JP3429974B2 - Flow type particle image analysis method and apparatus - Google Patents

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a particle image suitable for analyzing particles by taking an image of particles suspended in a flowing liquid such as cells or particles in blood or urine. The present invention relates to an analysis method and device.

[0002]

2. Description of the Related Art Classification and analysis of cells in blood and cells and particles in urine have hitherto been carried out by preparing a sample on a slide glass and observing with a microscope. In the case of urine, since the particle concentration in urine is low, the sample is observed after being centrifugally concentrated by a centrifugal separator.

In an apparatus for automating these observation and inspection operations, a sample such as blood is smeared on a slide glass, set on a microscope, and the microscope stage is automatically scanned to detect the position where particles are present. Stop the microscope stage and take a still image of the particles. Then, with respect to the photographed still image, the feature extraction by the image processing technique and the pattern recognition method are used to classify the particles in the sample.

However, in the above method, it takes time to prepare a sample, and it is necessary to find particles while mechanically moving the microscope stage and move the particles to an appropriate image capturing area. Therefore, there are problems that analysis takes time and the mechanical mechanism section becomes complicated.

Therefore, there is known a flow cytometer method in which a smear sample is not prepared, and a sample sample is allowed to flow in a flow cell while being suspended in a liquid for optical analysis. This method using a flow cytometer observes the fluorescence intensity and scattered light intensity from each particle in a sample, and has a processing capacity of several thousand per second. However, it is difficult to observe the feature quantity that reflects the morphological characteristics of the particles, and it is impossible to classify the particles by the morphological characteristics that are conventionally performed under a microscope.

Particle images in a continuously flowing sample sample are photographed, and the particles are analyzed from the individual particle images.
As an attempt to classify the materials, JP-A-57-500995, JP-A-63-94156, and JP-A-5-296
Japanese Patent Laid-Open No. 915 and Japanese Patent Laid-Open No. 4-72544 are known.

[0007] In Japanese Patent Publication No. 57-500995, a sample is passed through a channel having a special shape, particles in the sample are flowed into a wide imaging area, a still image is taken by a flash lamp, and the image is taken. A method for particle analysis using is shown. When a magnified image of sample particles is projected on a CCD camera using a microscope, a flash lamp, which is a pulse light source, periodically emits light in synchronization with the operation of the CCD camera. The emission time of the pulsed light source is short, a still image can be obtained even when particles are continuously flowing, and C
The CD camera can take 30 still images per second.

In Japanese Patent Laid-Open No. 63-94156, a particle detection optical system is provided upstream of a particle image capturing area in the sample flow, in addition to the still image capturing system. The method described in Japanese Patent Laid-Open No. 63-94156 is a method in which the particle detection unit knows the passage of particles in advance, and when the particles have just reached the particle image capturing area, the flash lamp is turned on at an appropriate timing. In this method, it is possible to capture a still image at a timing only when the passage of particles is detected without periodically emitting light from a pulsed light source, the particle images are efficiently collected, and a sample sample with a low concentration is sampled. It does not even process images without meaningless particles.

Further, Japanese Patent Laid-Open No. 5-296915 discloses a method in which a particle detection optical system is further incorporated into a particle image pickup system. That is, a method of irradiating a sample sample flow with a laser beam for particle detection through a microscope condenser lens of a microscope image pickup system is described. It is characterized in that it is not necessary to separately prepare an optical system for particle detection, and the particle detection position can be arranged as close to the particle image capturing area as possible.

Further, Japanese Patent Laid-Open No. 5-296915 proposes a particle number correction method for obtaining the number of particles and the particle concentration contained in the entire measurement sample from the number of particles subjected to image processing.

Further, in Japanese Patent Laid-Open No. 7-83818, a timing control method for controlling the lamp lighting timing and image capturing when the particle detection system, flash lamp and CCD camera are used in an interlaced system. Is described.

[0012]

Generally, in order to efficiently count and classify the number of plural kinds of particles in a sample by analyzing a still image of a particle sample that is flowing continuously, the above-mentioned method is used. As is done in the known example, a particle detection system is required to detect particles passing through in the particle still image imaging area or upstream thereof.

That is, the pulse light source is turned on only when the particles have passed, and a still image of the particles is taken. Since this method can perform analysis processing on a measurement sample having a low particle concentration very efficiently, it is possible to increase the amount of measurement sample, shorten the measurement time, and improve the measurement accuracy.

Further, in the case of processing a particle sample having a high density, particles are missed due to dead time associated with image input.
Japanese Patent No. 6915 proposes a particle number correction method for obtaining the number of particles and the particle concentration contained in the entire measurement sample from the number of particles subjected to image processing.

Further, in JP-A-5-296915 and JP-A-4-72544, a counter for counting all the particles that have passed through the particle detection system is used. In this case, the number of particles of each kind in the sample can be known from the value of the particle counter and the existence ratio of the number of particles subjected to image processing.

For particle detection, a method is generally used in which a laser beam is focused on a measurement sample flowing in a flow cell and irradiated, and light scattered light from particles traversing the laser beam is detected. Light scattered light generally produces an optical signal whose intensity is proportional to the effective scattering cross section of the particle. This optical signal is converted into an electric signal by a photodetector. The magnitude of the optical signal is affected by the optical refractive index of particles, absorption, size, internal state of particles, scattered light detection conditions, and the like. As another method of detecting particles, a method of forming a particle image on a one-dimensional image sensor and detecting the particles by signal processing is known.

However, as described above, in order to measure or classify the number of plural kinds of particles contained in the sample by analyzing the still images of the particles flowing continuously, However, there is a problem of dead time in measurement as described below.

