JP2002071548A - Flow type particle image analysis method and apparatus - Google Patents

Abstract

(57) [Summary] [Object] To accurately calculate a particle concentration even in a case of a sample containing particles which have a particle size spread and must be subjected to detection processing in two measurement modes. To realize a flow-type particle image analysis method that can be used. A first measurement mode is set so as to mainly analyze a particle component A having a small number of particles and a large number, and a second measurement mode is set so as to analyze a large number of particle components B having a small number of particles. ing. For this reason, it was difficult to measure an accurate particle concentration of the particle component C having a widely distributed particle size. Therefore, the first measurement mode is divided into regions 1 and 2 by the detection level Cs _TH2 , and the second measurement mode is set to the detection level Cs _TH2.
s Divide into areas 3 and 4 with _TH2 . By using the particle information detected in these regions 1 to 4 and processing the particle size from small to large as one continuous data, it is possible to accurately measure the particle concentration of the particle component C. Become.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a flow type particle image analysis method and a flow type particle image analysis apparatus.
The present invention relates to a flow type particle image analysis method and apparatus for capturing an image of a stationary particle suspended in a flowing liquid and analyzing the particles.

[0002]

2. Description of the Related Art In conventional particle image analysis, classification and analysis of cells in blood and cells and particles in urine have been performed by preparing a specimen on a slide glass and observing the specimen with a microscope. . In the case of urine, since the concentration of particles in the urine is low, the sample is observed after being centrifugally concentrated by a centrifuge in advance.

In such a method or apparatus for automating the observation and inspection work, a sample such as blood is spread on a slide glass, set on a microscope, the microscope stage is automatically scanned, and the presence of particles is determined. The microscope stage is stopped at a position where a static particle image is captured, and the classification and analysis of particles in the sample sample are performed by using feature extraction and pattern recognition techniques by image processing technology.

[0004] However, in the above method, it takes a long time to prepare a specimen, and furthermore, it is necessary to find particles while mechanically moving the microscope stage and to move the particles to an appropriate image capturing area. For this reason, there are drawbacks that the analysis requires time and the mechanical mechanism is complicated.

A particle image analyzing method or a particle image analyzing apparatus which does not produce a smear as described above includes a flow site in which a sample sample is suspended in a liquid and flown into a flow cell for optical analysis. The meter method is known.

[0006] The method using a flow cytometer observes the intensity of fluorescence and the intensity of scattered light from each particle in a sample, and has a processing capacity of several thousands per second.

However, it is difficult to observe a feature amount reflecting the morphological characteristics of the particles, and the particles cannot be classified by the morphological characteristics conventionally performed under a microscope.

As an attempt to take an image of a stationary particle in a continuously flowing sample, classify and analyze the particle from each static particle image, see Japanese Patent Application Laid-Open No. 57-50099.
No. 5, JP-A-63-94156, JP-A-4-94.
A technique described in, for example, Japanese Patent No. 72544 is known.

In the technique described in Japanese Patent Application Laid-Open No. 57-500995, a sample sample is passed through a specially shaped flow path, flows through a wide imaging area, and a static particle image is captured by a flash lamp. The method of particle analysis using the method is shown.

In this method, when an enlarged image of sample particles is projected on a CCD camera using a microscope, a flash lamp as a pulse light source emits light periodically in synchronization with the operation of the CCD camera.

With this arrangement, since the light emission time of the pulse light source is sufficiently short, a still image can be obtained even when particles are continuously flowing, and the CCD camera shoots 30 still images per second. be able to.

In the technique described in Japanese Patent Application Laid-Open No. 63-94156, a particle detection system is provided upstream of a particle image photographing area in a sample flow separately from a still particle image photographing system. This is a method in which a particle detection system detects the passage of particles in advance and turns on a flash lamp, which is a pulse light source, at an appropriate timing when the particles reach a particle image capturing area.

In this method, since the light emission of the pulsed light source is not periodically performed, the static particle images can be taken at the same timing only when the passage of the particles is detected, so that the static particle images can be efficiently collected. Therefore, even in the case of a sample sample having a low concentration, a meaningless image having no particles is not captured and image-processed.

Further, in the technique described in Japanese Patent Application Laid-Open No. 5-296915, similarly to the technique described in Japanese Patent Application Laid-Open No. 63-94156, means for detecting particles in a sample flow separately from a stationary particle image system is provided. Means for obtaining the actual number of particles in the sample particles and the number of particles for each classified type from the total number of particles subjected to image processing in the detected particle image is described.

In Japanese Patent Application Laid-Open Nos. 7-83817 and 9-72842, JP-A-5-29691 is disclosed.
No. 5 discloses a one-to-one correspondence method between a detected particle and a detected particle image, which is a problem.

[0016]

In the prior art particle image analyzing apparatus, generally, a still image of a continuously flowing sample particle is analyzed to determine the number of particles of a plurality of types in the sample. In order to perform the classification efficiently, a particle detection system that detects passing particles at a still particle image capturing area or upstream thereof is necessary as is performed in the above-described known example.

That is, the pulse light source is turned on only when the sample particles have passed, and a still image of the sample particles is taken.

The above method can process a measurement sample having a low particle concentration very efficiently, so that the amount of the measurement sample can be increased, the analysis time can be shortened, and the analysis accuracy can be improved.

However, in such a flow type particle image analyzer, a particle detection system for detecting passing particles at or upstream of the stationary particle image capturing area, a pulse light source is turned on only when the particles have passed, Although it has an image capturing system for capturing an image, the particle detection system and the image capturing system generally have different measurement principles.

As the above-described particle detection system, a method is generally used in which a measurement sample flowing in a flow cell is focused and irradiated with a laser beam, and scattered light from particles traversing the laser beam is detected. .

However, in a particle detection system, even if the particle detection condition is equal to or higher than a predetermined particle detection level, a still particle image obtained by an actual image pickup system has a small size and a small light scattering It is often the case that some particles do not reach particle detection levels.

As a result, the number of particles counted by the particle detection system and the number of particles subjected to image processing do not agree even if the particle count due to dead time which cannot be avoided in image capturing is considered, and the particle analysis accuracy and This may cause a decrease in reproducibility.

The technique described in Japanese Patent Application Laid-Open No. 7-83817 provides a method for properly coping with these problems. The method described in JP-A-7-83817 takes a one-to-one correspondence between particles captured in one image and particles detected by a particle detection system.

[0024] To this end, means for counting particles corresponding to detected particles out of particles detected on the screen when the flash lamp light source is turned on from the particle detection signal,
The present invention provides a means for associating the counted value with the detected particles among the particles in the image, and a means for distinguishing the detected particles from the particles not regarded as the detected particles.

In the above-described method, a flow-type particle image analysis method and a flow-type particle image are provided, each including a particle detection system and an image imaging system, wherein the principle of the particle detection system and the principle of particle image identification of the image imaging system are different. In the analyzer, the detection particles correspond to the particles in the still particle image, and even if particles other than the detection particles in the particle detection system are present in the still particle image in the image capturing system, the number of particles of each type in the sample is determined. An object of the present invention is to provide a flow type particle image analysis method and a flow type particle image analysis device which can correctly know the particle abundance ratio, the particle density, and the concentration information and have good analysis accuracy.

In this method, it is necessary to make a one-to-one correspondence between the detected particles and the particles in the particle image for each captured particle image, and a high-speed processing requires a complicated circuit configuration.

As a method of reducing this problem, there is a method of collectively associating the detected particles with the particle image particles after the measurement is completed, as disclosed in Japanese Patent Application Laid-Open No. 9-72842.

The method of correspondence between these two detected particles and the particle image allows the unnecessary particle image to be discarded, and corrects even if the particle image is counted down due to dead time in the image pickup system. The classification ratio of particles can be obtained, and as a result, a correct particle density can be obtained, and analytical data having a good correlation with human microscopy can be obtained.

However, the optical signal by the particle detection is converted into an electric signal by the photodetector. The magnitude of the optical signal depends on the optical refractive index of the particle, absorption, size, internal state of the particle,
It is affected by the scattered light detection conditions, the detection level, and the like, and does not always completely match the morphological information of the particles used in the image processing, particularly, the area, the particle diameter, and the perimeter as the size information.

For this reason, since the particle detection condition does not completely correspond to the particle size parameter based on the image, there is no guarantee that particles in the vicinity of the particle detection level have a correct correspondence.

There is no problem as long as the target can sufficiently treat all the particles by sufficiently lowering the particle detection level. However, when processing a sample containing various particle components and also having a particle component having a wide size distribution, However, setting the particle detection level itself becomes difficult.

Furthermore, for a sample having a plurality of measurement modes and a plurality of types under a plurality of different measurement conditions,
A particular problem arises when it is necessary to combine the measurement results into one sample result.

