JP2835692B2 - Device for analyzing particles in fluids - Google Patents

Description

Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a particle analyzer for capturing and analyzing a still image of particles in a fluid, for example, blood cells. [0002] Conventionally, in the examination of particles such as cells, particles (cells) smeared and stained on a slide glass have been used.
Was observed through a microscope. [0003] However, complicated observations such as smearing, fixing, and staining of cells are required before performing observation with a microscope. While present in the fluid,
There is a problem that the original cell morphology is not always correctly observed because observation can be performed only in a state where the cells are fixed. [0004] On the other hand, some attempts have been made recently to capture a microscope image of particles in a fluid. [0005] For example, Kachel et al., "The Journal of Histochemistry and Cytochemistry"
Histochemistry and Cytoc
The method described in “hemistry” Vol. 27, No. 1, p. 335 (1979) allows the particles in the suspension to pass through the pores in a row and changes in the electrical impedance at that time. Detecting, turning on the flash lamp at that moment, and photographing with a camera at the same time, to obtain a still image of the particles. The means described in Japanese Patent Publication No. 57-500995 and Japanese Patent Application Laid-Open No. 58-76740 is to pass a fluid sample through a flow path, turn on a strobe light at a constant period in an imaging area, and simultaneously A still image of particles in the fluid is obtained by photographing with a CCD camera. [0007] Among the attempts to take a microscopic image of particles in a fluid, the above-mentioned Kachel's method uses both the resistive and optical detection principles.
The structure of the detector is very complicated and adjustment is difficult. Also, in the apparatus described in Japanese Patent Publication No. 57-500995, the structure of the flow cell forming the imaging region is very complicated, and the flow of the sample fluid is also complicated. [0009] Furthermore, this method simply turns on the strobe light periodically, and does not detect particles flowing in the fluid and image them, and thus the imaging efficiency is poor. That is,
When detecting blood cells in urine, the number of particles is small,
The state without particles frequently occurs, and meaningless imaging is repeated. [0010] None of the conventional methods describes image analysis of particles and use of the information.
Not only is it impossible to perform full-scale particle analysis, nor is it aimed at it. According to the present invention, there is provided a flow cell for enclosing a liquid sample in which particles are suspended with a sheath liquid and causing the liquid sample to flow, and a trigger cell in a region of the liquid sample in which the particles flow. A trigger light source that constantly emits light, and an imaging pulse light source that emits pulsed light when imaging the particles,
A particle detector that detects that the particle traverses the light from the trigger light source and generates a particle detection signal, a delay circuit that delays the particle detection signal for a predetermined time, and based on a signal from the delay circuit. Means for causing the imaging pulse light source to emit light, a condenser lens that irradiates the particles with pulse light from the imaging pulse light source, an objective lens and an imaging unit for capturing a still image of the particles by the pulse light, An image processing unit that processes and analyzes the obtained still image;
An apparatus for analyzing particles in a fluid, comprising: In other words, a trigger light source that is always turned on, a particle detector that detects that a particle has crossed a light beam from the trigger light source and issues a particle detection signal, and delays the particle detection signal by a predetermined time. And a delay circuit for sending the pulse to the imaging pulse light source. First, the particles to be detected are detected by the trigger light source and the particle detector which are always turned on, and the detected particles reach the front of the objective lens on the downstream side in the flow direction of the sheath liquid. At the timing, the imaging pulse light source operates,
A still image of a given particle can be obtained. Then, by analyzing the image, information on the shape and internal state of the particles can be obtained. An embodiment of the present invention will be described with reference to the screen shown in FIG. This embodiment is an apparatus for analyzing blood cells as particles. First, a liquid sample in which cells are suspended in a diluent or a staining solution is supplied from a cell analyzer 10 through an inlet 14 at an upper portion of a chamber 12. Supplied.
On the other hand, from the entrance 16 on the upper west side of the chamber 12,
Physiological saline or distilled water (hereinafter referred to as sheath liquid)
Is supplied. The liquid sample and the sheath liquid are both chamber 1
2 flows downward and reaches a prismatic flow cell 18.
In the flow cell 18, the liquid sample flows in a sheath liquid in a pod-like shape, and a so-called sheath flow is formed. Next, the optical system of this embodiment will be described.