The dead time mentioned above occurs when a microscopic particle image is converted into an electric signal using a TV camera. When the sample particles pass through the particle detection system, the particle arrival times are random. On the other hand, in a general interlaced CCD / TV camera, two fields per frame, that is, an odd field and an even field, are extracted as image signals independently of the arrival of particles. The electric charge accumulated in the photosensitive portion of the CCD element is once transferred to the accumulating portion as a read pulse signal,
After that, video signals are sequentially output. One image reading pulse signal for one field is output for each vertical synchronization signal.

Until the two image reading pulse signals are output, the next image cannot be captured, that is, the flash lamp cannot emit light to obtain a particle image associated with particle detection. This is because if the next particle image is picked up even though the image transfer has not been completed, double exposure will result. This is where the dead time of particle measurement occurs.

Japanese Unexamined Patent Publication (Kokai) No. 7-83818 describes an interlaced CCD camera that enables lighting of a flash lamp and a method of controlling the lighting timing of the flash lamp in an arbitrary field. How to reduce it is not described.

In the method described in Japanese Patent Laid-Open No. 7-83818, considering that the arrival of particles is random, a minimum one field time is required when the particle concentration is small,
There is a maximum dead time of 2 field hours and 1.5 field hours on average. When the particle concentration is high, the flash lamp is turned on for each frame, and the average dead time approaches from 2 fields to 1.5 fields. In this case, the dead time is 1 field or more,
The repetition cycle as the lighting ability of the flash lamp is 1
It is enough to be within the field time.

By the way, it is conceivable to apply a non-interlaced CCD camera to the flow type particle image analyzer in order to increase the number of measured images. Since this non-interlaced CCD camera captures an image in one frame = 1 field, it can capture twice as many images as in the interlaced system. That is, typically, 60 images can be captured per second.

The interlaced flow-type particle image processing is partially described in Japanese Patent Laid-Open No. 5-296915, but the flash lamp lighting timing, double exposure measures, and the repetition cycle as the flash lamp lighting capability. In consideration of the above problem, the timing of particle image input of the CCD camera, and the like, the above-mentioned problem of how to minimize the dead time is not considered.

Even in the non-interlaced system, CC
The electric charge accumulated in the photosensitive portion of the D element is once transferred to the accumulating portion by a read pulse signal, and then the video signal is sequentially output. Therefore, one image reading pulse signal for one frame = 1 field is output for each vertical synchronizing signal.

When the flash lamp is turned on at an arbitrary time, the next image is photographed until the next image reading pulse signal is output, that is, the flash lamp is caused to emit light in order to obtain a particle image associated with particle detection. There is a problem with that. This is because if the next particle image is picked up even though the image transfer has not been completed, double exposure will result. Therefore, even in the non-interlaced method, dead time for particle measurement occurs. The maximum dead time is 1 field, the minimum is 0, and the average dead time when the particle concentration is small is 0.5 field.

In practice, however, the flash lamp has a limited cycle, and the lamp cannot be turned on until a fixed time elapses after the lamp is turned on. This is also a cause of dead time. In the interlace system, as described above, there is a dead time of one field time or more, and the dead time corresponding to the ramp repetition period is sufficiently absorbed, but in the non-interlace system, it may not be absorbed.

An object of the present invention is to shorten the dead time in the flow type particle image analysis using the above-mentioned non-interlaced type TV camera as compared with the interlaced type, to increase the number of measured particle images and to improve the measurement accuracy. It is an object of the present invention to realize a flow-type particle image analysis method and apparatus that can be improved and can shorten the measurement time and improve / improve the processing speed.

[0028]

[Means for Solving the Problems]

(1) In order to achieve the above object, the present invention is configured as follows. That is, a particle sample suspended in a liquid is caused to flow in a flow cell, particles that pass through a particle detection region in the flow cell are detected, and a still image of the detected particles that have passed through the imaging region in the flow cell is used by an imaging means. In a flow-type particle image analysis method for performing morphological classification of particles by imaging and performing an image analysis on the imaged particle image, the imaging means is a non-interlaced imaging means for detecting particles. After turning on the pulse lamp for irradiating the image pickup area with light, the pulse lamp is prohibited from turning on for particle detection until the next image reading pulse signal of the image pickup means is output. When the time until the image reading signal of the image pickup means is output after lighting is shorter than a predetermined minimum lighting period of the pulse lamp, The pulse lamp Lamp prohibition time after lighting the minimum lighting cycle determined by the lamp operating capability, the first to prohibit the lighting of the pulsed lamps
As the lighting prohibition time of, the still image of the detected particles is captured using the image capturing means.

(2) Preferably, in the above (1),
When the second lighting prohibition time determined by the characteristics of the image pickup means is set and the pulse lamp is turned on during the second lighting prohibition time, the timing of light emission of the pulse lamp is set to the second lighting prohibition time. Immediately after the lighting prohibition time of has elapsed.

(3) Further, preferably, in the above (1), a second lighting prohibition time which is determined from the characteristics of the image pickup means is set, and the pulse lamp is lit during this second lighting prohibition time. When the conditions are met, the pulse lamp is prohibited from lighting.