That is, in a plurality of measurement modes, the particle detection conditions are separately set, so that even if an attempt is made to obtain the particle concentration only with the particle detection conditions of the particle detection system, it is not possible to know the sample particle concentration correctly. There is a problem that there is.

The problem will be described with reference to FIG. Here, in order to simplify the description, a case where there are two measurement modes will be considered.

In the first measurement mode, the flow rate of the sample flow is reduced and the particle detection level is reduced to detect small particles, mainly for the purpose of analyzing the particle component A having a small particle size and a large number. .

On the other hand, in the second measurement mode, in order to process a large number of large particle components B, the number of detected particles is increased and the sample flow rate is increased to increase the measurement volume. It is assumed that

In the second measurement mode, the measurement volume is increased because the number concentration of the target particles is low.

Therefore, the small particle component A can be processed in the first measurement mode, and the large particle component B can be processed in the second measurement mode without any problem.

However, in a normal sample of a size between the small particle component A and the large particle component B, the size distribution has a spread, and the first and second separate measurement modes described above use In some cases, the analysis cannot be performed by the processing of (1).

That is, in the case of the particle components C which are widely distributed from a small size to a large size, a correct particle concentration cannot be measured only in each of the first measurement mode and the second measurement mode.

In the first measurement mode, since the measurement volume is smaller than that in the second measurement mode, the measurement result of the particle concentration has a statistically large variation. In addition, in the second measurement mode, a particle component smaller than the detection level is not detected, so that an originally correct particle concentration cannot be measured.

In the second measurement mode, it is difficult to determine where to set the particle detection level. That is, if the detection level is lowered to detect even small particles, the significance of providing two measurement modes is diminished.

In the case of processing even small particles in the second measurement mode, Japanese Patent Application Laid-Open Nos.
As described in JP-A-72842, the number of particles to be processed is reduced as a result of an increase in the number of particles counted due to dead time unavoidable in the image capturing stage, and the accuracy of particle analysis and reproducibility are reduced. May cause. This is because the processing ratio of the small particle component A, which has a large number to be processed, increases, and as a result, the number of processed images of the large particle component B decreases.

It is an object of the present invention to accurately determine the particle concentration even when a sample containing particles which must be subjected to detection processing in either of the two measurement modes due to the particle components having a wide particle size. It is an object of the present invention to realize a flow type particle image analysis method and apparatus which can calculate the particle size.

[0045]

In order to achieve the above object, the present invention is configured as follows. (1) Flowing a liquid sample in which particles are suspended into a flow cell and counting the number of particles in the liquid sample by a particle detection system; and detecting a still image of particles passing through an imaging region in the flow cell. In a flow type particle image analysis method having an imaging step of imaging by an imaging system and an analysis step of performing morphological classification of particles in the still image by image analysis processing, a plurality of measurement modes having different particle detection conditions are set. The setting step, the step of associating the particles detected in the particle detection step for each measurement mode with the particle images classified and identified by performing image analysis processing, and the analysis results obtained for each measurement mode A step of dividing into a plurality of size regions using characteristic parameters of the particle image, and a step of calculating a particle concentration of a plurality of particle components in the sample for each measurement mode. You.

(2) Preferably, in the above (1),
The plurality of measurement modes having different particle detection conditions are two measurement modes.

(3) Also, preferably, in the above (2), the two measurement modes having different particle detection conditions are combined as a first particle detection condition by combining these two measurement modes. The particle detection area is set such that all the particles are detected.

(4) Preferably, in the above (2), each of the two measurement modes having different particle detection conditions has a particle detection region preset as a second particle detection condition. When a particle detection signal higher than the set level is detected, it is regarded as a detected particle.

(5) Preferably, in the above (2) or (3), a step of processing under the first particle detection condition and classifying / identifying the associated particle image into a plurality of types of particles. Estimating the number of particles in a plurality of regions using the information on the number of particles detected under the first particle detection condition.

(6) Preferably, in the above (5), a first measurement mode and a second measurement mode for measuring particles having a particle diameter larger than the particle diameter of the object to be measured in the first measurement mode. The number of all particle images processed in the first measurement mode is Ng1, the number of particle images of the first particle component in the first region of the plurality of regions is NgA (1),
The number of particle images of the third particle component in the first area is NgC (1),
The number of particle images of the second particle component in the second region is represented by NgB (2),
The number of particle images of the third particle component in the second area is NgC (2),
Assuming that the total number of particles detected by the particle detection system is Nd1,
The number of particles NA (1) of the first particle component in the first region, the number of particles NC (1) of the third particle component in the first region, and the number of particles NC (1) in the second region The particle number NB (2) of the particle component 2;
The following equations (1-1) to (1-4) are used to calculate the number of particles NC (2) of the second particle component in the second region, NA (1) = NgA (1) / Ng1 × Nd1 ---- (1-1) NB (2) = NgB (2) / Ng1 × Nd1 ---- (1-2) NC (1) = NgC (1) / Ng1 × Nd1 ---- (1 -3) Use NC (2) = NgC (2) / Ng1 × Nd1 ---- (1-4).

(7) Preferably, in the above (4), the step of classifying and identifying the associated particle images processed under the second particle detection condition into a plurality of types of particle groups; Estimating the number of particles in each region using information on the number of particles detected under the particle detection conditions.

(8) Preferably, in the above (7), the measurement mode includes a first measurement mode for detecting the first particle component and a second measurement mode having a diameter larger than that of the first particle component. A second measurement mode for detecting the second particle component. The first measurement mode is divided into a first region and a second region, and the second measurement mode is divided into a third region and a third region. 4, the total number of particle images processed in the second measurement mode is Ng2, the number of particle images of the second particle component in the fourth region is NgB (4), and the number of particle images of the first particle component is NgB (4). The number of particle images of the third particle component having a diameter including the diameter of the second particle component from a part of the diameter is defined as NgC (4), and the total number of particles detected by the particle detection system is defined as Nd2.
Then, the estimated values of the number of particles NB (4) of the second particle component and the number of particles NC (4) of the third particle component processed in the fourth region are expressed by the following equations (2-1), (2-1). 2-2), NB (4) = NgB (4) / Ng2 × Nd2 ---- (2-1) NC (4) = NgC (4) / Ng2 × Nd2 ---- (2-2) Use to calculate.

(9) Preferably, in the above (1) or (2), a plurality of regions are divided for each measurement mode by using a particle image parameter relating to a predetermined size, and particles in each region are divided. A step of determining the number of particles for each type.

(10) Preferably, in the above (9), a step of using particle diameter information as a particle image parameter relating to a preset size is provided.

(11) Preferably, in the above (9), there is provided a step of using area information of particles as particle image parameters relating to a preset size.

(12) Preferably, in the above (9), a step of using the projection length of the particle as a particle image parameter relating to a preset size is provided.

(13) Preferably, the above (10)
Or in (12), the particle diameter or the projection length is a length along the flow direction of the sample.

(14) Preferably, in the above (5) to (8), the sample is included in the sample based on the measurement condition information, the number of each particle component obtained for each of a plurality of regions, and the counting result of the particle detection system. Calculating a particle concentration of the plurality of types of particles.

(15) Preferably, (14)
Has a first measurement mode and a second measurement mode for measuring particles having a particle diameter larger than the particle diameter of an object measured in the first measurement mode, wherein the first measurement mode is The first measurement area is divided into a first area and a second area, the second measurement mode is divided into a third area and a fourth area, and the number of particles of the first particle component in the first area is NA ( 1), the number of particles of the third particle component in the first region is NC (1), the number of particles of the second particle component in the second region is NB (2), and the third particle in the second region is The number of particles of the component is NC (2), the number of particles of the second particle component processed in the fourth region is NB (4), and the number of particles of the third particle processed in the fourth region is NB (4).
The number of particles of the particle component is represented by NC (4), and the volume of each sample measured in the first measurement mode and the second measurement mode is represented by Vm1.
And Vm2, the particle concentration DA of the first particle component, the particle concentration DB of the second particle component, and the particle concentration DC of the third particle component are represented by the following equations (3-1) to (3-3): DA = NA (1) / Vm1 ---- (3-1) DB = (NB (2) + NB (4)) / (Vm1 + Vm2) ---- (3-2) DC = NC (1 ) / Vm1 + (NC (2) + NC (4)) / (Vm1 + Vm2) ---- (3-3)
Calculated using

(16) Preferably, in the above (2) or (9), image particle size information corresponding to a particle detection area in the second measurement mode is used as a particle image parameter relating to a predetermined size. Used to divide the first measurement data into a plurality of regions.