As a laser pulse light source 20 for imaging, a nitrogen laser,
A semiconductor laser or a YAG laser is used. Note that the pulse light source is not necessarily a laser, and a xenon flash lamp can be used in practice. In FIG. 1, arrows indicate the traveling directions of light rays. The light emitted from the imaging laser pulse light source 20 is converged by a Hayamiko lens 22 and the focus thereof is
8 is connected to the position where the liquid sample of the sheath flow flows. This point is hereinafter referred to as an imaging point 38. The laser light transmitted through the flow cell 18 reaches the objective lens 24. When the cells in the liquid sample pass through the imaging point 38 when the laser pulse light is emitted, the whole image of the cells is transmitted to the still image input unit 28 by the CCD camera 26. The image is processed and analyzed by the system control / image analysis unit 30. The trigger light source 32 is always lit, and the passage of cells through the flow cell 18 is detected by the cell detector 34 to generate a cell detection signal.
After delaying the cell detection signal for a certain period of time in step 6, the cells can be photographed by causing the imaging laser pulse light source 20 to emit light. The delay circuit 36 controls the flow cell 18 from the time when the cell is detected by the cell detector 34 until the time when the cell reaches the imaging point 38.
This is for delaying the light emission of the imaging laser pulse light source 20 by the time flowing through the inside. The cell detector 3 depends on the size of the cell.
Since the pulse width of the pulse detected in step 4 is different, the imaging laser pulse light source 20 can be made to emit light only when a cell of a target size is detected, so that cells in the liquid sample can be selectively imaged. . When the imaging laser pulse light source 20 emits light continuously at a constant cycle, cells can be captured and imaged at a probability corresponding to the concentration of cells present in the liquid sample. The system control / image analysis unit 30 also functions to control the overall operation of the cell analyzer 10. Next, the trigger light source 32 and the cell detector 3
4 will be described in detail with reference to FIG. Reference numeral 18 denotes a flow cell having a rectangular cross section, which has a thickness of about 0.3 mm at the thinnest point perpendicular to the optical axis.
0.5 mm and the other side is 0.2 to 1 mm. These dimensions are variable depending on the sample to be analyzed. The inside diameter of the nozzle 19 for injecting the sample is made 0.2 to 0.5 mm. A semiconductor laser 40 emits visible light and has an output of 2 to 20 mW. Reference numeral 42 denotes a collimator lens, which is commercially available for an optical disk device. This lens 42 has a numerical aperture NA of 0.4
5 to 0.6, and outputs parallel light whose light beam has an elliptical cross-sectional shape. Reference numeral 44 denotes a condenser lens having an F number smaller than 30 and a focal length f of 10 mm or more. The position of the condenser lens 44 is adjusted by a fine movement position adjusting device (not shown) so that a focus is formed near the center of the flow cell 18. Reference numeral 46 denotes a cylinder lens.
(A) A radiation pattern having a cross-sectional shape is narrowed down as shown in (b). The dimension S of the cross section in FIG.
The dimension l is determined by the amount of astigmatism due to the cylinder lens 46. For cell detection, S = 7
２０20 μm, l = 100-300 μm is appropriate. The above-described semiconductor laser 40, collimating lens 42, condenser lens 44, and cylinder lens 46 constitute the trigger light source 32 in FIG. Reference numeral 48 denotes a beam stopper which directly blocks light and transmits forward scattered light in a range of 2 degrees or more with respect to the optical axis. It is known that the intensity of such low angle forward scattered light reflects the size of the cell. Reference numeral 50 denotes a collector lens, which is a low-magnification microscope objective lens, that is, a lens having a magnification of 4 to 20 times. The collector lens 50 collects forward scattered light at the center of a pinhole 52 described later. Reference numeral 52 denotes a pinhole, and the diameter of the pinhole is φ0.2 to φ2 m depending on the magnification and the thickness of the flow cell 18.
m. The collector lens 50 and the pinhole 52 are both fine movement position adjusting devices (not shown).
By adjusting the position. Collector lens 50
When the objective lens for a biological microscope is used as the collector lens 50, the distance between the
m is about 2 when using a metal microscope objective lens.
