JPH07218417A - Particle analyzing method - Google Patents

Abstract

PURPOSE:To analyze particles with high accuracy at a high speed by making a sample solution to flow in a thickness thicker than the depth of focus of an image pickup system and taking the image of the sample solution at the timing related to the passing depth of the particles. CONSTITUTION:While a sample solution 2 is made to flow in a thickness thicker than the depth of focus of an image pickup system, the solution 2 in a flow cell 1 is irradiated with laser light from a laser light source 4 and optical photodetectors 12a and 12b obtain the positions of particles in the solution 2 in both the depth and lateral directions against the optical axis by detecting scattered light 33 from the particles and measuring the direction and magnitude of the positional deviation of the distribution of the light 33. On the other hand, a CCD camera 6 is periodically actuated and an objective lens 3 is also periodically driven by means of an inching device 15. As a result, particle detecting signals are outputted whenever particles pass through a particle detecting area. When the particles pass through the particle detecting area, the images of the particles are picked up at the timing at which the depth signals of the passing particles and lens position signals coincide with each other.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a particle analyzer and method for picking up an image of particles suspended in a liquid and analyzing the particles, and particularly suitable for analyzing particles in blood or urine. The present invention relates to a particle analysis method.

[0002]

2. Description of the Prior Art For morphological examination of particles in urine,
Conventionally, in the visual observation, a urine sample is centrifuged, a sediment is stained to prepare a specimen on a slide glass, and the specimen is observed under a microscope. At that time, the concentration rate of centrifugation is always constant, and the amount of sample to be observed is also constant,
He was trying to know what kind of sediment and what concentration was contained in the original urine sample. In addition, the sediment contained particles of several μm such as blood cells and bacteria to particles of several hundred μm such as cylinders, and the microscope magnification was switched between high magnification and low magnification for observation. At that time, although the amount of the sample to be observed varies depending on the purpose, it is typically 5 μm at high magnification.
1, concentrated urine in an amount equivalent to 750 μl of raw urine at low magnification was observed, and the number of each sediment component was counted.

As an apparatus for automating these inspections, there is an apparatus which allows particles to be suspended in a liquid and flow in a flow cell for optical analysis. For example, special table Sho 57-500995
The publication discloses a device in which a fluid sample is passed through a channel of special shape, in which the particles in the sample are confined in a wide imaging region to create a static image. In this device, a magnified image of the flow is formed on the image pickup surface of the CCD camera using a microscope. As the light source, a pulsed light source that periodically emits light in synchronization with the operation of the CCD camera is used. Since the emission time of the pulsed light source is short, a still image can be obtained even when particles are flowing. Image analysis of the obtained static image enables morphological analysis of particles in the liquid. It is also disclosed that the particle concentration can be analyzed by counting how many particles are contained in the imaged sample volume.

Further, there is an apparatus described in Japanese Patent Application Laid-Open No. 63-94156, which detects the passage of particles without periodically emitting light from a light source and picks up a still image at the timing. In the publication, an optical system for particle detection is provided upstream of a particle passage, in addition to an optical system for capturing a still image, and a particle detector detects that a light beam of a trigger light source that is always turned on crosses a particle. It is described that a stationary image of a particle is obtained by causing a pulsed light source to emit light after a fixed delay time after detection.

Also, Japanese Patent Application No. 4-30080 filed by the present inventor.
Specification No. 8 describes a particle concentration analysis method in which the number of particles in an image obtained by detecting particles is counted and the particle concentration is calculated. In this method, a particle detection area having the same width as the imaging area is provided upstream of the imaging area, and the width of the fluid sample flowing is equal to or smaller than the width of the imaging area. Can be used to accurately obtain the particle concentration. Since a sample liquid having a volume significantly larger than the volume of the field of view to be imaged is flowed and particles are imaged while passing through the field of view, there is an advantage that analysis can be performed in a short time.