(17) Preferably, the above (16)
And determining as a particle image parameter relating to a preset size from a histogram of corresponding image parameters in the second measurement mode.

(18) Preferably, the above (17)
In, it is determined from the particle image parameters of the sample being processed.

(19) Preferably, the above (17)
In the above, it is determined from the particle image parameters of a plurality of samples.

(20) Further, preferably, (17)
In, the particle concentration in each of the plurality of regions in each measurement mode is calculated, and the state of the measurement system is checked by comparing the particle concentration between the measurement modes.

(21) Preferably, the above (20)
In the above, if an abnormality is found by checking the measurement state, the particle concentration in the sample is calculated using only the data in the measurement mode considered to be normal.

(22) Preferably, the above (20)
In the above, when an abnormality is found by checking the measurement state, a step of calculating a ratio of data of two measurement data and estimating a value of a particle concentration in an abnormal measurement mode is included.

(23) Preferably, in the above (1) or (2), correction of visual field vignetting due to inconsistency between the sample flow position and the imaging of the particle image and correction of sample thinning are performed in each measurement mode.

(24) Preferably, in the above (1) or (2), when the optical system magnification of each measurement mode is different, the size information is converted again by the magnification.

(25) A liquid sample in which particles are suspended flows into a flow cell, and a particle detection means for counting the number of particles in the liquid sample by a particle detection system; and a stationary state of particles passing through an imaging region in the flow cell. In a flow type particle image analyzer having an imaging unit for imaging an image by a particle imaging system and an analysis unit for performing morphological classification of particles in the image by an image analysis process, a plurality of measurements having different particle detection conditions are performed. Means for setting the mode, means for associating the particles detected by the particle detecting means for each measurement mode with the particle images classified and identified by image analysis processing, and analysis obtained for each measurement mode. Means for dividing the result into a plurality of size regions using the characteristic parameters of the particle image, and means for calculating the particle concentration of the plurality of particle components in the sample for each measurement mode. Provided with a door.

(26) Preferably, in the above (25), the plurality of measurement modes having different particle detection conditions are two measurement modes.

(27) Preferably, the above (26)
In, two measurement modes with different particle detection conditions are:
By combining these two measurement modes, the first
The particle detection area is set such that all the particles to be measured are detected.

(28) Preferably, (26)
Or in (27), means for classifying and identifying the associated particle image processed under the first particle detection condition into a plurality of types of particles, and using the number of particles detected under the first particle detection condition Means for estimating the number of each particle.

(29) Preferably, the above (25)
Or in (28), processing under the second particle detection condition,
A means for classifying and associating the associated particle images into a plurality of types of particle groups, and a means for estimating the number of each particle using the number of particles detected under the second particle detection condition.

(30) Preferably, the above (25)
Alternatively, in (26), based on the measurement conditions, the number of particles detected under the second particle detection conditions, and the counting result of the particle detection system, the particle concentration of a plurality of types of particles contained in the sample is calculated. Means.

(31) Preferably, the above (25)
Alternatively, in (30), the first measurement mode is divided into a plurality of regions using the image particle size information corresponding to the particle detection region in the second measurement mode as the particle image parameter relating to the preset size.

(32) Preferably, the above (31)
And means for determining from the histogram of the corresponding image parameter in the second measurement mode as the particle image parameter relating to the preset size.

(33) Preferably, the above (31)
In, it is determined from the particle image parameters of the sample itself being processed.

(34) Preferably, the above (31)
In the above, it is determined from the particle image parameters of a plurality of samples.

(35) Preferably, the above (31)
In, the particle concentration of each of the plurality of regions in each measurement mode is calculated, and the state of the measurement system is checked by means for comparing the particle concentration between the measurement modes.

(36) Preferably, the above (25)
From (35) to (35), the analysis target is a biological cell.

(37) Preferably, the above (25)
From (35) to (35), the analysis target is a blood cell component present in blood.

(38) Preferably, the above (25)
From (35) to (35), the analysis target is a urinary sediment component present in urine.

[0083]

DESCRIPTION OF THE PREFERRED EMBODIMENTS First, the principle of the present invention will be described with reference to FIG.
This will be described with reference to FIG. Note that, for simplicity, the description will be made assuming that the number of the particle measurement modes is two.

As shown in FIG. 4, in the measurement sample,
Assume that three types of particle components are included. The three types of particle components are a particle component A having a small size, a particle component B having a large size, and a particle component C which is widely distributed from a small size to a large size distribution. Hereinafter, these will be referred to as a particle component A, a particle component B, and a particle component C, respectively.

Since the particle detection area in the first measurement mode covers all particles contained in the sample, it is assumed that the detection level is at a level that can sufficiently detect the particle component A.

On the other hand, in the second measurement mode, the detection level is determined so that the particle component B can be sufficiently detected.
It is assumed that the particle component C is set under the condition that only a part of the particles can be detected as shown in FIG.

FIG. 4 shows the particle image area on the horizontal axis and the histogram of the image parameters on the vertical axis. The horizontal axis is not limited to the image parameters related to the particle image area, but the particle size information. You can replace it with the general. In FIG. 4, the particle detection region has a certain width, because the detection principle of the actual particle detection system does not always match the particle image size information. However, in this description, an image particle larger than the particle image area constant Cs _TH2 will be described as a detected particle.

As shown in FIG. 4, each of the two measurement modes is divided into two regions using the particle detection level Cs _TH2 of the second measurement mode. The first measurement condition is divided into the region 1 and the region 2, and the second measurement mode is divided into the region 3 and the region 4.

Although it is often thought that no particles are present in the region 3, it is often the case that small particles other than the detected particle image are photographed at the same time. It can be ignored by taking. This operation for detecting a detected particle and a detected particle image occurs both in the first measurement mode and when fine particles such as dust are present, and is a necessary operation in either measurement mode.

A method for obtaining this correspondence will be described. There are two ways to take this correspondence. The first method is a method described in JP-A-7-83817.
A one-to-one correspondence is established between the particles captured in one frame image and the particles detected by the particle detection system during the time when the flow passes between both ends of the image.

It is necessary to select a particle image using the number of particles detected within this time and the size information of the particle image in the image. This is because small undetected particles may also be present in the image at the same time.

For this purpose, it is considered that only the number of detected particles from the larger size parameter of all the particle images existing in one frame is regarded as detected particles. As the size parameter, particle diameter information, image area information, and particle projection information are used.

Further, from the attitude control effect of the flow, considering that the major axis direction tends to be aligned with the flow direction, it is better size information to use the diameter information of the particle in the flow direction. In this case, it also corresponds to the pulse width of the particle detection waveform.

However, in this method, it is necessary to establish a one-to-one correspondence between the detected particles and the particles in the particle image for each captured particle image, and a high-speed processing requires a complicated circuit configuration. .

As a method of alleviating this problem, there is a method of collectively associating the detection particles with the particle image particles after the measurement is completed, as disclosed in Japanese Patent Application Laid-Open No. 9-72842. That is, instead of taking the correspondence between the detected particles and the particle images in units of one image, at the end of the measurement, by taking into account the correspondence in units of samples, only the predetermined particle image is extracted from the larger size parameter of the whole particle image. Things.

The predetermined number of images can be known by calculating the average number of particle images from the measurement conditions and the number of detected particles counted by the particle detection system. Details are described in JP-A-9-72842. In this method, there is a step of calculating the average number of particle images, which does not take stricter correspondence than that taking correspondence for each frame image, but considering the statistical fluctuation due to counting down due to dead time, the difference between the two is different. Is small enough to be used as an estimate of particle concentration.

In these two methods, unnecessary particle images can be discarded, and even if particle images are counted down due to dead time in the image pickup system, the amount of the countdown is corrected by The correct particle classification ratio can be obtained. As a result, the correct particle density is obtained,
There is a characteristic that analytical data having a good correlation with human microscopy can be obtained.

However, the particle components are widely distributed from small to large in size, and the particle detection level of the particle component C present in the region in question is the second size in which only a certain size or more can be detected. In the measurement mode alone, a correct particle concentration cannot be obtained.

Next, the steps of estimating the number of components of each particle in each measurement mode will be described. Since there is particle image countdown due to dead time, the number of particles of each particle component measured in the measurement sample in each measurement mode does not match the number of processed particle images.

Therefore, by taking the product of the ratio of the number of particle images of each particle component A, B, and C to the number of all particle images and the number of particles detected by the particle detection system, the sample processed in each measurement mode is obtained. Estimate the number of each component particle that will be present in the volume.

The particle detection threshold in the first measurement mode is set so that all the particle components can be detected. The size (area) of the particle image corresponding to this detection threshold is Cs _TH1
It is. Further, the particle detection threshold used in the second measurement mode for separating the region 3 and the region 4 is Cs _TH2 .

