JP2008224465A - Method and instrument for measuring fine particle - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fine particle measuring instrument capable of easily and inexpensively acquiring a plurality of fine particle images photographed with identical timing. <P>SOLUTION: The fine particle measuring instrument is equipped with a flow channel pipe 10 equipped with a measuring region 12 having light transmission properties and permitting a fluid containing fine particles to pass; a supply means 14 for supplying the fluid to the flow channel pipe 10; a plurality of imaging means 20 and 30 for photographing the inside of the measuring region 12 to acquire a plurality of fine particle images; and a control means 50 for controlling the operations of the supply means 14 and the imaging means 20 and 30. The control means 50 is constituted so as to take a plurality of the fine particle images at the same time, in a state where the flow of the fluid in the measuring region 12 is temporarily stopped. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a fine particle measurement method and apparatus, and more particularly to a fine particle measurement method and apparatus capable of measuring characteristics by imaging fine particles such as cells, blood cells, microorganisms, powder, and dust.

As a device for imaging fine particles contained in a fluid, for example, a flow cytometer disclosed in Patent Document 1 is known. This flow cytometer is equipped with a flow cell that serves as a flow path for the sample liquid containing particles. By capturing the sample liquid that passes through the flow cell with a plurality of imaging means, a fluorescent image and a transmitted light image of the same particle are acquired. It is configured to be able to.
JP-A-6-235691

  However, since the conventional flow cytometer captures a still image of particles transported at a predetermined speed in the flow cell, a sufficient exposure time cannot be secured and image information can be output. As a result of the weakness, an image intensifier for amplifying the image is required, and there is a problem that measurement cannot be performed at low cost.

  In addition, in order to prevent an excessive input from being generated in the image intensifier due to the flash lamp light irradiated when the transmitted light image is captured, it is necessary to shift the imaging timing of the fluorescent image and the transmitted light image in time. There was also a problem that it was difficult to compare each other.

  Therefore, an object of the present invention is to provide a fine particle measuring method and apparatus capable of easily and inexpensively acquiring a plurality of fine particle images captured at the same timing.

  The object of the present invention is to supply a fluid containing fine particles to a flow path tube having a light-transmitting measurement region, and in a state where the flow of fluid in the measurement region is temporarily stopped, This is achieved by a particle measuring method including an imaging step of simultaneously imaging the inside of the measurement region by a plurality of imaging means and acquiring a plurality of particle images.

  In addition, the object of the present invention is to provide a channel tube having a light-transmitting measurement region through which a fluid containing fine particles can pass, supply means for supplying fluid to the channel tube, and imaging in the measurement region. And a plurality of imaging means for acquiring a plurality of fine particle images, and a control means for controlling the operation of the supply means and the imaging means, the control means temporarily stopped the flow of fluid in the measurement region This is achieved by a fine particle measuring apparatus that simultaneously captures a plurality of the fine particle images.

  According to the fine particle measurement method and apparatus of the present invention, a plurality of fine particle images captured at the same timing can be easily and inexpensively acquired.

Hereinafter, actual forms of the present invention will be described with reference to the accompanying drawings. FIG. 1 is a schematic configuration diagram of a particle measuring apparatus according to an embodiment of the present invention. As shown in FIG. 1, the particle measuring device 1 includes a flow cell 10 through which a fluid such as a liquid containing particles passes, a fluorescence image capturing device 20 and a dark field image capturing device 30 that are respectively arranged around the flow cell, and an image. Processing device 40
And a control device 50.

  The flow cell 10 is formed of a flat rectangular tube and includes a fluid inlet 10a and a fluid outlet 10b on both sides in the longitudinal direction. In the center of the flow cell, windows 11 made of a light-transmitting material such as glass or transparent plastic are formed on the front and back surfaces, respectively (only one is shown in FIG. 1), and the fluorescent image capturing device 20 and dark field image capturing are performed. A measurement region 12 for imaging the inside of the flow cell 10 by the device 30 is provided inside the window portion 11. The shape of the flow cell is not particularly limited as long as the fluid containing fine particles can be observed from the outside. In addition to the one of this embodiment, for example, other flow pipes such as a circular tube, an elliptical tube, and a deformed tube, Any configuration may be used as long as it includes a measurement region having light transmittance in part. However, it is preferable that the internal width is as small as possible within a range that does not hinder the passage of the fine particles so that the entire fine particles in the measurement region 12 can be imaged.

  A transparent supply pipe 2 is connected to the fluid inlet 10 a of the flow cell 10, and a particulate detection device 16 is disposed in the supply pipe 2. The fine particle detection device 16 includes a light source 17 that irradiates the supply pipe 2 with detection light such as laser light, and a light receiving unit 18 that receives the detection light. Detection is performed by changing the amount of received light due to blocking or scattering, or by detecting fluorescence, and a particulate passing signal is output so that the particulate image can be captured in the flow cell 10.