[0006]

According to the method described in Japanese Patent Application No. 4-300808, in order to obtain a clear image of a particle and perform highly accurate analysis, the particle must be within the depth of focus of the imaging system. The thickness of the flow range of the sample had to be within the depth of focus because it had to be included. Generally, in a high-resolution imaging system, the depth of focus is thinner than the width of the field of view,
It was necessary to run the sample over a very thin flat area. Therefore, in order to analyze a large amount of sample at high speed, it was necessary to increase the flow rate without increasing the thickness of the sample flow. However, there is a limit to the flow velocity that can be stably flowed in the flow cell, and it was impossible to increase the speed more than a certain level. Further, if the position where the sample flows is displaced due to the influence of the vibration of the apparatus or the foreign matter in the flow cell, the definition of the image is lowered and the accuracy of the analysis is lowered.

An object of the present invention is to provide a particle analyzer capable of performing analysis at high speed and with high accuracy.

[0008]

In order to achieve the above object, the particle analysis method of the present invention is an imaging method having a finite depth of focus, in which a sample solution containing particles is flown with a controlled width and thickness. In a particle analysis method in which an instantaneous image of particles is captured by the system, the thickness of the sample liquid is made thicker than the depth of focus of the imaging system, the focus position of the imaging system is changed oscillatingly, and the particles pass through. It is characterized in that the depth to be detected is detected and an image is taken at a timing related to the depth of passage of particles.

Further, it is characterized in that the optical signal diverging from the particles is converged, and the converged position is detected to obtain the passage depth of the particles.

Further, it is characterized in that the image pickup system repeats image pickup repeatedly and vibrates the focus position at a cycle shorter than the minimum interval of repetition.

In order to achieve the above object, the particle analyzer of the present invention comprises a flow cell for flowing a sample solution in which particles are suspended,
A first light source for irradiating continuous light to the first part of the flow cell, a detector for detecting an optical signal emitted from particles passing through the first part of the flow cell, and a pulsed light for irradiating the second part of the flow cell. A second light source, an image pickup device having a focus near the second part of the flow cell, and a particle analyzer for analyzing the characteristics and concentration of particles by analyzing an image picked up by the image pickup device. A mechanism is provided, which detects position information in the depth direction of particles passing through based on the optical signal of the detector with respect to the imaging device, and the relationship in which the depth information and the operation of the focus adjustment mechanism are determined. It is characterized in that pulsed light is emitted from the second light source at the following timing and an image is taken by the imaging device.

Further, the image pickup device is an object which repeats the image pickup repeatedly, and the focus adjusting mechanism is periodically operated at a cycle shorter than a minimum interval of the repetition of the image pickup device.

Further, the focus adjusting mechanism is a driving mechanism attached to the objective lens.

Further, it is characterized in that an optical signal diverging from particles passing through the first portion of the flow cell is detected by a plurality of optical position detectors.

[0015]

In the analyzer of the present invention, when the passage of particles is detected at the first portion of the flow cell, the depth at which the particles pass is simultaneously detected and a particle depth signal is output. The flow in the flow cell is a laminar flow, and the particles that have passed through the first portion flow to the second portion with the same depth. Since the focus adjusting mechanism provided in the image pickup system including the flow cell, the objective lens, and the image pickup device periodically operates, the depth of the image pickup surface where a clear image is obtained periodically changes. While the particles detected at the first portion are passing through the second portion, a sharp image is obtained because the pulsed light source emits light at the moment when the depth of the imaging surface and the depth of the particles match. Since the vibration cycle of the focus adjustment mechanism is shorter than the minimum interval at which the imaging system repeatedly images, there is a timing at which the imaging surface and the depth of the particles always match during the imaging period, so particles that pass through are rarely missed. . Therefore, a clear image of the particles can be obtained even when the sample is flown in a region thicker than the depth of focus of the imaging system, and a large amount of sample liquid can be flowed in a short time for analysis.

Further, even if the position where the sample flows is deviated due to the influence of the vibration of the apparatus or the foreign matter in the flow cell, since the image is picked up at the timing when the image pickup surface and the depth of the particles match, a clear image is always obtained. Highly accurate analysis is possible.