The number of all particle images processed in the first measurement mode is Ng1, and the number of particle images of the particle component A in the area 1 is NgA.
(1) The number of particle images of the particle component C in the region 1 is NgC (1), the number of particle images of the particle component B in the region 2 is NgB (2), and the number of particle images of the particle component C in the region 2 is NgC ( 2)
Processed in the measurement mode, the number of particles NA of the particle component A in the region 1 (1), the number of particles NC of the particle component C in the region 1 (1), the number of particles NB (2) of the particle component B in the region 2, Number of particles of particle component C in region 2
The estimated value of NC (2) is represented by equations (1-1) to (1-4). Note that the total number of particles detected by the particle detection system is Nd1.

NA (1) = NgA (1) / Ng1 × Nd1 ---- (1-1) NB (2) = NgB (2) / Ng1 × Nd1 ---- (1-2) NC (1 ) = NgC (1) / Ng1 × Nd1 ---- (1-3) NC (2) = NgC (2) / Ng1 × Nd1 ---- (1-4) As in the first measurement mode, The second measurement mode is as follows.

The particles in the region 3 do not exist originally because they are below the particle detection threshold, and are discarded at the stage of establishing a correspondence even if the corresponding particles below Cs _TH2 are present in the image.
There is no particle image.

The number of all particle images processed in the second measurement mode is Ng 2, and the number of particle images of the particle component B in the area 4 is Ng B
(4) Assuming that the number of particle images of the particle component C is NgC (4), the estimated value of the number of particles NB (4) of the particle component B in the region 4 and the estimated number of particles NC (4) of the particle component C in the region 4 are as follows. , And equations (2-1) and (2-2). The total number of particles detected by the particle detection system is expressed as Nd
And 2.

NB (4) = NgB (4) / Ng2 × Nd2 ---- (2-1) NC (4) = NgC (4) / Ng2 × Nd2 ---- (2-2) As described above, in the first measurement mode, the purpose is to mainly process the particle component A. Since the sample volume to be measured is small, the particle components B and C have a small number of images, and the statistical data This is a processing mode with large variations.

On the other hand, in the second measurement mode, the particle components B and C
However, as in the case of the particle component C, particles having a small size in the particle component C are not processed.

In order to deal with such a problem, the particle component A calculates the particle concentration per unit volume only from the first measurement mode.
The calculation is performed in consideration of both the first measurement mode data and the second measurement mode data.

The particle concentrations DA, D of the particle components A, B, and C
B and DC are calculated by equations (3-1) to (3-3). Here, Vm1 and Vm2 are the sample measurement volumes in the first measurement mode and the second measurement mode, respectively.

DA = NA (1) / Vm1 ---- (3-1) DB = (NB (2) + NB (4)) / (Vm1 + Vm2) ---- (3-2) DC = NC (1) / Vm1 + (NC (2) + NC (4)) / (Vm1 + Vm2) ---- (3-3) In general, in the above formulas (3-2) and (3-3) When the measurement volume satisfies the condition of Vm1 << Vm2, NB (2) and Vm1 may be ignored for the particle component B.
Similarly, with respect to the particle component C, even if the second item NC (2) and Vm1 in the equation (3-3) are ignored, the error in the concentration calculation is small.

As described above, a preset size
The second measurement mode is used as the particle image parameter
Threshold of particle image size information corresponding to the particle detection area
Cs _TH2Divides the first measurement mode into multiple areas
It is characterized by that.

The feature is that the particle image size information is divided into the first measurement mode and the second measurement mode based on the image parameter size information.

The reason is that the threshold value C of the particle image size information
In the method of dividing into two regions at the particle detection stage using the particle detection level corresponding to s _TH2 , the region of the first measurement mode can be divided into two, but the particle detection signal and the particle image size This is because information does not always match.

That is, although the particle detection signal and the particle image size approximately correspond to each other, the particle detection signal indicates the difference between the type of the particle and the optical property of the particle (for example, the refractive index).
This is because it largely depends on sample flow conditions, optical system adjustment state, and the like.

The flow conditions of the sample differ in each measurement mode. Particularly, the difference in the flow velocity is affected by the frequency characteristic of the detection system, and the waveform of the particle detection signal also changes. This is because the above-described problem does not occur when all processes are handled in a unified manner using image size information.

The determination of the threshold value Cs _TH2 of the particle image size information is performed as follows. One is to measure a large number of samples in advance under the second measurement mode condition, and obtain a histogram for the size information of the particle image as shown in FIG. The actually obtained histogram should be as indicated by the dotted line near the particle detection area.

The small-sized particles are not detected, and have a distribution different from the original size histogram. Threshold C
For s _{TH2, the} point at which the portion represented by the dotted line starts to decrease is defined as the particle image detection level. If the number of particle images to be measured is sufficiently large, the above operation can be performed for each measurement sample to determine the threshold value Cs _TH2 .

The threshold Cs is set below the section drawn by the dotted line.
_It should be noted that if _TH2 is set, the histogram shape of the region 4 may be incorrect, and the particle concentration value of the region 4 may not be accurate.

The particle concentration of the particle component B can be determined only in the first measurement mode or the second measurement mode. Using this fact, it is possible to check if there is a difference in particle concentration from statistical errors. The same applies to the region 2 and the region 4 for the particle component C.
It can also be checked by comparing the particle concentration of only the part.

By using this fact, the abnormal data can be corrected or the particle concentration can be calculated using only the measurement mode data under normal conditions.

At the stage of calculating the particle concentration, the phenomenon of thinning of the sample caused in the sample handling processing stage,
There may be a case where the correspondence between the number of detected particles and the number of images is insufficient due to the mismatch between the sample flow and the imaging region. These data correction methods are described in JP-A-11-947.
27.

In the above description, the measurement conditions in the two measurement modes, particularly the optical magnification, have been implicitly assumed to be constant. However, when the magnification is different, the difference in magnification is corrected and the particle size is corrected. Information needs to be handled. Therefore, the flow-type particle image analysis method according to the present invention may include a step of correcting a difference in magnification.

As described above, the analysis results in a plurality of measurement modes can be regarded as one continuous data on the particle size by using the particle image size information. As a result, for particle components widely distributed from small to large particle sizes, it is necessary to correctly estimate the particle concentration of each particle component per unit volume from the particle image classification results processed under a plurality of different measurement conditions. Can be done.

Next, an embodiment of the present invention will be described with reference to FIGS. 1, 3 and 5. FIG. 1 is a schematic configuration diagram of a flow-type particle image analyzer used in a flow-type particle image analysis method according to an embodiment of the present invention, and FIG.
It is a block diagram which shows the structure of a central control / arithmetic processing part in embodiment of FIG. FIG. 5 is a flowchart showing a processing flow of a particle number calculation operation of the apparatus and the processing unit shown in FIGS. 1 and 3.

In FIG. 1, 100 is a flow cell, 10
1 is an image capturing means, 102 is a particle analyzing means, 103 is a particle detecting means, 1 is a flash lamp, 1a is a flash lamp driving circuit, 2 is a field lens, 3 is a microscope condenser lens, 5 is a microscope objective lens, and 6 is a connection lens. An image position 7 is a projection lens.

Reference numeral 8 denotes a TV camera, 11 denotes a field stop,
Reference numeral 12 denotes an aperture stop, 14 denotes a laser beam, 15 denotes a semiconductor laser, 16 denotes a collimator lens, 17 denotes a cylindrical lens, 18 denotes a reflecting mirror, and 19 denotes a minute reflecting mirror.

Reference numeral 20 denotes a beam splitter, 21 denotes an aperture, 22 denotes a light detection circuit, 23 denotes a flash lamp lighting control circuit, 24 denotes an AD converter, 25 denotes an image memory, 26 denotes an image processing control circuit, and 27 denotes feature extraction. A circuit, 28 is an identification circuit, 29 is a central control / arithmetic processing unit, 40 is a particle detection processing unit, and 50 is a display unit.

Referring to FIG. 1, a flow type particle image analysis apparatus used in a flow type particle image analysis method according to an embodiment of the present invention includes a flow cell 100 to which a sample liquid in which particles are suspended is supplied, The apparatus includes an image capturing unit 101 that captures an image of the sample liquid in the sample 100, a particle detecting unit 103 that detects particles, and a particle image analyzing unit 102 that analyzes particles from a captured image.

The flow cell 100 is supplied with the sheath liquid 111 together with the sample liquid 110, and forms a flow in which the sample liquid 110 is wrapped in the sheath liquid 111.

The sample liquid flow 110 is a stable steady flow having a flat cross section perpendicular to the optical axis 9 of the microscope of the image pickup means 101, that is, a so-called sheath flow. The center of the flow cell 100 is positioned above the plane of the paper. It flows down from below.