  On the other hand, a discharge pipe 4 is connected to the fluid outlet 10 b of the flow cell 10, and a pump 14 that supplies fluid to the inside of the flow cell 10 by suction is interposed in the discharge pipe 4.

  Further, shutoff valves 26 and 27 are provided in the vicinity of the fluid inlet 10 a and the fluid outlet 10 b in the supply pipe 2 and the discharge pipe 4, respectively. By closing the shutoff valves 26 and 27, the fluid in the flow cell 10 is closed. The flow can be temporarily stopped. The pump 14 can also be arranged upstream of the flow cell 10 so as to supply fluid into the flow cell 10 by indentation pressure. It is also possible to use other power sources such as compressed air as means for supplying the fluid to the flow cell 10, and furthermore, the fluid is self-weighted by arranging a fluid storage tank above the flow cell 10. It can also be configured to be supplied to the flow cell 10. The shutoff valves 26 and 27 are not necessarily provided when the flow of fluid in the flow cell 10 can be stopped by stopping the operation of the pump 14, for example.

  The fluorescence imaging device 20 includes a cooled CCD camera (for example, 300,000 pixels) 21, an excitation light source 22 that irradiates excitation light to the fine particles in the flow cell 10, a lens 23 that collects the emitted light of the fine particles, A filter 24 that is disposed between the CCD camera 21 and the lens 23 and selectively transmits only fluorescence in a predetermined wavelength region is provided, and a fluorescence image of fine particles existing in the measurement region 12 of the flow cell 10 is captured. The excitation light source 22 is configured by arranging a plurality of LEDs (for example, green LEDs having a wavelength of 530 nm) 22 a at equal intervals along the inner peripheral surface of the ring-shaped member, and is the same as the CCD camera 21 with respect to the measurement region 12. It arrange | positions in the vicinity of the measurement area | region 12 so that uniform excitation light can be irradiated from the side.

  The dark field image pickup device 30 includes a CCD camera (for example, 1.4 million pixels) 31 and picks up a dark field image of fine particles existing in the measurement region 12 of the flow cell 10 by forward scattered light of the fine particles from the excitation light source 22. . The dark field image capturing device 30 is disposed so as to face the fluorescent image capturing device 20 with the measurement region 12 interposed therebetween, and the captured dark field image has a relationship between the fluorescent image and the front and back.

The image processing device 40 is connected to the fluorescence image capturing device 20 and the dark field image capturing device 30 and performs image processing so that the same particle can be identified by superimposing the captured fluorescence image and dark field image. Apply.

  The control device 50 controls the operation of the pump 14 so that the fluid passes through the flow cell 10 at a predetermined flow rate. When a particle passage signal is input from the particle detection device 16, the control device 50 has a predetermined time delay and shuts off the valve. By controlling to close the pumps 26 and 27 and stop the pump 14, the flow of the fluid is temporarily stopped in a state where the detected fine particles are included in the flow cell 10.

  Further, the control device 50 controls the imaging timing of the both so that the imaging by the dark field imaging device 30 is performed simultaneously with the imaging of the fluorescence imaging device 20. In the present embodiment, the control device 50 controls the operations of the fluorescence image capturing device 20 and the dark field image capturing device 30 so that both the exposure start time and the exposure end time coincide with each other. In addition to such a case, “capture simultaneously” means that the exposure time zones of a plurality of imaging devices overlap at least partially.

  Next, a method for measuring phytoplankton contained in seawater will be described as an example of a particle measuring method using the particle measuring apparatus 1 having the above configuration. For example, size measurement is effective when it is desired to know the amount of carbon fixed by the phytoplankton in the ocean, and a population coefficient is required for ballast water inspection of ships. It is required to examine the temporal variation and its spatial distribution. The fine particle measuring apparatus and the fine particle measuring method according to the present invention enable measurement of such phytoplankton.

  First, the control device 50 operates the pump 14 to supply seawater containing fine particles such as phytoplankton and suspended particles into the flow cell 10. And based on the detection of the phytoplankton by the microparticle detection apparatus 16, the pump 14 is stopped and the shutoff valves 26 and 27 are closed to temporarily stop the flow of seawater in the measurement region 12 of the flow cell 10. The size of the measurement region 12 may be appropriately set in consideration of the size of the target fine particles. For example, when a phytoplankton having a size of about 5 μm to several mm is a measurement target, the plankton of 5 μm or more is counted. In order to obtain a resolution as high as possible, the size of the measurement region 12 can be set to about 0.01 cc.

