camera 17 receives the visible light L
1 (488 nm) does not enter the side light generated from the cells, so that the video camera 17 can obtain a good cell image which is not affected by the light from the light source 1.

Similarly, only the side fluorescent light generated by the visible light L1 enters the second detector 11 by the action of the dichroic mirror 9, so that the second detector 11
A good particle signal which is not affected by light from the light source can be obtained.

The band pass filter 6 includes 48
Since the one that transmits light having a wavelength of 8 nm is used, only the forward scattered light generated by the visible light L1 enters the first photodetector 7, and the lateral light generated by the near-infrared light L2. Does not enter at all, the first photodetector can obtain a good particle signal which is not affected by the light from the light source 2.

It is also possible to substitute the near-infrared light source with an infrared lamp having a wide wavelength band and a narrow band filter for limiting the wavelength width.

In this embodiment, as shown in FIG. 1, the light collimating region between the lens 12 and the lens 14 has
A cold mirror 13 that transmits infrared light and reflects visible light is provided. Therefore, of the side scattered light and side fluorescent light generated from the cells irradiated by the visible light L1, the lens 1
The light incident on the fourth side is reflected by the cold mirror 13 and guided to the second photodetector 1. Thereby, the intensity of the detected light increases, and in particular, the collection efficiency of the second photodetector 11 improves.

It is possible to install such a cold mirror between the sheath flow cell 3 and the lens 14, but in this case, the cold mirror needs to be a concave mirror and the position of the cold mirror must be set. Is not easy.

On the other hand, since the cold mirror 13 in this embodiment is provided in the optical path collimating region, a flat mirror can be used and its position can be easily set.

[0039]

According to the present invention, a sample flow containing cells is irradiated with light using an imaging flow cytometer, the size of the cells is calculated from the captured cell image, and the detected fluorescence signal of the nucleus is detected. Since the size of the nucleus is calculated from, the ratio of the size of the nucleus to the size of the cell can be efficiently and accurately calculated.

[Brief description of the drawings]

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

FIG. 2 is a configuration diagram showing a signal processing system according to an embodiment of the present invention.

[Description of Signs] 1 continuous light source 2 pulse light source 2a optical fiber 3 sheath flow cell 4 condenser lens 5 condenser lens 6 band pass filter 7 first detector 8 collimating lens 9 dichroic mirror 10 condenser lens 10 11 second photo detector 12 Collimating lens 13 Cold mirror 14 Condenser lens 15 Condenser lens 16 Bandpass filter 17 Video camera

Claims (3)

[Claims] 1. A sheath flow cell that wraps a sample solution containing stained cells in a sheath solution and converts the sample solution into a sample flow, a first light source that irradiates the sample flow with light, and a cell that receives light from the first light source. An imager that captures an image to obtain an image, a second light source that irradiates the sample stream with light, a photodetector that detects a fluorescence signal generated from a nucleus of a cell that has received light from the second light source, and the image and the fluorescence signal An analysis unit that calculates a cell size C from the image, a nucleus size N from the fluorescence signal, and calculates an N / C ratio. N / C
Particle measuring device comprising an output section for outputting a ratio of 2. A staining process, wherein the cell material is stained with a first dye having an absorption wavelength in a near-infrared region, and a second dye capable of fluorescence excitation is dyed.
2. The particle measuring device according to claim 1, wherein the nuclear material is treated with a dye. 3. The light source according to claim 1, wherein the first light source is a near-infrared light source having a wavelength of 720 to 1500 nm, and the second light source is 400 to 70 nm.
3. The particle measuring device according to claim 2, comprising a visible light source having a wavelength of 0 nm.