(4) Further, a particle sample suspended in a liquid is caused to flow in a flow cell, particles passing through a particle detection area in this flow cell are detected, and the detection particles passing through the imaging area in the flow cell are stopped. An image is picked up using an image pickup means, and in a flow type particle image analysis method for performing morphological classification of particles by analyzing the imaged particle image, the image pickup means is a non-interlaced image pickup means, A first pulse lamp lighting charging circuit having a first discharge capacitor and a second pulse lamp lighting charging circuit independent of each other and having a second discharge capacitor The pulse lamp for irradiating the imaging region with light to detect particles is turned on, and the charging circuit for turning on the first pulse lamp is normally operated. It is used for lighting the pulse lamp in a certain situation.After the pulse lamp is lit, the pulse lamp lighting is prohibited until the next image reading pulse signal of the image pickup means is output, and after the pulse lamp is lit, the image reading of the image pickup means is performed. However, when the time until the pulse signal is output is shorter than the minimum lighting period determined by the ability of the pulse lamp, the energy charged in the second discharge capacitor is used to light the pulse lamp.

(5) Preferably, in the above (4),
For each field of the image captured by the image capturing means, the first
The charging circuit and the second charging circuit are alternately switched and used as the charging circuit for lighting the pulse lamp.

(6) Further, a particle sample suspended in a liquid is caused to flow in a flow cell, particles passing through a particle detection area in this flow cell are detected, and detection particles passing through an imaging area in the flow cell are stopped. An image is picked up using an image pickup means, and in a flow type particle image analysis method for performing morphological classification of particles by analyzing the imaged particle image, the image pickup means is a non-interlaced image pickup means, Light for capturing a particle image is emitted to the imaging region by either the first pulse lamp or the second pulse lamp, which are independent of each other, and the first pulse lamp is used as a normal pulse lamp. However, after the first pulse lamp is turned on, the next pulse lamp is turned off until the image reading signal of the image pickup means is output, and the first pulse lamp is turned on. After slump lighting, time until the image reading signal of the imaging means is outputted is shorter than the minimum lighting period determined by the pulse lamp abilities, turns on the second pulse lamp.

(7) Preferably, in the above (6),
For each field of the image captured by the image capturing means, the first
The pulse lamp and the second pulse lamp are alternately switched and used as a light source for capturing a particle image.

(8) Further, a particle sample suspended in a liquid is caused to flow in a flow cell, particles passing through a particle detection area in this flow cell are detected, and detection particles passing through an imaging area in the flow cell are stopped. In order to detect a particle in a flow-type particle image analysis device that images an image using an image capturing unit and performs morphological classification of particles by analyzing the captured particle image. After the pulse lamp for irradiating the imaging area with light and the pulse lamp are lit, the lighting of the pulse lamp for particle detection is prohibited until the next image reading pulse signal of the imaging means is output. After the pulse lamp is turned on, the time until the image reading signal of the image pickup means is output is a predetermined minimum lighting period of the pulse lamp. If it is short, the lamp lighting prohibition time after lighting the pulse lamp is set as the minimum lighting cycle determined by the lamp lighting ability, and the pulse lamp lighting control drive means is set as the first lighting prohibition time for prohibiting the lighting of the pulse lamp. Equipped with.

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042]

[0043]

[0044]

In the present invention, a non-interlaced image pickup means is used. In order to avoid the problem of double exposure in this case, after the pulse lamp is turned on, the pulse lamp is turned off until a read signal from the imaging means is output. Further, under the condition that the time from the lighting of the pulse lamp to the reading signal is shorter than the lighting repeating cycle due to the restrictions of the pulse lamp driving system and the like, the lighting of the pulse lamp is prohibited even after the reading signal of the imaging means is output. .

As a result, the particle image can be correctly captured without capturing an incorrect image due to double exposure of the particle image or insufficient light amount. By providing this first flash lamp emission prohibition time, correct particle image processing can be executed.

Further, as a characteristic of the image pickup means itself, by providing the second lamp turn-off prohibition time, the image pickup means malfunctions even if the timing of lighting the pulse lamp coincides with the second turn-on prohibition time. There is no such thing. When it is desired to light the pulse lamp by the particle detection during the second lighting prohibition time, it can be dealt with by lighting the pulse lamp immediately after the second lighting prohibition time.

As a result, the processing of particles under such conditions is not stopped, and correct image processing can be performed. As another countermeasure against this problem, it is possible to stop the lighting of the pulse lamp while the second lighting prohibition time is sufficiently small. Whichever method is adopted, it is possible to prevent the above-mentioned malfunction of the image pickup means.

The minimum time of the lighting cycle of the pulse lamp is 1
The minimum condition is to be less than or equal to the field, but the minimum value of the dead time is close to 1 field in the vicinity of 1 field. If the pulse lamp is too restrictive, by providing two discharge capacitor charging circuits and switching the discharge circuit for each field, the restriction due to the charging time of the discharge capacitor can be eliminated, and the minimum dead time can be reduced. It can be reduced to a value determined by the characteristics of the pulse lamp itself and the discharge circuit time constant.

Furthermore, if two pulse lamp lighting circuits including a pulse lamp are installed and both are switched for each field, the influence of the lamp characteristics and the discharge time constant can be ignored.

[0051]

BEST MODE FOR CARRYING OUT THE INVENTION Embodiments of the present invention will be described below with reference to the accompanying drawings. FIG. 1 is an overall schematic configuration diagram of a flow type particle image analysis apparatus for carrying out a flow type particle image analysis method according to a first embodiment of the present invention.

First, the structure of a flow type particle image analysis apparatus will be described with reference to FIG. As shown in FIG. 1, the flow-type particle image analyzer according to the first embodiment of the present invention is a flow cell 10 to which a sample liquid in which particles are suspended is supplied.
0, an image pickup means 101, a particle analysis means 102, and a particle detection means 103.