[0017]

Embodiments of the present invention will be described with reference to the drawings. FIG. 1 is a perspective view of the particle analyzer of the first embodiment.

The flow cell 1 has a sample liquid supply port 21, a sheath liquid supply port 22, and a discharge port 23. A pulsed light irradiation system including a pulsed light source 5 and a condenser lens 7 is arranged on one side of the flow cell 1, and an imaging system including an objective lens 8 attached to the fine movement device 15 and a CCD camera 6 is arranged on the opposite side. The laser light source 4 is obliquely provided on the side where the objective lens 8 is located. Further, there is a half mirror 9 between the condenser lens 7 and the pulse light source 5, and a condenser lens 10, a beam splitter 11, an optical position detector 12a, and
There is a particle detection system consisting of b.

The controller 13 includes an optical position detector 12
The a, b, pulse light source 5, and fine movement device 15 are connected. Further, the CCD camera 6 is connected to the image processor 14.

The sample liquid supply port 21 is supplied with a sample liquid containing particles from a sample liquid supply device (not shown) at a constant speed. A clean sheath fluid is supplied to the sheath fluid supply port 22 from a sheath fluid supplier (not shown) at a constant speed. Inside the flow cell, a sheath flow is formed in which the sample liquid 2 is wrapped in the sheath liquid 3 and flows. Flow cell 1
The sample liquid flows in a laminar flow state at a constant velocity in the parallel flow path portion of

The laser beam 32 emitted from the laser light source 4 continuously irradiates the particle detection region which is a part of the parallel flow path portion of the flow cell 1. Scattered light 33 from which particles in the sample liquid passing through the portion irradiated by the laser beam 32 diverge
Passes through the condenser lens 7, the half mirror 9, the condenser lens 10, and the beam splitter 11, and the optical position detector 12a,
Image on 12b. The signals output from the optical position detectors 12a and 12b include information on the positions where the particles pass.

The fine movement device 15 is a device whose position can be controlled by an electric signal like a piezo element or a voice coil, and the signal from the controller 13 causes the objective lens 8 to have an amplitude of several times to several tens of times the depth of focus of the objective lens 8. 8 vibrates in the optical axis direction.

The controller 13 uses the optical position detector 12
It has a table for converting the signals of a and b into the position where particles pass, a table for converting the signal sent to the fine movement device 15 into the focus position of the image pickup system, and also has a timer. When the passage of the particles in the sample liquid 2 is detected, a light emission signal is sent to the pulse light source 5 at the moment when the particle passage position converted by each table and the focal position coincide with each other within a certain period after a certain delay time.

When the pulsed light source 5 receives the light emission signal, the pulsed light source 5 outputs about 1
It emits microsecond pulsed light. The pulsed light 31 emitted from the pulsed light source 5 is applied to the imaging area which is a part of the parallel flow path portion of the flow cell 1 by the condenser lens 7.

The relationship between the imaging area and the particle detection area is shown in FIG. The particle detection region 25 is an elongated region that crosses the flow of the sample liquid 2. The imaging region 26 is the particle detection region 2
5 is a rectangular region downstream.

An image of particles in the sample liquid flowing through the image pickup region is condensed by the objective lens 8 and is formed on the light receiving surface of the CCD camera 6.

Although the CCD camera 6 operates in a fixed image pickup cycle, the light incident on the light receiving surface during the image pickup cycle can be accumulated and taken out as an image signal. Therefore, if the pulse lamp emits light during the imaging period, a static image of the particles at that moment can be obtained.

The picked-up image signal is analyzed by the image processor 14 and converted into information on the type and number of particles.

FIG. 3 is an explanatory view of the main part of the particle detection system of the first embodiment. The flow cell 1, the condenser lens 7, and the half mirror 9 in FIG. 1 are omitted. P, q, in the figure
r and s represent the positions where the particles pass.

When the particles pass, the scattered light 33 is divided into two by the beam splitter 11 immediately after the condenser lens 10 and forms an image on the optical position detectors 12a and 12b. FIG. 4 shows a state of image formation at that time.