Set to 00 mm. The flow cell 18 and the collector lens 50
The distance between the collector lens 50 and the pinhole 52 is adjusted according to the distance between. Reference numeral 54 denotes a photodiode or a pin photodiode. A reverse bias voltage is applied to the photodiode or the pin photodiode 54 to reduce the junction capacitance, thereby increasing the response speed of the element. If the response speed of the element is not increased as described above,
The laser pulse light source may not be able to emit light until the cell reaches the imaging point 38 after the passage of the cell is detected. The above-described collector lens 50, pinhole 52, and photodiode 54 correspond to a part of the cell detector 34 in FIG. 56 is a pulse laser diode,
This includes, for example, L2376 (wavelength 890 nm, output 10 W) manufactured by Hamamatsu Photonics or GA manufactured by SANDERS
AP-12 (wavelength 870-904 nm, output 12W) is used. Reference numeral 58 denotes a condenser lens, which constitutes a magnifying optical system on the imaging light source side. The focal position F of the condenser lens 58 is slightly farther than the center position of the flow cell 18 when viewed from the pulse laser diode 56, as shown in FIG. That is, the focus position F is set such that when the image is taken by the CCD camera 64, a sufficiently large image of the cell is obtained in the screen. Further, a distance q between the condenser lens 58 and the focal position F and a distance P between the pulse laser diode 56 and the condenser lens 58
And q / D と 10. The pulse laser diode 56 and the condenser lens 58 substantially correspond to the imaging laser pulse light source 20 and the condenser lens 22 in FIG. 1, respectively. Reference numeral 60 denotes a coherency reducing component, for example, a ground glass or a bundle of optical fibers is used. An imaging lens 62 has a magnification of 20 times or more.
A 40 × microscope objective lens is used and corresponds to the objective lens 24 in FIG. This imaging lens 62
The position is also adjusted by a fine movement position adjusting device (not shown). 64 is a CCD camera. Next, an example in which an N ₂ laser, a YAG laser, an N ₂ / Dye laser, a combination of a YAG laser and a harmonic oscillator, or the like is used as a light source instead of the pulse laser diode 56 in FIG. FIG.
Shown in In this case, since the light from the laser 66 is parallel, the condenser lens 58 shown in FIG. 2 is not required, and the laser light is projected onto the flow cell 18 as it is. FIG. 6 shows a case in which side scattered light is detected in addition to forward scattered light as shown in FIG. 2 for cell detection. In this case, reference numeral 68 denotes a collector lens which detects side scattered light. By detecting the side scattered light in accordance with the forward scattered light, cell detection can be performed more reliably and versatilely. Because the side scattered light reflects the size of the cell and the surface or internal state of the cell, the amount of information increases compared to the case where only forward scattered light mainly reflecting the size of the cell is detected, and the passage of the cell Can be detected more reliably. Further, since information reflecting the surface or internal state of the cell can be obtained, it is possible to accurately determine the type of cell that could not be identified only by the size of the cell, and to determine the type of cell required. It is also possible to take an image only when passing. Next, the case where a nitrogen laser is used will be described with respect to the optical resolution of the cell analyzer 10 of this embodiment and the limit of the flow rate of the sheath flow for promoting the cells as a still image. The nitrogen laser has a wavelength of 337.1 nm and a pulse width of about 10 ns, and can provide a strong pulse oscillation (some of which has a peak output of 100 KW or more). In the case of a nitrogen laser, since it is ultraviolet light, it has excellent resolution as described below. The resolution δ is determined by the numerical aperture NA of the objective lens and the wavelength λ of the light, and the equation δ = λ / NA holds. In this embodiment, the numerical aperture N of the objective lens 24 is
Since A was 0.85, the resolution δ was δ = λ / NA = 337.1 / 0.85 = 396.6 [n
m], which is about 0.4 μm. On the other hand, the pulse width Δx of the nitrogen laser is about 1
0 nS and the resolution is about 0.4 μm, so that the maximum flow velocity V of the sheath flow for the cell to be viewed as a still image
V = δ / Δx = 0.40 × 10 ⁻⁶ / 10 × 10 ⁻⁹ =
40 [m / S]. That is, a still image can be obtained by flowing cells at a speed of about 40 m / s or less. However, if the flow velocity is too high, the sheath flow will be disturbed. Therefore, the flow velocity must be set to about 6 m / s or less in practice. Next, an example is shown in which leukocytes of a healthy person are imaged using the cell analyzer 10 of the present embodiment, and the leukocytes are grouped from the obtained images. First, leukocytes are stained so that the state of intracellular granules and the like can be recognized well. The staining method here is characterized in that, in the smear method, dried blood is stained, whereas the collected blood is stained in a living state. The method for staining and diluting the cells is as follows. a. One drop of Giemsa stain is added to 1 ml of distilled water to prepare a Giemsa diluent. b. The Giemsa diluent and the blood stock solution are mixed at a ratio of 4: 1 and stained for about 30 minutes with occasional stirring. The sample prepared by the above-described method (hereinafter, this method is referred to as “super vital staining method”) flows into the cell analyzer 10 through the inlet 14. The image of the cell passing through the imaging point 38 is prompted by the CCD camera 26, and the still image input unit 2
8 to the system control / image processing unit 30.