The image pickup means 101 has a function as a microscope, and includes a flash lamp (pulse lamp) 1 which is a pulse light source, a flash lamp driving circuit 1a for causing the flash lamp 1 to emit light, and a pulse light flux from the flash lamp 1 to be parallel. A field lens 2 for controlling the flow of the parallel pulsed light flux 10 from the field lens 2
A microscope condenser lens 3 for focusing on the sample liquid flow 110 in 00.

Further, the image pickup means 101 projects the pulsed light flux irradiated on the sample liquid flow 110 in the flow cell 100 via the microscope objective lens 5 and the projection lens 7 for focusing the light flux on the image forming position 6. It has a TV camera 8 that takes in the image at the imaging position 6 and converts it into an image data signal that is an electric signal, a field stop 11 that limits the width of the pulse beam 10, and an aperture stop 12.

As the TV camera 8, a CCD camera or the like with a small afterimage is generally used. In the present invention, a CCD type TV camera, particularly a non-interlaced type TV camera is used.

The particle analyzing means 102 is an AD converter 24 for converting the image data signal transferred from the TV camera 8 into a digital signal, and an image memory for storing data based on the signal from the AD converter 24 at a predetermined address. 25,
The image processing control circuit 26 for controlling writing and reading of data in the image memory 25, the image memory 2
A feature extraction circuit 27 and a discrimination circuit 28 that perform image processing based on the signal from 5 to count the number of particles and classify the particles.
Have.

Further, the particle analysis means 102 includes a particle number analysis unit 40 for determining the number of particles in the sample liquid and the TV camera 8.
Imaging conditions, sample liquid flow conditions of the flow cell 100, control of the image processing control circuit 26, storage of image processing results from the identification circuit 28, exchange of data with the particle number analysis unit 40, and display on the display unit 50. It has a central control unit 29 for performing.

The particle detecting means 103 is a semiconductor laser 15 which is a detection light source for emitting a laser beam as a detection beam, and the laser beam from the semiconductor laser 15 is a parallel laser beam 1.
The collimator lens 16 has a size of 4, a cylindrical lens 17 that focuses only one direction of the laser light beam from the collimator lens 16, and a reflecting mirror 18 that reflects the light beam from the cylindrical lens 17.

The particle detecting means 103 is provided between the microscope condenser lens 3 and the flow cell 100,
A minute reflecting mirror 19 that guides the laser light flux from the reflecting mirror 18 to a position on the sample liquid flow 110 on the upstream side of the image capturing area and close to the image capturing area, and collects the scattered light of the laser light flux by the particles. A microscope objective lens 5, a beam splitter 20 that reflects scattered light collected by the microscope objective lens 5, and a beam splitter 2
A light detection circuit 22 that receives scattered light from 0 through a diaphragm 21 and outputs an electric signal based on its intensity, and flash lamp lighting that operates a flash lamp drive circuit 1a based on the electric signal from this light detection circuit 22. Control circuit 2
Have three. The microscope objective lens 5 is also used as the image pickup means 101.

Further, the sheath liquid is supplied to the flow cell 100 together with the sample liquid, and a flow in which the sample liquid is wrapped in the sheath liquid is formed in the flow cell 100. Then, the sample liquid flow 110 is the image capturing means 1
A stable steady flow (sheath flow) having a flat cross-sectional shape in a direction perpendicular to the optical axis (01) of the optical axis 01 (microscope optical axis) of 01 is sent to the center of the flow cell 100 toward the lower side of the paper surface. The flow velocity of the sample liquid flow 110 is controlled by an appropriate flow velocity control means (not shown) according to the conditions set by the central controller 29.

In addition, the flash lamp drive circuit 1a,
The flash lamp lighting control circuit 23 constitutes flash (pulse lamp) lighting control drive means.

Next, the basic operation of the flow type particle image analyzer will be described with reference to FIG. Semiconductor laser 15
Always oscillates continuously. This is because it is always possible to observe particles in the sample passing through the detection area. The laser light flux from the semiconductor laser 15 is converted into a parallel laser light flux 14 by the collimator lens 16, and is focused by the cylindrical lens 17 in only one direction. This laser beam is reflected by the reflecting mirror 18 and the minute reflecting mirror 19.
Is reflected by the sample liquid flow in the flow cell 100.
0 is illuminated. This irradiation position is a particle detection position where the laser light beam is focused by the cylindrical lens 17, is an upstream side of the image capturing region on the sample liquid flow 110, and is a position close to the image capturing region.

When the particle to be measured crosses the laser beam for particle detection, the laser beam is scattered. Then, the scattered light is reflected by the beam splitter 20, received by the photodetector circuit 22, and converted into an electric signal based on the intensity thereof by the photodetector circuit 22.

Further, in the photodetector circuit 22, it is judged whether or not the converted electric signal is above a predetermined signal level, and if it is above a predetermined signal level, it is considered that the image processing target particles have passed. Then, the electric signal is sent to the flash lamp lighting control circuit 23 and the particle number analysis unit 40.

The flash lamp lighting control circuit 23 controls the particle detection position and the image capturing area so that the flash lamp 1 emits light to capture an image when the particles have just reached a predetermined position in the image capturing area of the TV camera 8. After a predetermined delay time determined by the distance between and and the flow rate of the sample solution,
The detection signal is sent to the flash lamp drive circuit 1a.

However, the delay time is very short because the distance between the particle detection position and the image capturing area is very short, and the accuracy of particle detection and analysis is influenced by the flow velocity of the sample solution and the particle concentration. I will not receive it.

Further, the flash lamp lighting control circuit 23
Then, a light emission ready signal is sent to the flash lamp drive circuit 1a at the same time as the above detection signal, and the light emission timing of the flash lamp 1 is controlled based on the timing of the field signal. Further, the detection signal from the flash lamp lighting control circuit 23 is also sent to the image processing control circuit 26.