The flow rate of the sample liquid flow 110 is controlled by the central control / arithmetic processing unit 29.

The image pickup means 101 has a function as a microscope, and a flash lamp 1 as a pulse light source, a flash lamp driving circuit 1a for emitting the flash lamp 1, and a pulse light beam from the flash lamp 1 are made parallel. Field lens 2 and microscope condenser lens 3 for focusing parallel pulsed light beam 10 from field lens 2 to sample liquid flow 110 in flow cell 100
And a microscope objective lens 5 for condensing a pulse light beam applied to the sample liquid flow 110 in the flow cell 100 to form an image at the image forming position 6, and a particle image at the image forming position 6 projected through the projection lens 7. , A TV that converts an image signal into an image data signal, which is an electric signal captured by an interlace method
Camera 8 and field stop 1 for limiting the width of pulsed light beam 10
1 and an aperture stop 12. Note that, as the TV camera 8, a CCD camera or the like having little afterimage is generally used.

The particle detecting means 103 is a semiconductor laser 15 serving as a detection light source for emitting laser light as detection light.
A collimator lens 16 for converting the laser beam from the semiconductor laser 15 into a parallel laser beam 14, a cylindrical lens 17 for focusing only one direction of the laser beam 14 from the collimator lens 16, and a cylindrical lens 1.
7 is located between the microscope condenser lens 3 and the flow cell 100, and reflects the laser beam 14 from the mirror 18 upstream of the image capturing area on the sample liquid flow 110. And a minute reflecting mirror 19 for guiding to the vicinity.

Further, the particle detecting means 103 is provided with a microscope objective lens 5 for condensing the scattered light of the laser beam 14 by the particles (this microscope objective lens 5 is shared with the microscope objective lens 5 for image formation of the image pickup means 101). Is performed), a beam splitter 20 that reflects the scattered light condensed by the microscope objective lens 5, and receives the scattered light from the beam splitter 20 via a diaphragm 21 and outputs an electric signal based on the intensity thereof. A light detection circuit 22; and a flash lamp driving circuit 1a based on an electric signal from the light detection circuit 22.
And a flash lamp emission control circuit 23 for operating the flash lamp.

The particle analyzing means 102 converts the image data signal transferred from the TV camera 8 into a digital signal, and stores the data based on the signal from the AD converter 24 at a predetermined address. An image processing control circuit 26 for controlling writing and reading of data in the image memory 25;
A feature extraction circuit 27 that performs image processing based on a signal from the image memory 25 to perform the particle count and classification, an identification circuit 28,
A central control / arithmetic processing unit 29; a particle detection system processing unit 40 that determines the number of particles in the sample liquid 110, controls the particle detection system, and calculates a particle image; A display unit 50 for displaying the result.

The central control / arithmetic processing unit 29 is provided with a TV
Setting of photographing conditions of the camera 8 and flow conditions of the sample liquid in the flow cell 100, control of the image processing control circuit 26, storage of image processing results from the identification circuit 28, transmission and reception of data with the particle detection processing unit 40, and display The display unit 50 is configured to perform display and the like.

Next, the configurations of the central control / arithmetic processing unit 29 and the particle detection processing unit 40 will be described with reference to FIG. 3, the same reference numerals as those in FIG. 1 denote the same parts, and a detailed description thereof will be omitted.

In FIG. 3, the particle detection processing section 40 includes a particle counting section 51, a particle image number calculation section 52, and a particle detection control section 53. The central control / arithmetic processing unit 29
Are the image correspondence unit 54, the region 1 particle processing unit 55, the region 2 particle processing unit 56, the region 3 particle processing unit 57, and the region 4
A particle processing unit 58, a measurement condition setting unit 59, a particle concentration calculation 60, and a central control unit 61 are provided.

Next, the operation of the flow type particle image analysis method having the above configuration and the flow type particle image analysis apparatus used for the method will be described.

First, the operations of the image pickup means 101 and the particle detection means 103 will be described. The semiconductor laser 15 constantly oscillates continuously, and always observes that particles in the sample 110 pass through the detection region.

The laser beam 14 from the semiconductor laser 15 is converted into a parallel laser beam by the collimator lens 16, and the laser beam 14 is focused by the cylindrical lens 17 in only one direction.

The laser beam 14 is reflected by the reflecting mirror 18 and the micro-reflecting mirror 19 and is irradiated onto the sample liquid flow 110 in the flow cell 100. This irradiation position is a particle detection position where the laser beam 14 is focused by the cylindrical lens 17, and is a position near the upstream side of the image capturing area on the sample liquid flow 110.

When a particle to be analyzed crosses the laser beam 14, the laser beam 14 is scattered by the particle. The scattered light of the laser beam 14 is reflected by the beam splitter 20, received by the photodetector circuit 22, and converted into an electric signal based on the intensity.

Further, an output signal from the light detection circuit 22 is sent to a particle detection processing section 40, and a signal having a predetermined level and a predetermined pulse width or more of the sent signal is sent to the particle detection processing section 40.
It is regarded as a detected particle, and the result is transmitted to the flash lamp lighting control circuit 23.

As described above, when the particles are detected, the flash light emission signal is transmitted from the flash lamp lighting control circuit 23 to the flash lamp drive circuit 1a, assuming that the particles to be processed have passed. The predetermined delay time is determined by the distance between the particle detection position and the image capturing area, the flow rate of the sample liquid 110, and the like.

When a flash emission signal is transmitted to the flash lamp drive circuit 1a, the flash lamp drive circuit 1a causes the flash lamp 1 to emit light. The pulse light emitted from the flash lamp 1 travels on the microscope optical axis 9, becomes parallel light by the field lens 2, is focused by the microscope condenser lens 3, and is focused on the flow cell 100.
The sample liquid stream 110 is irradiated on the inside.

The width of the pulse light beam 10 is limited by the field stop 11 and the aperture stop 12. Flow cell 10
Pulse light flux 1 applied to sample liquid flow 110 in 0
Numeral 0 is condensed by the microscope objective lens 5, passes through the beam splitter 20, and forms an image at an image forming position 6. The image at the image forming position 6 is projected on the imaging surface of the TV camera 8 by the projection lens 7, and is converted into an image data signal by an interlace method.

Thus, a static particle image of the particles in the sample liquid 110 has been captured.

The imaging conditions of the TV camera 8 are preset in the central control unit 29, and the imaging operation of the TV camera 8 is controlled by the imaging conditions.

Next, the particle analyzing means 102 will be described. An image data signal imaged by the TV camera 8 and converted by the interlace method is converted into a digital signal by the AD converter 24, and data based on this is output to the image memory 25. Then, the image memory 25 is controlled by the image processing control circuit 26, and the data is stored at a predetermined address of the image memory 25.

The data stored in the image memory 25 is read out under the control of the image processing control circuit 26, input to the identification circuit 28 via the feature extraction circuit 27, and is subjected to image processing. The result is stored in the unit 29. The contents stored in the central control / arithmetic processing unit 29 include:
9 shows particle identification feature parameters and particle classification result data used for particle classification.

The classification and identification processing of particles is automatically performed by the usual pattern recognition processing. The image processing result, the measurement conditions, and the information on the number of images subjected to the image processing are
It is sent from the image processing control circuit 26 to the particle detection processing section 40.

The details of the operations of the central control / arithmetic processing unit 29 and the particle detection processing unit 40 will be described below with reference to FIG.

In FIG. 3, a photodetector 22 detects a particle detection signal having a signal level higher than a predetermined signal level. The particle detection level is set by the particle detection control unit 53, and a signal detected when an electric signal exceeding the set value is detected is set as a detected particle signal. Since there are a plurality of measurement modes, the detection level differs for each measurement mode, and the detection level is designated by the instruction of the measurement condition setting unit 59 via the central control unit 61.

When the particles pass through the particle detection area in the flow cell 100, whether the particle image can be captured, that is, the image captured immediately before is being captured by the TV camera 8, or the image processing is being performed. The determination is performed by the particle detection control unit 53.

If image processing is not being performed, the flash lamp 1 is turned on via the flash lamp lighting control unit 23 and the flash lamp driving circuit 1a. In this case, since the position of the particle detection position and the position of the particle image imaging are generally different, the time delay until the lamp is turned on calculated from the distance between the two points and the sample flow rate is determined by the measurement condition setting unit 5.
9 is set.

[0157] The particle detection signal is supplied to the particle counting section 51, and based on the supplied particle detection signal, the particle counting section 5 receives the signal.
In step 1, the number of detected particles is counted. At the end of the measurement, the total number of detected particles in each measurement mode is counted. Based on the number of detected particles and the measurement conditions such as the sample flow rate, the flow velocity, and the measurement time, the average number of particle images that would have been captured is calculated.