When the particles are at the position of q which is the focus position of the condenser lens 10, the scattered light distribution on the light position detectors 12a and 12b has a steep peak. When the passing position of the particle is at the position p far from the condensing lens 10 or at the position q approaching, the peak of the scattered light distribution becomes gentle and the peak position shifts. The direction in which the peak position shifts is 12
The directions are opposite in a and 12b. When the particle is at the position s shifted laterally, the shift of the peak position of scattered light is 12a.
And 12b shift in the same direction.

A one-dimensional line sensor or a semiconductor optical position detector is used as the optical position detectors 12a and 12b.
The intensity and position of the scattered light distribution can be measured simultaneously. Therefore, by measuring the direction and magnitude of positional deviation of the scattered light distribution on the optical position detectors 12a and 12b, the particle position in the depth direction and the lateral direction with respect to the optical axis can be obtained. The calculation is performed by the table included in the controller 13.

FIG. 5 is a diagram showing the operation timing of the first embodiment. The CCD camera operates with a fixed imaging period as a cycle. The objective lens 8 is periodically driven by the fine movement device 15. When the particles pass through the particle detection area, a particle detection signal is issued. The gate opens for a time B after a delay time A from the particle detection signal. While the gate is open, an imaging pulse is generated at the timing when the depth signal of the passed particle and the lens position signal match each other to perform imaging. Even if two particles are detected during one imaging period, the imaging pulse is performed only once.

The delay time A corresponds to the time for particles to flow from the particle detection area to the upper end of the imaging area. Gate opening time B
Corresponds to the time for the particles to pass through the imaging area. Since the cycle of lens position movement is less than twice the gate opening time B,
The lens position and the particle depth always intersect within the time when the gate is open. The gate opening time B is less than or equal to the imaging cycle.

In the present embodiment, the depth at which the particles pass is detected, and the focus position of the image pickup system is picked up at the moment when the focus is achieved within the changing cycle, so a clear image without defocus can be obtained. Highly accurate image analysis is possible, and highly accurate particle analysis is possible.

Further, even if the sample flow is made thicker than the depth of focus of the imaging system, highly accurate image analysis can be performed,
The flow rate of the sample stream can be increased, and analysis can be performed in a short time. In particular, even in the case of a sample in which the concentration of particles is low and the probability that particles are included in the volume of the field of view of the imaging system is low, a large amount of sample liquid is flowed to detect passage of particles and focus is achieved. Since it is possible to capture images, it is possible to efficiently obtain an image of particles and shorten the analysis time. Also,
Since the amount of analysis can be increased by increasing the flow rate of the sample, the number of particles contained in a certain volume can be accurately obtained.

Further, in this case, it is possible to make the thickness of the sample flow larger than the amplitude of the fine movement device 15, and when the particle is within the range of the change of the focal position, the image is taken and the image analysis is performed. In the case of particles passing outside the range, only particle counting can be performed without imaging, and the particle concentration can be accurately analyzed.

Further, in the case of this embodiment, the increase of the sample flow rate can be made more effective by combining the increase of the sample flow thickness and the increase of the flow velocity. If the flow velocity is increased so that the time for particles to pass through the imaging range is a fraction of the imaging period or less, analysis is performed by the product of the multiplication factor of the sample flow thickness and the multiplication factor of the flow velocity. The amount of sample can be increased.

Compared with the case where the sample amount is increased only by increasing the flow velocity, there are many merits when the increase of the sample amount is attempted by combining the increase of the sample thickness. First, since a relatively low flow velocity is highly effective, a stable sheath flow can be obtained even when the processing accuracy of the flow cell 1 is low, so that the cost of the device can be reduced. Further, since the image shift is small even if the light emission time of the pulse lamp is extended, an inexpensive pulse lamp can be used, and if the light emission time is extended and the light amount is increased, an inexpensive CCD camera with low sensitivity can be used. Further, since the sheath flow rate can be reduced, the amount of sheath liquid consumption can be reduced and the drive system can also be downsized.