  The moving speed of the microparticles in the flow cell 10 immediately decreases (for example, in about 0.2 seconds) due to the stop of the fluid, and the movement is extremely slow compared to the conventional measurement. For this reason, a sufficient exposure time can be ensured when the fluorescent image capturing device 20 and the dark-field image capturing device 30 capture fine particles (for example, about 0.2 seconds), so that the excitation light source 22 composed of a plurality of LEDs 22a can be used. The excitation light can be used as illumination light for capturing a dark field image, and simplification of the configuration and reduction of power consumption can be achieved. Further, even if a high-sensitivity camera is not used as the imaging means, a clear image capable of sufficiently discriminating fine particles can be obtained using a general or cooled CCD camera.

  The control device 50 controls the imaging timing so that the fluorescent image capturing device 20 and the dark field image capturing device 30 capture images simultaneously. The fluorescence image capturing apparatus 20 captures a fluorescence image by chlorophyll fluorescence emitted from phytoplankton by irradiation with excitation light. In the present embodiment, since the excitation light source 22 irradiates the measurement region 12 with excitation light from the same side as the fluorescence imaging device 20, the excitation light source 22 is not affected by forward scattered light, and thus is emitted from the phytoplankton. Only fluorescence can be efficiently imaged, and a clear fluorescence image can be obtained.

Further, the dark field image capturing device 30 captures a dark field image with forward scattered light of phytoplankton or suspended particles by excitation light. The image processing device 40 performs image processing so that the fluorescence image and the dark field image captured at the same time can be measured by superimposing each of them. The filter 24 of the fluorescence imaging device 20 can be appropriately replaced according to the fluorescence wavelength emitted by the target phytoplankton.

  The image processing device 40 inverts one of the images in consideration of the imaging direction of the fluorescent image and the dark field image, determines the search range from the size and coordinates of the fine particles on the dark field image, and the same on the fluorescent image Search for particles. Thus, after grasping the correspondence relationship of each particle in the fluorescent image and the dark field image, the distortion caused by the geometric arrangement of the imaging device and the distortion caused by the lens mechanism are corrected. For example, for distortion due to the inclination of the imaging axis of each of the imaging devices 20 and 30, the reference image can be captured and the inclination can be corrected by two-dimensional image conversion (affine transformation). It can be corrected by acquiring a reference image moved in the depth direction and measuring a distortion parameter.

  An example of the image corrected in this way by the image processing apparatus 40 is shown in an enlarged manner in FIG. In FIG. 2, (a) is a fluorescent image, and (b) is a dark field image. While phytoplankton appears in both fluorescent and dark field images, suspended particles other than phytoplankton appear only in dark field images, so by comparing the two, phytoplankton can be compared with other suspended particles. Can be identified. The image processing device 40 automatically distinguishes phytoplankton and suspended particles by superimposing the images after performing processing to change the density and color tone of the fine particles between the fluorescent image and the dark field image. In addition, the size and distribution density of each can be automatically calculated. FIG. 3 shows an example of results obtained by classifying phytoplankton and suspended particles for each size and measuring the population density for each size.

According to the fine particle measurement method of the present embodiment, fine particles are measured in a state where the flow of fluid is stopped. Therefore, it is possible to take an image of the movement of the fine particles themselves by increasing the exposure time. FIG. 4 is a fluorescent image obtained by capturing red tide plankton (scientific name: Karenia mikimotoi) with an exposure time of 5 seconds, and the exposure time is shortened (for example, 0.2 seconds) in order to measure the shape of the fluorescent image and plankton. By comparing with the dark field image, the plankton that moves can be identified. The speed of movement can be measured from the length of the spirally extending locus, and features such as the type of plankton can be known from the shape features of the locus. In this way, the mobility of the fine particles can be measured by making the exposure start times or the exposure end times of the two imaging devices coincide and making the exposure times different from each other.

  Moreover, it is also possible to comprise so that LED22a arrange | positioned at the excitation light source 22 may irradiate excitation light of a different wavelength (for example, 8 wavelengths), respectively, and the difference of the measured fluorescence wavelength or fluorescence intensity of phytoplankton It is also possible to measure the seed composition.

  Further, the fine particle measuring apparatus shown in FIG. 1 can be configured to be mounted on an insidious body or the like and to be measured in the actual sea by being housed in a pressure-resistant casing. FIG. 5 shows a schematic configuration of the vacuum vessel 61, an imaging system 62 including the flow cell 10, the fluorescence imaging device 20, and the dark field imaging device 30, and a control board 63. The personal computers 64 and 65 and the battery 66 are accommodated, seawater is sucked into the vacuum vessel 61 from the outside of the casing 60, and a series of operations of stopping the fluid, imaging, image processing, and image storage are performed by driving the battery 66. By performing automatically, measurement of phytoplankton in the sea can be performed.