When the detection signal is sent from the flash lamp lighting control circuit 23 to the flash lamp driving circuit 1a, the flash lamp driving circuit 1a causes the flash lamp 1 to emit light. The pulsed light emitted from the flash lamp 1 travels on the microscope optical axis 9, becomes parallel light at the field lens 2, is focused by the microscope condenser lens 3, and is irradiated onto the sample liquid flow 110 in the flow cell 100. The field stop 11 and the aperture stop 12
This limits the width of the pulsed light flux 10.

Sample liquid flow 11 in flow cell 100
The pulsed light flux irradiated to 0 is condensed by the microscope objective lens 5 and forms an image at the image forming position 6. The image at the image forming position 6 is projected on the image pickup surface of the TV camera 8 by the projection lens 7 and converted into an image data signal. This means that a still image of the particles has been captured. The image capturing conditions of the TV camera 8 are preset in the central control unit 29, and the shooting operation of the TV camera 8 is controlled by this.

The particle analysis means 102 operates as follows. The image data signal output from the TV camera 8 is A
The digital signal is converted by the D converter 24, and the data based on the digital signal is stored in a predetermined address of the image memory 25 under the control of the image processing control circuit 26. Image memory 2
The data stored in 5 is read out under the control of the image processing control circuit 26, and the feature extraction circuit 27 and the identification circuit 2 are read.
8, the image processing is performed, and the result is stored in the central control unit 29.

What is stored in the central control unit 29 is a particle classification result and particle identification feature parameter data used for particle classification. The classification and identification processing of particles is automatically performed by the pattern recognition processing which is usually performed. The result of the image processing, the measurement condition, and the information on the number of images subjected to the image processing are sent from the central controller 29 to the particle number analyzer 40. In the particle number analysis unit 40, the information from the central control unit 29, the particle detection signal from the photodetection circuit 22, and the image processing control circuit 26.
Based on the control signal from, the correspondence between the detected particles and the particle classification results is examined, and the final classification and classification results of the particle images are summarized, that is, the morphological classification of particles is performed.

When measuring a sample in a plurality of measurement modes, the above-mentioned operation steps are executed in each measurement mode,
At the final stage, combine the measurement data of each mode to obtain the final result.

The flow-type particle image analysis method and the flow-type particle image analysis apparatus of the present invention include biological samples and cells suspended in a liquid, blood cell components such as red blood cells and white blood cells in blood,
Alternatively, it is effective for classification and analysis of urinary sediment components existing in urine.

In the present invention, a CCD type TV camera, particularly a non-interlaced type TV camera is used. In the non-interlaced TV camera, one image information is output as a video signal every one field time. The charges accumulated on the imaging surface are transferred to the accumulation unit by a field read signal (image reading pulse signal), and then output to the outside as a video signal. Therefore, the flash lamp 1 cannot be lit by particle detection until the field read signal is output.

If the flash lamp 1 is turned on, double exposure is performed, and normal particle image data cannot be obtained. In the interlace method, the lamp light emission must be prohibited until two read signals are output, whereas in the non-interlace method, the light emission must be prohibited until the next read signal is output.

However, since it takes time to store the charge in the discharge capacitor for the flash lamp, the time sufficient to store the charge cannot be secured immediately before the read signal is output. Therefore, it is necessary to extend the lighting prohibition time of the flash lamp 1 by a predetermined time after the field read signal is output, in consideration of the time for charge accumulation.

FIG. 2 is a diagram showing the output timing of the lighting prohibition signal of the flash lamp 1. With reference to FIG. 2, the flash lamp lighting prohibited section when the flow type particle image processing is performed by the non-interlaced CCD TV camera will be described.

As shown in FIG. 2A, the field read signal is output at substantially the same timing as the vertical synchronizing signal. When a particle passes through the particle detection area and the particle is detected as an electric signal by the light detection circuit 22, the particle detected at the specified position in the imaging area is delayed by a certain time. A detection delay signal is created ((B) in FIG. 2).

This delay signal operates as a flash lamp lighting signal. Whether or not the flash lamp 1 is actually lit depends on whether the flash lamp lighting prohibition signal ((D) in FIG. 2) is ON or OFF. Depends on When the flash lamp lighting prohibition signal is ON, lighting of the flash lamp is prohibited, and when it is OFF, lighting of the lamp is possible.

Next, how to set the flash lamp lighting prohibited section for the measurement particles that randomly pass through the particle detection system will be described. When the flash lamp is turned on by the particle detection delay signal, the charge on the imaging surface is once transferred to the storage unit, and the flash lamp turn-on prohibited section is a period until the next field read signal is output (((D) in FIG. 2). a) Section).

However, as described above, since it takes time for the electric charge to be stored in the discharge capacitor for the flash lamp, the time from the lighting of the flash lamp until the field read signal is output is the charging time of the discharge capacitor (see FIG. 2). When it is shorter than Tc of (C), the charging time is set as the lighting prohibited section (section (b) of (D) of FIG. 2). By performing these two operations, it is possible to maintain a state in which the light emission intensity does not change due to double exposure and insufficient charging time.

The above flash lamp lighting prohibited section is referred to as a first lighting prohibited section. In order for the flash lamp lighting prohibition shown in FIG. 2 to work effectively, it is necessary that at least the charging time Tc of the discharge capacitor is shorter than the repetition period (one field time) of the field read signal. This is because if the dead time is longer than one field time, there is no difference from the interlaced method.