In calculating the number of particle images, there are two methods of estimating and calculating the number of frame images from the number of detected particles and the method of using the number of lighting of the flash lamp 1. The use can estimate the number of particle images closer to the actual measurement conditions.

The number of times the flash lamp 1 is turned on can be known by counting the number of times the lamp is turned on by the particle detection control unit 53. The number of times the flash lamp 1 is turned on can also be known by counting the number of frames subjected to image processing by the image processing control circuit 26.

Usually, the number of particle images actually processed is larger than the number obtained as described above. This is because no particles are detected in the image, but many small-sized particles are present.

For this reason, a distinction is made between the particle image detected by the image correspondence unit 54 and the image that is not. The particle image here is obtained by the feature extraction circuit 27 and the identification circuit 28 shown in FIG.
And the particle classification is performed. By the feature extraction circuit 27 and the identification circuit 28, the size parameter information of each particle image can be known. Using this size parameter information, the correspondence with the particle detected image is obtained as follows. This is because it is considered that the size information of the particles corresponds to the size of the particle detection signal.

Therefore, the reordering process is performed for all the processed particle images in descending order of the size parameter, and only the number of the particle images obtained by the particle image number calculation unit 52 calculated earlier from the larger one is extracted from the particle images. Correspondence with detection particles can be taken.

The details of the correspondence between the detected particles and the particle images described above are described in detail in JP-A-9-72842.

In the above-mentioned method of associating the detected particles with the particle images, the method of processing one sample as a whole has been described. No. 83817. In flow-type particle image processing having a particle detection system, an operation for obtaining the correspondence between such detected particles and particle images is required.

From the corresponding particle images, the region 1 particle processing unit 55, region 2 particle processing unit 56, or region 3 particle processing unit 57 and region 4 particle processing unit 58 exist in each region for each measurement mode. Count the number of particles for each particle type. The estimation of the number of particles is performed by using the expressions (1-1) to (1-4) or (2) using the number of detected particles in each measurement mode, the total number of images, and the number of images of each particle component subjected to image processing. -1), (2-
2) Calculate the amount. Normally, since the particles in the region 3 do not exist, the process may be set to 0 from the beginning and not performed.

The flow of the above processing will be described with reference to FIG. Since the processing procedure in each measurement mode is the same, only the first measurement mode will be described.

First step: The measurement conditions in the first measurement mode are set. As the measurement conditions, a measurement time, a sample flow condition, and a value of a particle detection level are set. In the first measurement mode, since all the measurement target particle components are targeted, the particle detection level is sufficiently reduced so that even small particles are detected.

Second stage: A stage for determining whether or not the end state of the first measurement mode has been reached. If the end stage has not been reached, the process proceeds to the next third stage, and if the end stage has been reached. If so, proceed to the sixth stage.

Third stage: The particle detection process is continued, and when the particles are detected, the flash lamp 1 is turned on when the detected particles reach the particle imaging position after a certain period of time.
When the lamp 1 is turned on, a particle image is taken by the TV camera 8 and particle image processing required for pattern recognition is performed. Normally, correction of uneven brightness of an image, a cutout level for cutting out a particle image are determined, and further processing until cutting out of a particle image is performed.

Fourth stage: Extraction of characteristic parameters necessary for pattern recognition from the cut-out particle image, and automatic classification of particle images using the characteristic parameters.

Fifth step: Among the characteristic parameters of the particle image obtained in the fourth step, data of characteristic parameters relating to the particle size are sequentially stored in the memory area. When the above processing is completed for one frame image, the process returns to the second stage.

Sixth step: The total number of particles detected by the particle detection system at the end of the measurement is taken out (check of the number of detected particles).

Seventh step: The number of times the flash lamp 1 has been turned on or the number of all frame images obtained by capturing the particle image is extracted.

Eighth step: Based on the data obtained in the sixth and seventh steps, the total number of particle images that would have been processed in the entire measurement mode is calculated.

Ninth step: Correspondence between detected particles and particle images is performed. Every time a particle image is formed in the fifth stage, the list of parameter data relating to the particle size stored therein is rearranged in descending order of value (larger in size). From the rearranged parameter list, only the number of particle images calculated in the eighth step are extracted from the larger size.
The extracted particle image corresponds to a detected particle in a particle detection system.

Step 10: Particles taken out in step 9
Of the images, the particle image size is Cs _TH2Smaller particles
Let the image be the particles in region 1, and the number of particles in each of the following particle components
Is calculated. Next, the total number of particle images obtained in the eighth stage,
The obtained number of particle images of each particle component and the inspection obtained in the sixth step
Using the above formulas (1-1) and (1-3) from the total number of emitted particles
In the first measurement mode, the number of particles belonging to the area 1 is calculated.
It is calculated for each particle component.

Eleventh stage: The same process as in the tenth stage is performed for region 2, and in the first measurement mode, the number of particles belonging to region 2 is calculated using the above equations (1-2) and (1-4). It is calculated for each particle component. This completes the processing in the first measurement mode.

The same processing as the above-described processing in the first measurement mode is also performed in the second measurement mode. At this time, the particle detection level is set to Cs _TH2 , no particles originally exist in the region 3, and the number of particles of each particle in the region 4 is calculated by the above equations (2-1) and (2- Note that 2) is used.

After calculating the number of particles of the particle components in each region in each of the above-described measurement modes, the particle concentration calculation section 60 finally performs a particle concentration calculation process on the measurement sample. The actual calculation procedure is obtained by substituting the number of particles obtained for each region in each measurement mode described above into the above equations (3-1), (3-2), and (3-3).

In the above description, the case where only three types of particles A, B and C exist in the measurement sample has been described. If the target is a sample other than three types of particles, for example, if there are many types of particles such as only two types of A particles and C particles or more than four types, if particle classification by pattern recognition can be performed, For example, it is possible to apply the above-described embodiment of the present invention, and there is no problem.

Furthermore, although the number of particle measurement modes has been considered as two, the same concept can be used even if the number of measurement modes is increased. However, it is necessary to consider that increasing the number of measurement modes complicates the processing itself and increases the processing time.

Using the threshold Cs _TH2 of the particle image size information corresponding to the particle detection region of the second measurement mode as the particle image parameter relating to the preset size, the first measurement mode is divided into a plurality of regions. Is a feature of the embodiment of the present invention.

The feature is that the division of the first measurement mode and the second measurement mode in the particle image size information is performed based on the size information of the image parameters.

The reason is that the threshold value C of the particle image size information
Although the area of the first measurement mode can be divided into two at the particle detection level corresponding to s _TH2 , it may happen that the particle detection signal and the particle image size information do not always match as described above. This is because there is no problem if all processes are handled in a unified manner using image size information.

Since the particle detection conditions are affected by the optical properties of the particles to be measured, the laser irradiation conditions, the frequency characteristics of the detection system, circuit noise, etc., the light scattering detection electric signals are always the same. Because it cannot be.

On the other hand, the size parameter of the particle image is a comparatively stable comparison target parameter as long as the focus adjustment of the image operates correctly.

The threshold value Cs _TH2 of the particle image size information is determined as follows. One is a second measurement mode condition,
A large number of samples are measured in advance, and a histogram for the size information of the particle image as shown in FIG. 6 is obtained. The actually obtained histogram should be as indicated by the dotted line near the particle detection area.

The smaller size particles are not detected and have a distribution different from the original size histogram.
In FIG. 6, the threshold Cs _TH2 is a point where the portion indicated by the dotted line starts to decrease, and this point is set as the particle image detection level.
If the number of particle images to be measured is sufficiently large, the above operation can be performed for each measurement sample to determine the threshold value Cs _TH2 . It should be noted that if a threshold value is set below the above-described threshold value in the section drawn by the dotted line, the value of the particle concentration in the area 4 may not be accurate.

The particle concentration of the particle component B can be determined only in the first measurement mode or the second measurement mode. Using this fact, it is possible to check if there is a difference in particle concentration from statistical errors. The same applies to the region 2 and the region 4 for the particle component C.
It can also be checked by comparing the particle concentration of only the part.

By using this fact, the abnormal data can be corrected, and the particle concentration can be calculated using only the measurement mode data under normal conditions.

A sample thinning phenomenon caused by sample handling before the sample flows through the flow cell, or a phenomenon in which the number of detected particles and the number of images are not sufficiently corresponded due to a mismatch between the sample flow and the imaging area may occur. . These data correction methods are described in JP-A-11-947.
Since it is described in Japanese Patent Publication No. 27, data can be corrected based on the description.