The obtained image is subjected to processing for removing flicker and noise. The white blood cell image obtained as described above
Divided into two groups. Table 1 shows the characteristics and appearance rates of each group. 7 to 10 show images of each group. One side of the image is about 30 μm for group A, group C and group D, and group B
Is about 15 μm. [0048] The results of comparing these white blood cell images with white blood cell images obtained by microscopic observation of a sample prepared by taking a smear sample subjected to May-Kimsa combined staining or blood subjected to super vital staining on a slide glass and sealing with a cover glass , Group A was identified as neutrophils, group B as lymphocytes, group C as monocytes, and group D as eosinophils and eosinophils. Therefore, the apparatus of this embodiment images white blood cells, analyzes the obtained white blood cell image, and recognizes the size of the white blood cells and the state of the granules, thereby obtaining the characteristics shown in Table 1. Classify leukocytes into various cells
Can be identified. Next, FIG. 11 shows an example in which red blood cells in the flow of the flow cell in the apparatus of this embodiment are imaged. The peripheral ring-shaped fringes in the figure are diffraction fringes by red blood cells. If the diffraction fringes hinder the image analysis of red blood cells, the diffraction phenomenon may be reduced by reducing the coherence of the laser light. For this purpose, for example, it is appropriate to dispose a glass between the condenser lens 22 and the flow cell 18. According to the present invention, the following specific effects can be obtained. (1) Rather than simply turning on the strobe periodically as in the conventional example, the particles in the fluid are accurately caught and only the caught particles are imaged. Efficiency is improved. (2) By analyzing this still image, information on the shape and internal state of the particles can be obtained. (3) The morphology of the particles when they are present in a stream that could not be obtained by microscopic observation of the particles on the slide glass can be directly observed. (4) If the flow cell used in the apparatus is prismatic, it is easy to manufacture and inexpensive. Also, when detecting the passage of particles and controlling the light emission of the imaging pulse light source,
The device according to the invention is very simple in construction, easy to manufacture and easy to maintain and adjust, since it is only necessary to provide the particle detection optics on top of the imaging optics.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a schematic diagram showing one embodiment (cell analyzer) of a particle analyzer according to the present invention. FIG. 2 is an explanatory diagram of an optical system of one embodiment using a semiconductor laser in the particle analyzer according to the present invention. FIG. 3 is an explanatory diagram showing a light emission pattern in the particle detection system according to the embodiment of the present invention. FIG. 4 is an explanatory diagram showing an enlarged optical system on the imaging light source side in the embodiment of FIG. 2 of the present invention. FIG. 5 is a partial explanatory view of an optical system of one embodiment using an N ₂ laser or the like as a light source for imaging in the particle analyzer according to the present invention. FIG. 6 is a partial explanatory view of an optical system when a side scattered light detection system is added to the embodiment of FIG. 2 of the present invention. FIG. 7 is a micrograph showing white blood cells of Group A. FIG. 8 is a micrograph showing white blood cells of Group B. FIG. 9 is a micrograph showing white blood cells of Group C. FIG. 10 is a micrograph showing white blood cells of Group D. FIG. 11 is a micrograph showing red blood cells. [Description of Signs] 10 Cell analyzer 12 Chamber 14 Sample inlet 16 Sheath liquid inlet 18 Flow cell 20 Laser pulse light source 22 for imaging 22 Condenser lens 24 Objective lens 26 CCD camera 28 Still image input unit 30 System control / image analysis unit 32 Trigger Light source 34 Cell detector 36 Delay circuit 38 Imaging point

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Claims (1)

(57) [Claims] A flow cell for surrounding and flowing the liquid sample in which the particles are suspended with the sheath liquid, a trigger light source for constantly irradiating light for trigger to an area of the liquid sample in which the particles flow, and a pulse when imaging the particles An imaging pulse light source that emits light, a particle detector that detects that the particles have traversed the light from the trigger light source and generates a particle detection signal, and a delay circuit that delays the particle detection signal for a predetermined time Means for causing the imaging pulse light source to emit light based on a signal from the delay circuit; a condenser lens for irradiating the particle with pulse light from the imaging pulse light source; and capturing a still image of the particle by the pulse light An apparatus for analyzing particles in a fluid, comprising: an objective lens and an imaging unit for performing the processing, and an image processing unit that processes and analyzes the obtained still image. 2. 2. The apparatus according to claim 1, wherein the imaging pulse light source is a laser pulse light source.