FIG. 3 is a diagram showing a second flash lamp lighting prohibited section and processing after the lighting is prohibited. Figure 3
In, the field read pulse appears immediately after the vertical synchronizing signal is output (after several tens of microseconds) ((A) and (B) in FIG. 3). However, there is a second lamp lighting prohibited section before and after this field reading pulse in the operation of the TV camera. That is, the high-speed shutter operation of the TV camera is prohibited. Even if there is a timing of lighting the flash lamp due to particle detection in this section ((D) of FIG. 3), it is prohibited to light the flash lamp 1 by the lamp lighting prohibition signal ((C) of FIG. 3). It

However, since the particles to be measured pass through at random times, the probability of arriving at the second flash lamp lighting prohibited section has a finite value. Therefore, it is necessary to perform a corresponding process for the case where the measurement particles arrive at the imaging region in the section where the flash lamp is prohibited from being lit in this section.

As shown in FIG. 3, there is shown a case in which it is detected that particles have passed during the second flash lamp lighting prohibition time, and the flash lamp is lit after the prohibition section is delayed (FIG. 3). (E)).

Since the lamp lighting prohibited section is usually short, which is several horizontal times, particles existing in this section may be negligible as a probability. Therefore, even if the particles pass through the second flash lamp lighting prohibited section, it is possible to process the particles without delaying the lighting of the flash lamp. Which of these methods is adopted can be determined by the conditions of the measurement sample.

As described above, according to the first embodiment of the present invention, after the flash lamp 1 is turned on, the flash lamp 1 is prohibited from being turned on until the read signal of the CCD camera is output. Therefore, double exposure is prevented. In addition, after the flash lamp 1 is turned on, after the charge accumulation time of the discharge capacitor has elapsed, the flash lamp is prohibited from being turned on until the next field read signal is output. When the time is shorter than the charging time of the discharge capacitor, the charging time is configured as the lighting prohibited section, so that the shortage of light quantity due to the shortage of the charging time is avoided,
The capturing of an inappropriate image is suppressed.

Therefore, the non-interlaced T
The dead time in flow type particle image analysis using a V camera is shortened compared to the interlaced method, the number of measured particle images is increased, the measurement accuracy is improved, the measurement time is shortened, the processing speed is improved. A flow type particle image analysis method and apparatus that can be improved can be realized.

By the way, in the above-described flow type particle image processing apparatus according to the first embodiment, the first and second flash lamp lighting prohibited sections are provided even if the measured particles pass through the particle detection system in these sections. Since the image is not processed,
Although it is shorter than the interlaced method, there is still dead time in particle measurement.

It is desirable to make this dead time as small as possible, but it is inevitably necessary from the flash lighting to the field read signal in order to avoid double exposure. However, the time for accumulating charges in the discharge capacitor of the lamp power supply is shortened, and it is desirable to reduce the dead time due to charge accumulation. To shorten the charging time, it is conceivable to use a flash lamp with a large power capacity. However, a flash lamp having a large power capacity may not be practical.

FIG. 4 is an internal block diagram of a flash lamp drive circuit 1a of an apparatus for carrying out the flow type particle image analysis method according to the second embodiment of the present invention. The second embodiment is an example of reducing the dead time due to the charge accumulation described above.

In FIG. 4, the flash lamp drive circuit 1a has two charging circuits. These two
The two charging circuits have the same configuration.
30 and 33 are the main power source 1 and the main power source 2, and 31
And 34 are resistor 1 (Rm1) and resistor 2 (Rm1
2). 32 and 35 are discharge capacitors 1 (Cm
1) and discharge capacitor 2 (Cm2), 36 is a switch, and 37 is a trigger circuit. The first charging circuit comprises a main power supply 30, a resistor 31, and a discharging capacitor 32, and a second charging circuit
The charging circuit of is composed of a main power supply 33, a resistor 34, and a discharge capacitor 35.

The flash lamp lighting control circuit 23, as shown in FIGS. 5A, 5B and 5C, alternately switches the main power source 1 and the main power source 2 in accordance with the field read signal. To supply the signal for switching to the switch 36.

The charging time Tc of each charging circuit is shorter than at least one field time, and when the main power supply 30 or 33 is turned on, sufficient charge (energy) is stored in the discharge capacitor 32 or 35. It is in the state of being. Then, when the particle detection delay signal is output from the flash lamp lighting control circuit 23 to the trigger circuit 37 ((D) of FIG. 5), the trigger circuit 37 starts discharging the flash lamp 1.

The flash lamp lighting prohibition section is from the output of the particle delay signal to the output of the next field read signal in order to avoid double exposure ((E) of FIG. 5). In this case, since the first main power supply circuit and the second main power supply circuit are switched each time the field signal is output, it is not necessary to consider the prohibition of the flash lamp lighting by the charging time Tc. Since the particles flow randomly, the maximum dead time is close to one field time in this method, but the minimum dead time can be almost zero. Note that other configurations are the same as those in the example described in FIG. 1, and thus description thereof will be omitted.

As described above, according to the second embodiment of the present invention, the same effect as that of the first embodiment can be obtained, and the output from the first main power supply circuit and the second main power supply circuit can be obtained. Since the output from the main power supply circuit is switched and supplied to the flash lamp 1 each time a field read signal is output, there is no need to consider the flash lamp lighting prohibition time due to the charging time Tc, and there is no dead. A flow type particle image analysis method and apparatus in which the time is further shortened can be realized.

By the way, also in the above-described second embodiment of the present invention, the dead time cannot be set to 0 in consideration of the conditions peculiar to the lamp and the discharge time. Therefore,
In order to further reduce the minimum dead time, it is possible to use two independent light sources including a flash lamp.