Note that these data can be corrected at the stage of calculating the particle concentration.

In the above-described example, the measurement conditions in the two measurement modes, particularly the optical magnifications, have been implicitly assumed to be the same. However, if the optical magnifications are different in the two measurement modes, the magnifications may be different. It is necessary to correct the difference and handle the particle size information. For this reason, it is also possible to configure so as to include a step of correcting a difference in magnification.

The flow-type particle image analysis method and the flow-type particle image analysis apparatus according to one embodiment of the present invention can be used for biological samples and cells suspended in a liquid, blood cell components such as red blood cells and white blood cells in blood, or urine. It is effective for the classification and analysis of urinary sediment components present in the soil.

In particular, in counting and classifying particles of urine sediment components in urine, the number of particles present in each sample may differ by more than several orders of magnitude. The treatment is effective in obtaining information on the number of particles in the sample liquid.

In the urine sediment component, the types and sizes of the particles are extremely varied, and it is not possible to accurately correspond the detection particles of the particle detection system to the particles in the still particle image.
According to embodiments of the present invention, accurate particle counting, particle classification,
Particle concentration can be analyzed.

As described above, in the first measurement mode, the particle component A having a small particle size and a large number is mainly analyzed, and in the second measurement mode, the particle component B having a small number and a large size is analyzed. It is set as follows.

For this reason, it was difficult to accurately measure the particle concentration of the particle component C in which the particle size was widely distributed.

[0199] Therefore, according to one embodiment of the present invention, the first measurement mode is divided regions 1 detection level Cs _{TH 2} to 2, region 3 and the second measuring mode at detection level Cs _TH2
And 4. By using the particle information detected in these regions 1 to 4 and processing the particle size from small to large as one continuous data, it is possible to accurately measure the particle concentration of the particle component C. Become.

That is to say, even when the target is a sample containing particles which have to be detected in either of the two measurement modes because the particle components have a wide particle size, it is necessary to accurately calculate the particle concentration. And a flow type particle image analysis method and apparatus that can perform the method.

According to the present invention, the analysis results in a plurality of measurement modes can be considered as one continuous data on the particle size by using the particle image size information. As a result, for particle components widely distributed from small to large particle sizes, it is necessary to correctly estimate the particle concentration of each particle component per unit volume from the particle image classification results processed under a plurality of different measurement conditions. Can be done.

There is also an effect that particle measurement data from a plurality of measurement modes can be used effectively.

[0203] Also, due to such a background, there is an effect that it is not necessary to strictly adjust the detection level of the particle detection system and adjust the optical system for particle detection.

[0204] Although the case where the laser beam from the semiconductor laser is used as detection light and the laser beam scattered by the particles is used for the particle detection of the particle detection system has been described, the present invention is not limited to this. Transmitted light can also be used.

[0205]

According to the present invention, even when a particle component has a particle size spread and a sample containing particles that must be subjected to detection processing in either of the two measurement modes is targeted, the particle concentration can be reduced. And a flow-type particle image analysis method and apparatus that can accurately calculate the particle size.

Further, the following effects can be obtained.

1. The analysis results in a plurality of measurement modes can be considered as one continuous data on the particle size by using the particle image size information.

2. In flow type particle image processing having a particle detection system, the particle concentration of each particle component per unit volume can be correctly estimated from the particle image classification results processed under a plurality of different measurement conditions.

[0209] 3. There is also an effect that particle measurement data from a plurality of measurement modes can be effectively used.

[0210] 4. Since it is considered based on the particle image size, it is not necessary to strictly adjust the detection level of the particle detection system.

[0211] 5. 4 above. Similarly to the above, it is not necessary to strictly adjust the optical system.

6. Data can be compared between a plurality of measurement modes and used to check whether the measurement system is operating normally.

[Brief description of the drawings]

FIG. 1 is a schematic configuration diagram of a flow type particle image analyzer used in a flow type particle image analysis method according to an embodiment of the present invention.

FIG. 2 is an explanatory diagram of a problem in the related art.

FIG. 3 is an internal block diagram of a central processing / arithmetic processing unit in the example of FIG. 1;

FIG. 4 is a diagram illustrating the principle of the present invention.

FIG. 5 is an operation flowchart according to the embodiment of the present invention.

FIG. 6 is an explanatory diagram of how to determine a particle image detection level in a second measurement mode according to the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Flash lamp 1a Flash lamp drive circuit 2 Field lens 3 Microscope condenser lens 5 Microscope objective lens 6 Image formation position 7 Projection lens 8 TV camera 11 Field stop 12 Opening stop 15 Semiconductor laser 16 Collimator lens 17 Cylindrical lens 18 Reflecting mirror 19 Micro reflection Mirror 20 Beam splitter 21 Aperture 22 Light detection circuit 23 Flash lamp lighting control circuit 24 A / D converter 25 Image memory 26 Image processing control circuit 27 Feature extraction circuit 28 Identification circuit 29 Central control / arithmetic processing unit 40 Particle detection processing unit 51 Particle counting Unit 52 particle image number calculation unit 53 particle detection control unit 54 image correspondence unit 55 region 1 particle processing unit 56 region 2 particle processing unit 57 region 3 particle processing unit 58 region 4 particle processing unit 59 measurement condition setting unit 60 particle densitometer Part 61 central control unit 100 the flow cell 101 imaging unit 102 particle detection means 103 particle analysis means 110 samples the flow 111 sheath liquid

 ──────────────────────────────────────────────────続 き Continued on the front page F term (reference) 2F065 AA26 AA45 AA58 AA61 BB15 CC00 DD06 FF04 GG06 GG08 HH04 JJ03 JJ15 JJ26 LL04 LL12 LL30 LL46 QQ03 QQ08 QQ24 QQ42 QQ43 2G059 AA01 GG01 JJ01 GG04 JJ01 GG04 JJ01 GG04 JJ01 GG04 GG01 MM01 MM02 MM03 MM05 MM09 MM10 PP04

Claims (38)