Further, in the case of this embodiment, it is not necessary to make the sample flow as thin as the depth of focus of the image pickup system.
The surface of particles in the sample liquid does not come into contact with the sheath liquid to cause a reaction, and therefore it is not necessary to use a special buffer solution or physiological saline for the sheath liquid to prevent the reaction with the particles.

Further, in the case of this embodiment, even if the position of the sample flow shifts due to disturbance such as foreign matter in the flow cell or vibration of the apparatus, it can be covered as long as it is within the range of the fine movement amplitude of the objective lens. High-precision analysis is possible.

Further, in the case of this embodiment, since the position of the particle in the depth direction and the position of the particle in the lateral direction can be detected at the same time, an image is picked up when the particle passes inside the width of the imaging visual field. And efficient analysis is possible.

FIG. 6 is an explanatory view of the main part of the second embodiment of the present invention. In this case, the laser beam 32 is emitted from the condenser lens 7 side. The scattered light 33 from the particles is reflected by the half mirror 9 behind the objective lens 8, separated by the beam splitter 11, and detected by the optical position detectors 12a and 12b. In this case, the objective lens 8 is also used as a lens for collecting scattered light.

FIG. 7 shows the relationship between the image pickup area and the particle detection area in the case of this embodiment. The particle detection area 25 is an area that substantially overlaps with the imaging area 26.

Particles in the sample liquid 2 are particle detection areas 25.
When it flows through, the scattered light of the particles forms an image on the optical position detectors 12a and 12b, but since the objective lens 8 is vibrating by the fine movement device 15, the image forming position moves. When the particles have just come into focus, the imaging positions on the optical position detectors 12a and 12b are the same. Therefore, the optical position detectors 12a, 12b
A focused particle image can be obtained by detecting the scattered light image forming position above and emitting an image by emitting a pulse lamp at the moment of coincidence.

In the case of this embodiment, since the condenser lens and the objective lens which are common to the particle detection system and the image pickup system are used, they are hardly displaced from each other.

Further, since the laser beam 32 does not enter the objective lens 8 after passing through the flow cell 1, the laser beam does not adversely affect the image pickup by the CCD camera 6.

FIG. 8 is a sectional view of the main part of the third embodiment of the present invention. In this case, the wedge plate 18 is placed between the objective lens 8 and the CCD camera 6 and rotated by the motor 17. Since the optical path length of the image pickup system changes due to the rotation of the motor 17, the depth of the focus position changes.

The relationship between the particle detection area and the imaging area in this case is the same as in FIG. The operation of the entire system is the same as that of the first embodiment except that the method of changing the focal position is different.

In the case of this embodiment, since the condenser lens and the objective lens which are common to the particle detection system and the image pickup system are used, they are hardly displaced from each other. Further, since the scattered light 33 is imaged by the objective lens 8 having high imaging performance, the particle position can be detected with high accuracy.

FIG. 9 is a sectional view of the main part of the fourth embodiment of the present invention. In this case, the method of detecting the particle passage position is different. Moreover, the magnification of imaging can be switched.

The laser light source 4 obliquely irradiates the flow cell 1 with the laser beam 32. The condenser lens 10 is installed on the side of the flow cell 1 and collects the scattered light 33 from the particles. The image forming position of the scattered light on the light position detector 12 corresponds to the depth of the particle passing position in the optical axis direction of the imaging system. Therefore, if the imaging position is detected, it can be directly converted into the particle passing position.

A switching lens 16 is provided between the objective lens 8 and the CCD camera 6. The switching lens 16 contains two types of lenses having different focal lengths, and one of them enters the optical path. By switching the lens, the imaging magnification can be switched between high magnification and low magnification.

In the device using this embodiment, the flow rates of the sample liquid and the sheath liquid are changed at the same time as the imaging magnification is changed to change the flow velocity. Also, the intensity of the pulsed light is changed.

In the case of this embodiment, even if the sample liquid contains various particles ranging from large particles to small particles, by changing the magnification during one measurement,
Highly accurate analysis with suitable magnification is possible.

Further, in the case of the present embodiment, since the image pickup system and the particle detection system are completely separated, they do not interfere with each other and the adjustment is easy.