FIG. 6 is an explanatory view of the main part of an apparatus for carrying out the flow type particle image analysis method according to the third embodiment of the present invention. In FIG. 6, 80 and 82 are flash lamps 1 and 2, and 81 and 83 are field lenses 1 and 2. A beam splitter 84 is provided to combine the light fluxes from the two flash lamps 80 or 82 into one. As the beam splitter 84, there is a beam splitter 84 having a constant reflectance and transmittance, and a special one having a reflectance and reflectance that changes every one field time. The latter has an advantage that the light quantity of the lamp light source can be effectively used.

The flash lamps 80 and 82 are supplied with power for lighting from a separate main power source and discharge capacitor, respectively, and in the same manner as in the second embodiment described above,
The flash lamp that can be turned on is switched for each field. That is, the basic idea is the same as in the second embodiment described above.

As described above, according to the third embodiment of the present invention, the same effect as that of the first embodiment can be obtained, and in addition, two flash lamps are provided and a field read signal is output. The charging time Tc is changed because the flash lamp that can be turned on is switched each time
It is not necessary to consider not only the flash lamp turn-off prohibition time, but also the conditions unique to the flash lamp and the discharge time (minimum lighting cycle determined by the lamp capacity), and the dead time is further shortened. And a device can be realized.

In the embodiment described above,
Although a CCD type image pickup device has been taken as an example of the image pickup device, the present invention is not limited to the CCD type image pickup device, and is applicable to other types of image pickup devices performing similar operations.

In the embodiment described above,
Although the flash lamp has been described as a representative of the light source, the same effect as that of the above-described embodiment can be expected even when a general pulse light source is used.

The particle image analysis method and apparatus of the present invention can be applied to image analysis of various particles, but is suitable for analysis of biological cells. The biological cells include blood cell components present in blood and urinary sediment components present in urine.

[0104]

Since the present invention is constructed as described above, it has the following effects. After the pulse lamp is turned on, the pulse lamp is prevented from being turned on until the reading signal of the image pickup means is output, so that double exposure is prevented. Further, after the pulse lamp 1 is turned on, after the charge accumulation time of the discharge capacitor has elapsed, the pulse lamp is prohibited from being turned on until the next field reading signal is output. When the time is shorter than the charging time of the discharge capacitor, the charging time is set to the lighting prohibited section, so that the shortage of the light amount due to the shortage of the charging time is avoided and the capturing of the inappropriate image is suppressed.

Therefore, the dead time in the flow type particle image analysis using the non-interlaced type image pickup means is shortened as compared with the interlaced type, and the number of measured particle images is increased, the measurement accuracy is improved, and the measurement is performed. It is possible to realize a flow type particle image analysis method and apparatus capable of shortening the time and improving / improving the processing speed.

Further, the first main power supply circuit for lighting the pulse lamp and the second main power supply circuit are provided, and the output from the first main power supply circuit and the output from the second main power supply circuit are read by field reading. If it is configured such that it is switched and supplied to the pulse lamp each time the output signal is output, it is not necessary to consider the pulse lamp lighting prohibition time due to the charging time, and the dead time is further shortened, and the flow type particle image analysis method is reduced. And a device can be realized.

If two pulse lamps are provided and the pulse lamps that can be turned on are switched each time a field read signal is output, not only the pulse lamp lighting prohibition time due to the charging time but also the pulse lamp specific It is not necessary to consider the conditions and the discharge time, and it is possible to realize the flow type particle image analysis method and apparatus in which the dead time is further shortened.

The dead time in particle imaging, which is a problem in the flow type particle image analysis apparatus using the non-interlaced imaging means, can be minimized. This dead time can be reduced to about 1/2 of the dead time of the interlaced system.

As a result, it can be expected that the volume of processed sample is increased, the processing time is shortened, and the measurement accuracy is improved.

Further, although the use of a pulse lamp having a capability that the lighting cycle of the pulse lamp has a minimum time of one field or less maximizes the above-mentioned effect, a method using two discharge capacitors, or In the system using two pulse lamps, the system is switched for each field, so that it is not necessary to extremely shorten the minimum time of the lamp lighting cycle, and even if the value is close to one field time, it operates normally.

[Brief description of drawings]

FIG. 1 is an overall schematic configuration diagram of a flow type particle image analysis apparatus for carrying out a flow type particle image analysis method according to a first embodiment of the present invention.

FIG. 2 is an explanatory diagram showing a processing timing relationship of particle image analysis in the example of FIG.

FIG. 3 is an explanatory diagram of a timing of a second flash lamp lighting prohibited section in the example of FIG. 1.

FIG. 4 is an internal block diagram of a flash lamp drive circuit 1a of an apparatus for carrying out a flow type particle image analysis method according to a second embodiment of the present invention.

5 is an explanatory diagram showing an operation timing of the flash lamp drive circuit in the example of FIG. 4. FIG.

FIG. 6 is an explanatory view of a main part of an apparatus for carrying out a flow type particle image analysis method according to a third embodiment of the present invention.