[Claims] A liquid sample in which particles are suspended flows into a flow cell, and a particle detection system counts the number of particles in the liquid sample, and a still image of particles passing through an imaging region in the flow cell. A flow-type particle image analysis method having an imaging step of imaging particles by a particle imaging system and an analysis step of performing morphological classification of particles in the still image by image analysis processing, wherein a plurality of measurements having different particle detection conditions are performed. A mode setting step, a step of associating the particles detected in the particle detection step for each measurement mode with a particle image classified and identified by image analysis processing, and an analysis obtained for each measurement mode. Dividing the result into a plurality of size regions using characteristic parameters of the particle image, and calculating a particle concentration of a plurality of particle components in the sample for each measurement mode A flow type particle image analysis method, comprising: 2. The flow type particle image analysis method according to claim 1, wherein the plurality of measurement modes having different particle detection conditions include:
A flow type particle image analysis method, wherein the method is in two measurement modes. 3. The flow type particle image analysis method according to claim 2, wherein the two measurement modes having different particle detection conditions include:
By combining these two measurement modes, the first
A particle detection area is set such that all particles to be measured are detected as the particle detection conditions. 4. The flow type particle image analysis method according to claim 2, wherein the two measurement modes having different particle detection conditions include:
Flow type particle images, each having a particle detection area set in advance as a second particle detection condition, and detecting a particle detection signal at or above this set level as detected particles. analysis method. 5. A flow-type particle image analysis method according to claim 2, wherein the particle image is processed under the first particle detection condition, and the associated particle image is classified and classified into a plurality of types of particles. Estimating the number of particles in a plurality of regions using information on the number of particles detected under the first particle detection condition. 6. The flow type particle image analysis method according to claim 5, wherein the first measurement mode and particles having a particle diameter larger than the particle diameter of the object measured in the first measurement mode are measured. A second measurement mode, wherein the total number of particle images processed in the first measurement mode is Ng1, and the number of particle images of the first particle component in the first region of the plurality of regions is NgA.
(1) The number of particle images of the third particle component in the first area is represented by NgC
(1) The number of particle images of the second particle component in the second region is NgB
(2) The number of particle images of the third particle component in the second region is represented by NgC
(2) Assuming that the total number of particles detected by the particle detection system is Nd1,
The number of particles NA (1) of the first particle component in the first region, the number of particles NC (1) of the third particle component in the first region, the second region processed in the first measurement mode Particle number NB of the second particle component of
(2), the following formulas (1-1) to (1-4) NA (1) = NgA (1) / Ng1 × Nd1 ---- (1-1) NB (2) = NgB (2) / Ng1 × Nd1 ---- (1-2) NC (1) = NgC (1) / Ng1 × Nd1 --- -(1-3) NC (2) = NgC (2) / Ng1 × Nd1 ---- A flow type particle image analysis method characterized by using (1-4). 7. The flow type particle image analysis method according to claim 4, wherein the associated particle images processed under the second particle detection condition are classified and classified into a plurality of types of particle groups. Estimating the number of particles in each region using information on the number of particles detected under the particle detection conditions of (1). 8. The flow-type particle image analysis method according to claim 7, wherein the measurement mode includes a first measurement mode for detecting the first particle component and a measurement mode having a larger diameter than the first particle component. A second measurement mode for detecting a second particle component, wherein the first measurement mode is divided into a first region and a second region, and the second measurement mode is defined as a third region. The number of all particle images processed in the second measurement mode is divided into Ng2, the number of particle images of the second particle component in the fourth region is NgB (4), and the first particle component is divided into the fourth region. The particle image number of the third particle component having a diameter including the diameter of the second particle component from a part of the diameter of the particle is NgC (4), and the total number of particles detected by the particle detection system is NgC (4).
Assuming that d2, the estimated value of the number of particles NB (4) of the second particle component processed in the fourth region and the estimated number of particles NC (4) of the third particle component are given by the following equation (2-1): (2-2) NB (4) = NgB (4) / Ng2 × Nd2 ---- (2-1) NC (4) = NgC (4) / Ng2 × Nd2 ---- (2-2) A flow type particle image analysis method, wherein the method is used to calculate. 9. The flow type particle image analysis method according to claim 1, wherein the particle type is divided into a plurality of regions for each measurement mode using a particle image parameter relating to a predetermined size. A flow type particle image analysis method, comprising a step of calculating the number of particles for each particle. 10. The flow type particle image analysis method according to claim 9, further comprising the step of using diameter information of particles as particle image parameters relating to a predetermined size. . 11. A flow-type particle image analysis method according to claim 9, further comprising the step of using area information of the particles as particle image parameters relating to a predetermined size. . 12. The flow type particle image analysis method according to claim 9, further comprising the step of using a projection length of the particle as a particle image parameter relating to a predetermined size. . 13. The flow type particle image analysis method according to claim 10, wherein the particle diameter or the projection length is a length along a flow direction of the sample. . 14. The flow-type particle image analysis method according to claim 5, wherein the measurement condition information, the number of each particle component obtained for each of a plurality of regions, and a particle detection system count result are used. And calculating a particle concentration of a plurality of types of particles contained in the sample. 15. A flow type particle image analysis method according to claim 14, wherein the first measurement mode and particles having a particle diameter larger than the particle diameter of the object measured by the first measurement mode are measured. A second measurement mode, wherein the first measurement mode is divided into a first area and a second area,
Is divided into a third region and a fourth region, the number of particles of the first particle component in the first region is NA (1), and the number of particles of the third particle component in the first region is Is processed by NC (1), the number of particles of the second particle component in the second region is NB (2), the number of particles of the third particle component in the second region is NC (2), and the fourth region is processed. The second
The number of particles of the particle component of NB (4), the number of particles of the third particle component processed in the fourth region is NC (4), and the respective samples in the first measurement mode and the second measurement mode Assuming that the measured volumes are Vm1 and Vm2, the particle concentration D of the first particle component
A, the particle concentration DB of the second particle component, and the particle concentration DC of the third particle component are represented by the following equations (3-1) to (3-3). DA = NA (1) / Vm1 ---- (3 -1) DB = (NB (2) + NB (4)) / (Vm1 + Vm2) ---- (3-2) DC = NC (1) / Vm1 + (NC (2) + NC (4)) / (Vm1 + Vm2) ---- (3-3)
A flow type particle image analysis method, wherein the method is used to calculate. 16. The flow type particle image analysis method according to claim 2, wherein image particle size information corresponding to a particle detection area in the second measurement mode is used as a particle image parameter relating to a predetermined size. And dividing the first measurement data into a plurality of regions. 17. A flow-type particle image analysis method according to claim 16, further comprising the step of determining, as a particle image parameter relating to a predetermined size, from a histogram of a corresponding image parameter in the second measurement mode. Characteristic flow-type particle image analysis method. 18. The flow type particle image analysis method according to claim 17, wherein the flow type particle image analysis method is determined from the particle image parameters of the sample being processed. 19. The flow type particle image analysis method according to claim 17, wherein the flow type particle image analysis method is determined from particle image parameters of a plurality of samples. 20. The flow-type particle image analysis method according to claim 17, wherein the state of the measurement system is determined by calculating the respective particle concentrations of a plurality of regions in each measurement mode and comparing the particle concentrations between the measurement modes. A flow type particle image analysis method characterized by checking. 21. The flow-type particle image analysis method according to claim 20, wherein, when an abnormality is found by checking the measurement state, the particle concentration in the sample is calculated only by the data of the measurement mode considered to be normal. Flow type particle image analysis method. 22. The flow-type particle image analysis method according to claim 20, wherein when an abnormality is found by checking the measurement state, a data ratio of the two measurement data is calculated,
A flow type particle image analysis method, comprising the step of estimating a particle concentration value in an abnormal measurement mode. 23. The flow-type particle image analysis method according to claim 1, wherein in each measurement mode, correction of a visual field vignetting due to a mismatch between a sample flow position and imaging of a particle image and correction of a sample thinning are performed. Flow type particle image analysis method. 24. The flow type particle image analysis method according to claim 1, wherein when the optical system magnification of each measurement mode is different, the size information is converted back to the magnification. analysis method. 25. Particle detection means for flowing a liquid sample in which particles are suspended into a flow cell and counting the number of particles in the liquid sample by a particle detection system, and a still image of particles passing through an imaging region in the flow cell. In a flow type particle image analyzer having an imaging unit for imaging the particles by a particle imaging system and an analysis unit for performing morphological classification of particles in the image by image analysis processing, a plurality of measurement modes having different particle detection conditions are provided. Means for setting, the means for associating the particles detected by the particle detecting means for each measurement mode with the particle images classified and identified by image analysis processing, and the analysis results obtained for each measurement mode Means for dividing the particle into a plurality of size regions using the characteristic parameters of the particle image, and means for calculating the particle concentration of the plurality of particle components in the sample for each measurement mode. A flow type particle image analysis device, comprising: 26. The flow-type particle image analyzer according to claim 25, wherein the plurality of measurement modes having different particle detection conditions are two measurement modes. 27. The flow-type particle image analyzer according to claim 26, wherein the two measurement modes having different particle detection conditions are combined by combining these two measurement modes.
A flow type particle image analyzer, wherein a particle detection area is set as a first particle detection condition so that all particles to be measured are detected. 28. A flow type particle image analyzing apparatus according to claim 26, wherein the means for classifying and identifying the associated particle image processed under the first particle detection condition into a plurality of types of particles, Means for estimating the number of each particle using the number of particles detected under one of the particle detection conditions. 29. The flow type particle image analyzer according to claim 25, wherein the processing is performed under the second particle detection condition,
Means for classifying and identifying the associated particle images into a plurality of types of particle groups, and means for estimating the number of each particle using the number of particles detected under the second particle detection condition. Characteristic flow type particle image analyzer. 30. A flow-type particle image analyzer according to claim 25, wherein the sample is determined based on the measurement conditions, the number of particles detected under the second particle detection conditions, and the counting result of the particle detection system. A flow type particle image analysis apparatus, comprising: means for calculating the particle concentration of a plurality of particle types included in the above. 31. The flow type particle image analyzer according to claim 25, wherein image particle size information corresponding to a particle detection area in the second measurement mode is used as a particle image parameter relating to a predetermined size. Wherein the first measurement mode is divided into a plurality of regions. 32. The flow-type particle image analyzer according to claim 31, further comprising means for determining, as a particle image parameter relating to a predetermined size, from a histogram of a corresponding image parameter in the second measurement mode. Characteristic flow type particle image analyzer. 33. A flow type particle image analysis apparatus according to claim 31, wherein the flow type particle image analysis apparatus is determined from the particle image parameters of the sample itself being processed. 34. A flow type particle image analysis apparatus according to claim 31, wherein said flow type particle image analysis apparatus is determined from a particle image parameter of a plurality of samples. 35. The flow type particle image analyzer according to claim 31, wherein the particle concentration in each of a plurality of regions in each measurement mode is calculated, and the state of the measurement system is checked by means for comparing the particle concentration between the measurement modes. A flow type particle image analysis apparatus, comprising: 36. The flow type particle image analyzer according to claim 25, wherein the analysis target is a living cell. 37. The flow-type particle image analyzer according to claim 25, wherein the analysis target is a blood cell component present in blood. Analysis device. 38. The flow type particle image analyzer according to claim 25, wherein the analysis target is a urinary sediment component present in urine. Image analysis device.