Further, in the case of this embodiment, when the imaging system is changed to a low magnification, the depth of focus becomes deep, so that it is possible to further increase the thickness of the sample flow and improve the analysis efficiency. is there.

Further, in the case of the present embodiment, not only the passage position of particles but also the size can be detected by the optical position detector 12, so that only small particles are imaged at the time of high magnification measurement, and the measurement of low magnification is carried out. Sometimes, imaging only large particles can further improve analysis efficiency.

Although the first to fourth embodiments have been described by the operation of periodically moving the focus, the controller 13 sends a control signal to the fine movement device 15 or the motor 17 after detecting the particles. It is also possible to take an image at the moment when the focus is achieved. It is also possible to enable both a mode for performing periodic operation and a mode for performing random operation with the same device. In this case, an appropriate mode can be selected depending on the amount of sample liquid and the concentration of particles that need to be measured. For example, when the particle concentration is relatively high, the random operation mode can be used to select only particles that do not require long focus movement from among a plurality of passing particles and can pick up an image.

In the above embodiments, the case where the focus is moved at high speed in a short cycle has been described, but it is not always necessary to move the focus at high speed. It is also possible to fix the focus position during one measurement, average the passing positions of the individual particles, and move the focus when the average position is deviated.

In this case, even if the sample flow passage position shifts due to foreign matter in the flow passage of the flow cell or thermal expansion, it is possible to automatically compensate.

[0062]

According to the present invention, the focus position of the image pickup system is periodically changed, and at the same time, the depth at which particles pass is detected, and the image is taken at the moment when the focus position and the depth of passage are detected. Provided is a particle analyzer capable of obtaining a focused and clear image of a particle even when a sample solution is made to flow thicker than the depth of focus of an imaging system and capable of analyzing a large amount of sample in a short time. Is possible.

[Brief description of drawings]

FIG. 1 is a perspective view of a first embodiment of the present invention.

FIG. 2 is a sectional view showing an image pickup area according to the first embodiment of the present invention.

FIG. 3 is an explanatory diagram showing a basic configuration of a particle detection system of the first embodiment.

FIG. 4 is a characteristic diagram showing a scattered light distribution of the first embodiment.

FIG. 5 is a timing chart showing the operation of the first embodiment.

FIG. 6 is a sectional view showing the basic configuration of a second embodiment.

FIG. 7 is a cross-sectional view showing an imaging region of a second embodiment.

FIG. 8 is a sectional view showing the basic structure of a third embodiment.

FIG. 9 is a sectional view showing the basic structure of a fourth embodiment.

[Explanation of symbols]

1 ... Flow cell, 2 ... Sample liquid, 3 ... Sheath liquid, 4 ...
Laser light source, 5 ... Pulse light source, 6 ... CCD camera, 7 ...
Condenser lens, 8 ... Objective lens, 9 ... Half mirror, 10 ... Condensing lens, 11 ... Beam splitter, 12
... Optical position detector, 13 ... Controller, 14 ... Image processor, 15 ... Fine movement device, 21 ... Sample liquid supply port, 22 ...
Sheath liquid supply port, 23 ... Ejection port, 25 ... Particle detection region,
26 ... Imaging area, 31 ... Pulsed light, 32 ... Laser beam, 33 ... Scattered light.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Hidenori Asai 882 Ichimo, Katsuta, Ibaraki Prefecture 882 Ichige, Hitachi Ltd. (72) Inventor Hideyuki Horiuchi 882, Ichige Katsuta, Ibaraki 882 Hitachi, Ltd. Factory Measuring Instruments Division

Claims (1)

[Claims] 1. A particle analysis method in which a sample solution containing particles is flowed while controlling the width and thickness, and an instantaneous image of the particles is captured by an imaging system having a finite depth of focus for analysis.
The thickness of the sample solution is made thicker than the depth of focus of the imaging system, the focus position of the imaging system is oscillatingly changed, the depth at which the particles pass is detected, and the depth at which the particles pass is detected. A particle analysis method, characterized in that imaging is performed at related timings.