As mentioned above, although one Embodiment of this invention was explained in full detail, the specific aspect of this invention is not limited to the said embodiment. For example, in the present embodiment, the measurement of phytoplankton contained in seawater has been described as an example, but the distribution density, size, fluorescence, etc. of cells having autofluorescence characteristics or artificially imparted fluorescence, etc. The present invention can also be applied to the measurement of strength and the like. For example, a substance contained in a cell can be estimated from the obtained cell size. It is also possible to examine the presence or absence of fluorescence of the fine particles. The fluid passing through the flow cell 10 may be a gas. In this case, it is preferable that the flow of the fluid is stopped so that the flow cell 10 is disposed horizontally and the fine particles are allowed to remain on the measurement region 12.

  In the present embodiment, the fluorescent image capturing device 20 and the dark field image capturing device 30 are disposed so as to face each other with the measurement region 12 interposed therebetween in order to facilitate the superimposition of the fluorescent image and the dark field image. By grasping each imaging axis, even a plurality of images captured in other arrangements can be corrected by the image processing device 40 so that they can be superimposed. For example, it is possible to arrange three or more fluorescent imaging devices 20 having different transmission wavelengths of the filter 24 around the flow cell 10 at equal intervals and superimpose the respective fluorescent images, and measure various phytoplankton. Can be done simultaneously.

  In the present embodiment, the fluorescent image and the dark field image are captured by the fluorescence image capturing device 20 and the dark field image capturing device 30. In addition to these images, a bright field image, a phase difference image, and a polarization image are also captured. Two or more imaging devices may be arranged around the flow cell 10 so that differential interference images can be simultaneously captured. Thereby, the shape, optical characteristics (for example, spectroscopic properties), mechanical characteristics (for example, the density of microparticles), population density, the presence or absence of motility, and physiological properties of the microparticles can be measured. Each imaging device may be the same type of image. For example, by capturing and synthesizing dark field images from the front and back surfaces of the flow cell 10, a clear tertiary image can be obtained even when the depth of field of each image is small. An original image can be obtained.

  When a plurality of imaging devices are used, measurement can be performed by changing the observation wavelength region, exposure start time, exposure end time, observation region (lens magnification, field size), observation region (depth), and the like. It is also possible to perform measurement by changing the wavelength, intensity, illumination time, irradiation angle, etc. of the illumination light. For example, using two cameras (A, B) and two illuminating devices (a, b), illuminating illumination light from the illuminating device a, and simultaneously capturing and superimposing images with the cameras A and B, and the illuminating device Illumination light having a different wavelength, intensity, irradiation time, and the like of illumination light from a is emitted from the illumination device b, and the fine particles are measured by comparing the images captured and superimposed simultaneously with the cameras A and B. It is possible to measure optical properties such as photosynthetic activity.

1 is a schematic configuration diagram of a fine particle measuring apparatus according to an embodiment of the present invention. It is a figure which shows an example of a fluorescence image and a dark field image. It is a figure which shows an example of the relationship between the size of microparticles | fine-particles, and a population density. It is a figure which shows an example of the fluorescence image which imaged the motion state of microparticles | fine-particles. It is a schematic block diagram of the fine particle measuring apparatus which can be used in the sea.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Fine particle measuring device 10 Flow cell 12 Measurement area | region 14 Pump 16 Fine particle detection device 20 Fluorescence image pick-up device 22 Excitation light source 26, 27 Shut-off valve 30 Dark field image pick-up device 40 Image processing device 50 Control device

Claims (7)

A supply step of supplying a fluid containing fine particles to a channel tube having a measurement region having light permeability;
A particulate measurement method comprising: an imaging step of capturing a plurality of particulate images by simultaneously capturing the interior of the measurement region by a plurality of imaging means in a state where the flow of fluid in the measurement region is temporarily stopped. The particle measurement method according to claim 1, further comprising an image processing step of correcting the plurality of particle images so as to be superposed. The fine particle measurement method according to claim 1, wherein the imaging step captures a fluorescent image and a dark field image. The fluorescence image is generated by receiving fluorescence from particles by irradiation of excitation light into the measurement region,
The fine particle measurement method according to claim 3, wherein the dark field image is generated by receiving scattered light from particles by irradiation with the excitation light. The fine particle measurement method according to claim 4, wherein the irradiation of the excitation light into the measurement region is performed by an excitation light source disposed on the same side as the imaging unit that captures a fluorescence image and the measurement region. The fine particle measurement method according to claim 1, wherein the plurality of imaging units are arranged to face each other with the measurement region interposed therebetween. A channel tube having a light-transmitting measurement region through which a fluid containing fine particles can pass;
Supply means for supplying fluid to the flow path tube;
A plurality of imaging means for capturing the inside of the measurement region and acquiring a plurality of fine particle images;
Control means for controlling the operation of the supply means and the imaging means,
The control means is a particle measuring apparatus that simultaneously captures a plurality of the particle images in a state where the flow of fluid in the measurement region is temporarily stopped.