[Explanation of symbols]

1 flash lamp 1a Flash lamp drive circuit 2 field lens 3 Microscope condenser lens 5 Microscope objective lens 6 Imaging position 7 Projection lens 8 TV camera 11 Field stop 12 Aperture stop 15 Semiconductor laser 16 Collimator lens 17 Cylindrical lens 18, 19 Reflector 20 beam splitter 21 Aperture 22 Photodetector circuit 23 Flash lamp lighting control circuit 24 AD converter 25 image memory 26 Image processing control circuit 27 Feature extraction circuit 28 Identification circuit 29 Central control unit 30 Main power supply 1 31 resistor 1 32 discharge capacitor 1 33 Main power supply 2 34 Resistor 2 35 discharge capacitor 2 36 switch 37 Trigger circuit 40 Particle Count Analysis Section 50 display 80 flash lamp 1 81 Field lens 1 82 flash lamp 2 83 Field lens 2 84 beam splitter 100 flow cell 101 image capturing means 102 Particle analysis means 103 Particle detection means 110 sample flow

Claims (8)

(57) [Claims] 1. A particle sample suspended in a liquid is caused to flow in a flow cell, particles passing through a particle detection region in the flow cell are detected, and a still image of the detection particle passing through the imaging region in the flow cell is captured. In the flow-type particle image analysis method of performing morphological classification of particles by image-capturing using the means and performing image analysis of the captured particle images, the image-capturing means is a non-interlaced image-capturing means and detects particles. In order to do so, after turning on the pulse lamp for irradiating the image pickup area with light, the turning on of the pulse lamp for particle detection is prohibited until the next image reading pulse signal of the image pickup means is output. When the time until the image reading signal of the image pickup means is output after the pulse lamp is turned on is shorter than a predetermined minimum lighting period of the pulse lamp. Is the minimum lighting cycle determined by the lamp lighting ability after the pulse lamp has been lit, and a first image of the pulse lamp is prohibited as the first lighting inhibition time. A flow-type particle image analysis method, characterized in that the image is captured by a method. 2. A flow-type particle image analysis method according to claim 1, wherein a second lighting prohibition time is set, which is determined from the characteristics of the image pickup means, and the pulse lamp is turned on at the second lighting prohibition time. A flow type particle image analysis method, characterized in that, when a condition for lighting is reached, the timing of light emission of the pulse lamp is set immediately after the second lighting prohibition time has elapsed. 3. A flow-type particle image analysis method according to claim 1, wherein a second lighting prohibition time is set, which is determined from the characteristics of said image pickup means, and a pulse lamp is lit during this second lighting prohibition time. The flow particle image analysis method is characterized in that the lighting of the pulse lamp is prohibited when the conditions are met. 4. A particle sample suspended in a liquid is caused to flow in a flow cell, particles passing through a particle detection area in the flow cell are detected, and a still image of the detection particle passing through the imaging area in the flow cell is captured. In the flow-type particle image analysis method of performing morphological classification of particles by performing image analysis using the means and performing image analysis of the captured particle image, the imaging means is a non-interlaced imaging means, and A first pulse lamp lighting charging circuit having a discharge capacitor and a second pulse lamp lighting charging circuit having a second discharge capacitor are independent of each other In order to detect particles, a pulse lamp that irradiates the imaging area with light is lit, and the first pulse lamp lighting charging circuit is operated under normal conditions. It is used for slump lighting, and after lighting the pulse lamp, prohibits the lighting of the pulse lamp until the next image reading pulse signal of the image pickup means is output,
If the time from the lighting of the pulse lamp until the image reading pulse signal of the image pickup means is output is shorter than the minimum lighting cycle determined by the ability of the pulse lamp, the second
A method of analyzing a flow-type particle image, characterized in that a pulse lamp is turned on by using the energy charged in the discharge capacitor. 5. The flow type particle image analyzing method according to claim 4, wherein the first charging circuit and the second charging circuit are provided for lighting the pulse lamp for each field of the image captured by the imaging means. The flow type particle image analysis method is characterized in that the charging circuits are alternately switched and used. 6. A particle sample suspended in a liquid is caused to flow in a flow cell, particles passing through a particle detection area in the flow cell are detected, and a still image of the detection particles passing through the imaging area in the flow cell is captured. In the flow-type particle image analysis method of performing morphological classification of particles by image-capturing using a means and performing image analysis of the captured particle image, the image-capturing means are non-interlaced image-capturing means and are independent of each other , Either the first pulse lamp or the second pulse lamp irradiates the imaging region with light for capturing a particle image, and the first pulse lamp is used as a normal pulse lamp. After the first pulse lamp is turned on, the next pulse lamp is turned off until the image reading signal of the image pickup means is output, and the first pulse lamp is turned on. When the time until the image reading signal of the image pickup means is output after lighting is shorter than the minimum lighting cycle determined by the capability of the pulse lamp, the second pulse lamp is turned on. Particle image analysis method. 7. The flow type particle image analysis method according to claim 6, wherein a particle image is picked up by the first pulse lamp and the second pulse lamp for each field of the image picked up by the image pickup means. A flow-type particle image analysis method, which is characterized in that the light sources are alternately switched and used. 8. A particle sample suspended in a liquid is caused to flow in a flow cell, particles passing through a particle detection area in the flow cell are detected, and a still image of the detection particle passing through the imaging area in the flow cell is captured. In a flow-type particle image analyzer that performs morphological classification of particles by performing image analysis using the means and performing image analysis of the captured particle image, a non-interlaced imaging means and the above-mentioned imaging area for detecting particles A pulse lamp for irradiating light on the pulse lamp, and after turning on the pulse lamp, prohibit the turning on of the pulse lamp for particle detection until the next image reading pulse signal of the imaging means is output, and the pulse When the time until the image reading signal of the imaging means is output after the lamp is lit is shorter than the predetermined minimum lighting period of the pulse lamp A pulse lamp lighting control drive means for setting the lamp lighting prohibited time after the pulse lamp lighting as a minimum lighting cycle determined by the lamp lighting ability, and as a first lighting prohibited time for prohibiting lighting of the pulse lamp. A flow-type particle image analyzer characterized by: