WO2016024430A1 - Microparticle detection device - Google Patents

Microparticle detection device Download PDF

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Publication number
WO2016024430A1
WO2016024430A1 PCT/JP2015/065303 JP2015065303W WO2016024430A1 WO 2016024430 A1 WO2016024430 A1 WO 2016024430A1 JP 2015065303 W JP2015065303 W JP 2015065303W WO 2016024430 A1 WO2016024430 A1 WO 2016024430A1
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Prior art keywords
microparticles
light
microparticle
same
detection
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PCT/JP2015/065303
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French (fr)
Japanese (ja)
Inventor
岡橋　哲秀
優紀 田中
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シャープ株式会社
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Publication of WO2016024430A1 publication Critical patent/WO2016024430A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/02—Investigating particle size or size distribution
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated

Definitions

  • the present invention relates to a microparticle detection apparatus.
  • microparticle detection device As a microparticle detection device, light is irradiated to microparticles developed in a liquid or on a membrane or slide glass, and fluorescence or scattered light generated from the microparticles is detected, and particle counting or property inspection is performed.
  • the fine particles include inorganic particles, microorganisms, cells, erythrocytes in blood, leukocytes, platelets, vascular endothelial cells, fine cell fragments of the tissue, and the like.
  • the microparticles become a microparticle suspension when in the liquid.
  • the fine particle suspension is flowed into the capillary together with the sheath liquid. Then, the type of particle and the size of the particle are classified by irradiating a part of the capillary with laser light and detecting scattered light or fluorescence generated when the fine particle is irradiated with light. For example, by labeling particles with a fluorescent reagent that binds to specific particles, the number of fluorescent particles can be counted to count only the specific particles. With this flow cytometer, particles of about submicron to 10 ⁇ m can be detected, and highly accurate detection is possible.
  • Imaging methods include imaging with a microscope and a digital camera, and methods of detecting scattered light or fluorescence while scanning an optical head two-dimensionally and imaging.
  • the laser light from the optical head is focused on the particle and irradiated, and while detecting scattered light or fluorescence generated from the particle, The optical head is scanned two-dimensionally to form an image.
  • the laser spot diameter is equal to or smaller than the particle size. More than that. For this reason, the image obtained as a result of the two-dimensional scan is not an image in which each particle is resolved, and it is difficult to directly measure the size of the particle from the image.
  • the laser beam irradiation spot is larger than the particle size, the intensity of the scattered light generated from the particle varies depending on the particle size, so that the particle diameter can be determined from the intensity of the scattered light. The reason is that there is a correlation between the particle diameter and the scattered light intensity.
  • a laser light source because light cannot be sufficiently collected by an LED (Light Emitting Diode) or a lamp light source.
  • a semiconductor laser is advantageous because it is small and inexpensive.
  • the wavelength band it may be expensive or only a solid laser having a volume larger than that of the semiconductor laser may be obtained. Therefore, depending on the wavelength band, there may be an expensive system.
  • the disk rotation method can greatly reduce the measurement time, but the measurement time is still long. Further, since it is generally a monitor such as a personal computer that performs imaging, it is necessary to transmit a large amount of acquired data to the personal computer or the like at high speed.
  • binarization processing is performed by reading scattered light or fluorescence.
  • the background and noise and the detection target particle are discriminated based on whether the brightness of scattered light or fluorescence is higher or lower than the threshold value, and the particle count is performed by counting the places where the brightness is higher than the threshold value. It is.
  • Patent Document 1 Japanese Patent Application Laid-Open No. 2004-325091
  • the scanned scattered light or fluorescent binary image is scanned using a scanning window having a particle detection window and a size discrimination window. The number of cells is counted for each cell size.
  • the conventional cell counting method it is possible to count for each particle size by dividing into a particle detection window and a size determination window so that the same particles are not counted repeatedly.
  • the amount of data increases because it is necessary to obtain the entire map data once. Furthermore, since the particle size is detected and the size is discriminated by operating the scanning window, the burden of calculation processing becomes large. Therefore, there is a problem that a necessary data amount and a data processing load increase.
  • an object of the present invention is to provide a microparticle detection apparatus capable of reducing the amount of data when counting is performed by detecting microparticles from a specimen containing microparticles.
  • a microparticle detection apparatus includes: An irradiation optical system for irradiating a specimen containing fine particles with light emitted from a light source; A light detection optical system for detecting light emitted from the microparticles in the specimen by the light irradiation; A detection unit that detects the microparticles based on the intensity of light from the microparticles detected by the photodetection optical system; The irradiation optical system and the light detection optical system are configured to relatively two-dimensionally scan the specimen in a first scanning direction and a second scanning direction that intersects the first scanning direction, The detection unit is Based on the intensity of light from the microparticles when the irradiation optical system and the light detection optical system scan in the first scanning direction, an existence region where the microparticles are present is determined for each scanning line. , By comparing the existing areas of the microparticles in the scanning lines adjacent to each other, it is determined whether or not the set of the existing areas
  • the “existing region where the microparticles exist” refers to a region sandwiched between the start point and the end point of the section where the intensity of light from the microparticles exceeds the threshold in each scanning line.
  • the detection unit determines that the set of the existing regions in both the scan lines is caused by the same microparticle, the detection unit is adjacent to one of the scan lines and the scan line. By comparing the existing area of the microparticles in the new scanning line, it is determined whether the set of the existing areas in the new adjacent scanning line is caused by the same microparticle. Yes.
  • the detection unit When the positions in the first scanning direction overlap each other with respect to the existence area of the microparticles in the scanning lines adjacent to each other, the detection unit has the same combination of the existence areas in both the scanning lines. It is determined that the region is a region where microparticles exist.
  • the detection unit In the case where the existence area determined to be caused by the same minute particle is present in the scanning lines adjacent to each other, the detection unit detects the same minute particle based on the existence area caused by the same minute particle. Position information is set.
  • the detection unit In the case where the existence area determined to be caused by the same minute particle is present in the scanning lines adjacent to each other, the detection unit detects the same minute particle based on the existence area caused by the same minute particle. Shape information or size information is set.
  • the microparticle detection apparatus of the present invention is For each scanning line when scanned in the first scanning direction, whether the group of the existing regions is caused by the same minute particles by a simple method of comparing the existing regions of the minute particles in the adjacent scanning lines. It is determined whether or not.
  • the comparison itself may be a very simple process. And the comparison regarding all the scanning lines can be performed by sequentially performing the comparison with respect to the adjacent scanning lines.
  • the comparison process can be performed simultaneously with the detection of the region where the microparticles exist in each scanning line, and the microparticles can be counted efficiently.
  • FIG. 1 It is a figure which shows schematic structure in the microparticle detection apparatus of this invention. It is a schematic diagram which shows the relationship between each track
  • FIG. 1 is a figure which shows schematic structure of the microparticle detection apparatus of this Embodiment.
  • the microparticle detection apparatus includes a disk into which a specimen is injected, a rotation drive system that rotates the disk, a light detection optical system that detects scattered light or fluorescence, and a drive mechanism that drives the detection optical system in a radial direction. And a detection unit that receives the signal from the optical detection optical system and detects the fine particles.
  • 1 is a light source device
  • 2 is an objective lens
  • 3 is a first detection device
  • 4 is a second detection device.
  • the light source device 1, the objective lens 2, the first detection device 3, and the second detection device 4 are housed in a frame to constitute an optical module 5.
  • a circular disk 6 is disposed above the optical module 5 so as to face the objective lens 2.
  • the disk 6 for example, a suspension, a gel support, or a membrane in which fine particles labeled with a fluorescent substance are distributed. Or the like is encapsulated as a sample (the specimen) 7.
  • the light source device 1 of the optical module 5 is provided with a first semiconductor laser 8 as a light source.
  • a first lens 9, a spot size adjusting lens 10, and a first The apertures 11 are arranged in this order.
  • a second semiconductor laser 12 that emits laser light having a second wavelength different from the first wavelength of laser light emitted from the first semiconductor laser 8 is disposed.
  • a second lens 13 for collimating the laser beam from the second semiconductor laser 12 is disposed.
  • a first dichroic mirror that transmits the laser light having the first wavelength and reflects the laser light having the second wavelength is transmitted to the intersection between the optical axis of the first semiconductor laser 8 and the optical axis of the second semiconductor laser 12.
  • the light source device 1 which is an example of the irradiation optical system is configured.
  • a prism 15 that reflects the light transmitted through the first dichroic mirror 14 toward the objective lens 2 is disposed.
  • a second dichroic mirror 16 that reflects the light from the prism 15 so as to enter the objective lens 2 is disposed at the intersection of the light reflected by the prism 15 and the optical axis of the objective lens 2.
  • the second dichroic mirror 16 transmits the fluorescence from the sample 7 and reflects the scattered light.
  • the “scattered light” referred to in the present invention is light in which the light emitted from the first semiconductor laser 8 or the second semiconductor laser 12 is isotropically scattered from the irradiated position of the sample 7 to the surroundings. Yes, the light has the same wavelength as the emitted light.
  • “fluorescence” means that the light emitted from the first semiconductor laser 8 or the second semiconductor laser 12 irradiates the sample 7 to excite the fluorescent substance labeling the microparticles, and irradiates the sample 7. It is fluorescence scattered isotropically from a location to the surroundings, and is light having a wavelength different from that of outgoing light.
  • the objective lens 2 is stored in a lens holder (not shown), and is moved in the optical axis direction by a drive unit (not shown) such as a stepping motor, so that the focal position is adjusted. It can be changed.
  • the spot size adjustment lens 10 is stored in a lens holder (not shown), and is moved in the optical axis direction by a drive unit (not shown) so that the spot size can be adjusted.
  • a sample 7 that is condensed by the objective lens 2 and converted into parallel light is sequentially from the second dichroic mirror 16 side below the second dichroic mirror 16 on the optical axis of the objective lens 2.
  • Bandpass filter 17 that attenuates light (light having a wavelength different from that of fluorescence)
  • a third lens 18 that collects fluorescence that has passed through bandpass filter 17, and stray light of fluorescence that has passed through third lens 18
  • the 2nd aperture 19 which cuts is arranged.
  • a first detector 20 including a detection element such as a photomultiplier tube (PMT) that detects fluorescence that has passed through the second aperture 19 is disposed below the second aperture 19 on the optical axis of the objective lens 2.
  • the second aperture 19 and the first detector 20 are housed in one case and constitute a first detection device 3 which is an example of the light detection optical system.
  • ND (attenuating) filter 21 that attenuates scattered light from the ND filter
  • fourth lens 22 that condenses the scattered light that has passed through the ND filter 21
  • a first lens that cuts stray light from the scattered light that has passed through the fourth lens 22.
  • Three apertures 23 are arranged.
  • a second detector 24 including a detection element such as the PMT for detecting scattered light that has passed through the third aperture 23 is disposed.
  • the 3rd aperture 23 and the 2nd detector 24 are stored in one case, and constitute the 2nd detection device 4 which is an example of the above-mentioned optical detection optical system. Note that three or more detection devices may be added by adding dichroic mirrors.
  • the several light source of the said 1st semiconductor laser 8 and the 2nd semiconductor laser 12 is mounted, a several light source is not necessarily required.
  • the disk 6 is configured to be transparent and circular, and is accommodated in a circular dish-shaped holder 26 fixed to the central shaft 25 and fixed to the central shaft 25.
  • the central shaft 25 can be rotated by a spindle motor 27 as an example of the rotational drive system.
  • the optical module 5 can be moved stepwise by the drive mechanism in the radial direction of the disk formed by the disk 6.
  • the drive mechanism of the optical module 5 is not particularly limited.
  • the frame of the optical module 5 is configured to be movable by being guided by the guide rail disposed in the radial direction by a timing belt or the like reciprocated in the radial direction by a stepping motor or the like.
  • the disk 6 is formed in a circular shape as described above.
  • the optical module 5 is set to move one step each time the disk 6 makes one revolution.
  • a band-like non-detection area having a certain width and extending in the radial direction is set on the disc 6 and the objective is set. It is necessary to move the optical module 5 by one step while the spot of the excitation light from the lens 2 moves in the non-detection region.
  • the optical module 5 may be continuously moved while the disk 6 is continuously rotated to scan in a spiral manner.
  • the optical module 5 when detecting light, the optical module 5 is moved in the radial direction of the disk 6 while rotating the disk 6 to detect fluorescence or scattered light from the microparticles in the sample 7.
  • a semiconductor laser emitting a laser beam having a wavelength reflected by the second dichroic mirror 16, for example, the first wavelength from the first semiconductor laser 8.
  • the laser beam is emitted.
  • the excitation light (laser light) emitted from the first semiconductor laser 8 is converged by the first lens 9, the spot size adjusting lens 10 and the first aperture 11, and passes through the first dichroic mirror 14.
  • the light is reflected by the prism 15 and the second dichroic mirror 16, passes through the objective lens 2 and the disk 6, and is collected at one point on the lower surface of the sample 7.
  • the length of the prism 15 in the longitudinal direction (horizontal direction) is short, the width in the direction orthogonal to the longitudinal direction is narrow, and the excitation light from the first semiconductor laser 8 is near the optical axis of the objective lens 2. It passes through only (excitation light transmission part).
  • scattered light isotropically scattered from the portion irradiated with the focused light to the surroundings.
  • the scattered light is isotropically emitted from the portion of the sample 7 irradiated with the focused light to the surroundings.
  • the component of the emitted scattered light that has passed through the disk 6 and entered the objective lens 2 passes through the objective lens 2 and is reflected by the second dichroic mirror 16, and the ND filter 21, the fourth lens 22, and the like. It passes through the third aperture 23 and is detected by the second detector 24.
  • the signal detected by the second detector 24 is subjected to processing such as AD conversion by a built-in AD converter and the like, and then sent to a PC (personal computer) 40, which is an example of the detection unit.
  • the fluorescence intensity distribution at each measurement point on the sample 7 is recorded in the internal memory or the like. Further, when the particle count is performed based on the detection signal, the particle count data is recorded in the internal memory or the like.
  • the third aperture 23 is arranged to cut spatial stray light. It also functions as a confocal aperture and removes unnecessary reflected light and stray light from areas other than the surface where the sample 7 exists. For example, since the reflected light generated on the surface of the disk 6 or the lens surface is deviated from the focal position of the objective lens 2, it becomes light spread at the position of the third aperture 23 by the optical system following the objective lens 2. It cannot pass through the third aperture 23 well.
  • the scattered light intensity at each measurement point is recorded in the internal memory of the PC 40 or the like.
  • the scattered light detection by the first wavelength laser light from the first semiconductor laser 8 has been described.
  • the second wavelength laser light from the second semiconductor laser 12 can also be detected by the first wavelength. Except for being reflected by the one dichroic mirror 14, the same is true.
  • fluorescence detection for detecting the fluorescence the semiconductor laser that emits laser light having a wavelength that passes through the second dichroic mirror 16 is used, and the fluorescence that has passed through the second dichroic mirror 16 is detected by the first detection device 3. It is exactly the same except for detecting at.
  • the specimen including the microparticles as the sample 7 is sealed in the disk 6. Then, the optical intensity data is acquired by the optical module 5 while the disk 6 is rotated.
  • the belt-like portion on the disk 6 where the above-mentioned data for one round which can be read by the optical module 5 is recorded is called a track.
  • the track is an example of the scan line.
  • the circumferential direction is an example of the first scanning direction
  • the radial direction is an example of the second scanning direction.
  • the optical module 5 is moved in the radial direction of the disk 6 to move the track for reading the light intensity data.
  • the optical module 5 may be gradually moved while the disk 6 rotates once, and the data of the light intensity may be acquired in a spiral shape.
  • the outer peripheral surface 26a of the holder 26 is provided with an encoder ring (not shown) in which reflecting surfaces are arranged at a constant angular pitch in the circumferential direction. Furthermore, a head 28 on which a pair of light emitting elements and light receiving elements are mounted is disposed at a position facing the outer peripheral surface 26 a of the holder 26.
  • the rotational displacement of the central shaft 25, that is, the rotational displacement of the disk 6 can be detected by the encoder signal from the head 28. Therefore, in synchronization with the encoder signal from the head 28, it is possible to acquire the scattered light intensity data at every fixed angle in the tangential direction.
  • the encoder signal is not limited to the encoder ring and the head 28, and may be generated by a rotary encoder attached to the spindle motor 27.
  • the optical module 5 can simultaneously detect scattered light and fluorescence as described above.
  • the scattered light is imaged and used to determine the size of the particles.
  • the fluorescence from the sample 7 is emitted only from the specific type of microparticles. Therefore, the fluorescence from the sample 7 is used to count the number of specific particles.
  • both the type (fluorescence) and size (scattered light) of a certain particle can be determined by searching for a point where the coordinates match in the detection images obtained by the two detectors.
  • This method can be applied, for example, when it is desired to exclude fluorescent particles having a size equal to or larger than an arbitrary threshold from the count.
  • advantages of enabling the optical module 5 to detect scattered light and fluorescence at the same time include low cost due to downsizing of the optical module 5, space saving, and shortening of measurement time due to simultaneous detection. It is done.
  • the optical module 5 irradiates the sample 7 with light, and detects the scattered light and fluorescence emitted from the microparticles included in the sample 7, thereby detecting the microparticles included in the sample 7.
  • the disk 6 enclosing the sample 7 is accommodated in the holder 26 and fixed to the central shaft 25.
  • the scanning procedure by the optical module 5 is as follows. (1) As described above, the disk 6 is placed at the initial rotation position while the disk 6 is received and fixed in the holder 26. (2) The disk 6 is rotated by rotationally driving the central shaft 25 with a spindle motor 27. The rotation angle ⁇ is detected by the encoder ring and the head 28. (3) The optical module 5 is emitted from the fine particles on the disk 6 by scanning the disk 6 every rotation (one track) while moving the optical module 5 by one track in the radial direction of the disk 6. Detects scattered light and fluorescence. (4) From the detected intensity of scattered light or fluorescence, the existence area where the microparticles exist for each track is determined.
  • FIG. 2 is a schematic diagram showing the relationship between each track on the disk 6 and the fine particles a and b.
  • FIG. 2A is a schematic diagram showing the entire disk 6.
  • the microparticles straddling a plurality of tracks are those having a substantially circular shape and having a diameter itself straddling the plurality of tracks, such as a microparticle a, As described above, the shape is an elongated ellipse, and the small width is substantially the same as that between the tracks.
  • FIG. 2B is an enlarged schematic view of a region including the microparticles a.
  • FIG. 2 (c) is an enlarged schematic view of a region including the fine particles b.
  • a method is proposed that enables accurate detection and counting in the case of the fine particles a and the fine particles b.
  • the determination of the fine particles a and the fine particles b is performed by comparing the light intensity detected by the first detector 20 or the second detector 24 with a threshold value.
  • the threshold setting method is not particularly limited. For example, in FIG. 2B, it is assumed that light having an intensity stronger than the threshold is detected in the track n between the tangential directions ⁇ 1 and ⁇ 2. In that case, the PC 40 determines that there are fine particles in the angle region between ⁇ 1 and ⁇ 2. The angles ( ⁇ 1, ⁇ 2) between the start point and the end point are recorded in the internal memory of the PC 40 or the like.
  • the track (n + 1) is measured in the same manner with the detection position of the disk 6 moved from the track n by one track in the radial direction.
  • FIG. 3 is a diagram in which the measurement data of the positions of the microparticles obtained in FIGS. 2B and 2C are replaced with a two-dimensional coordinate system of the track number and the rotation angle ( ⁇ ). In the following, FIG. 3 is used in order to easily understand the comparison of the region where the microparticles exist.
  • the existence area of the detected microparticles in the tangential direction is compared between the track n and the track (n + 1).
  • minute particles are detected in both the track n and the track (n + 1) in the angle region from ⁇ 3 to ⁇ 2, and this is an overlapping region.
  • the microparticles detected in the track n and the track (n + 1) are regarded as the same microparticles with an overlapping region.
  • the detection position of the disk 6 is moved by one track to determine the area where the microparticles exist, and the presence or absence of corresponding microparticles that can be determined as the same microparticles between adjacent tracks. Determine. When there is no corresponding microparticle that can be determined as the same microparticle, the microparticles that have been regarded as the same are counted as one microparticle.
  • the presence areas of the microparticles detected in the track (n + 1) and the track (n + 2) adjacent to each other are compared. In the angle region from ⁇ 9 to ⁇ 10, the existing regions of the microparticles detected in each track overlap. In such a case, in addition to the microparticles of the track n and the track (n + 1) that have been determined to be the same microparticles earlier, the microparticles detected in the track (n + 2) are further included from the track n. All the fine particles up to the track (n + 2) are determined to be the same fine particle b.
  • the presence areas of the microparticles detected in the track (n + 2) and the track (n + 3) are compared. Then, while the fine particles are detected in the angle region from ⁇ 9 to ⁇ 10 in the track (n + 2), the fine particles are not detected in the corresponding angle region from ⁇ 9 to ⁇ 10 in the track (n + 3). Therefore, at that time, the number of particles is counted with the minute particles detected from track n to track (n + 2) as one minute particle.
  • the region where the fine particles are present is determined. Then, the area of the microparticles is sequentially compared for each adjacent track, and if there is no corresponding microparticle that can be determined as the same microparticle between adjacent tracks, the number of particles as one microparticle at that time Count.
  • the same minute particle determination process described above can be performed if there is information on the existence area of minute particles between adjacent tracks, the detection of the existence area of minute particles for all tracks can be performed without ending. it can.
  • the microparticle existence area information after the comparison of the microparticle existence area may be sequentially deleted, the amount of data to be saved can be greatly reduced.
  • the sample 7 in which the fine particles are distributed is enclosed in a transparent and circular disk 6, and is rotated by accommodating the disk 6 in a holder 26 fixed to the central shaft 25. It is possible.
  • the present invention is not limited to this, and the sample 7 may be directly fixed to the surface of the glass stage fixed to the central shaft 25.
  • the PC40 of the microparticle detection apparatus acquires the position information of the microparticles.
  • fine particles are detected in each track.
  • the difference from the first embodiment in that case is that not only the microparticle existing area in each track is recorded in the internal memory of the PC 40 but also the representative coordinate information of each microparticle.
  • the representative coordinates are the coordinates on the rotation angle-track number coordinates shown in FIG. 3 of the start point or the end point in the microparticle existence region, or the coordinates of the intermediate point between the start point and the end point. is there. Note that all coordinates from the start point to the end point in the existence area may be recorded. In that case, the amount of data to be handled is increased, but more detailed information on the fine particles can be obtained.
  • the representative coordinates are set so that one representative coordinate corresponds to the microparticles determined to be the same.
  • each of the plurality of existence area information (angle area information) determined to be the same minute particle has unique representative coordinate information. Therefore, it is for avoiding that several representative coordinate information will respond
  • the existence area of the track n when it is determined by comparing the track n and the track (n + 1) that the microparticle existence area information detected in each track is based on the same microparticle, the existence area of the track n
  • the representative coordinates of the information are set to be the representative coordinates of the microparticles regarded as the same. Then, the representative coordinates of the existing area information detected in the track (n + 1) are deleted.
  • one representative coordinate can be associated with a minute particle that is regarded as the same.
  • the adjacent tracks are sequentially compared, and when there are no more microparticles corresponding to the adjacent tracks, they are counted as one microparticle,
  • the representative coordinates of the fine particles are set and recorded in the internal memory of the PC 40 or the like.
  • the configuration of the microparticle detection device in the present embodiment is the same as the schematic configuration of the microparticle detection device shown in FIG. 1 in the first embodiment. Therefore, a detailed description of the microparticle detection apparatus in this embodiment is omitted, and FIGS. 1 to 3 are used as appropriate in the following description.
  • the PC40 of the microparticle detection apparatus acquires information on the shape and size of the microparticles.
  • fine particles are detected in each track.
  • the detected microparticle existence regions are compared between adjacent tracks.
  • the difference from the first embodiment is that not only the microparticle existing area in each track is recorded in the internal memory of the PC 40 but also the shape and size of each microparticle is determined and recorded. That is.
  • the adjacent regions of the microparticles detected in the respective tracks are compared with each other to determine whether or not they are the same microparticles. Then, if it is determined by the above comparison that they are the same microparticles, the area obtained by adding the microparticle existence areas detected in the respective tracks is set as the same microparticle existence area. .
  • the adjacent tracks are sequentially compared, and when there are no more microparticles corresponding to the adjacent tracks, they are counted as one microparticle, Information on the shape and size of the microparticles is set and recorded in the internal memory of the PC 40 or the like.
  • the information on the shape is information on the shape of the entire region of the microparticles regarded as the same.
  • the size information is the size information obtained from the entire area of the microparticles, such as the area of the entire area of the microparticles, because the amount of data increases when the information about the shape is recorded in the internal memory or the like. Means the above-mentioned size information in the case of recording.
  • the method for counting the number of fine particles in the first embodiment is applied to a two-dimensional scanning method in which a sample or an optical module is two-dimensionally scanned in two directions orthogonal to each other.
  • a sample or an optical module is two-dimensionally scanned in two directions orthogonal to each other.
  • FIG. 4 and 5 show, as an example, a microparticle detection apparatus that scans in the X direction while moving the sample in the Y direction.
  • 4 is a perspective view showing the whole
  • FIG. 5 is a schematic sectional view of the optical module.
  • 1 is a light source device
  • 2 is an objective lens
  • 3 is a detection device.
  • the light source device 1, the objective lens 2, and the detection device 3 are housed in a frame to constitute the optical module 5.
  • a glass stage 29 is disposed above the optical module 5 so as to face the objective lens 2.
  • a transfer support such as the above is set as a sample (the specimen) 30.
  • the glass stage 29 has a rectangular shape, and scans in a two-dimensional direction of a first scanning direction in the long side direction and a second scanning direction in the short side direction orthogonal to the first scanning direction. It is configured.
  • the scanning method in that case is not particularly limited. In short, it is only necessary to include a first operation unit that reciprocates the glass stage 29 in the first scanning direction and a second operation unit that reciprocates in the second scanning direction.
  • the optical module 5 side may be scanned in a two-dimensional direction.
  • the semiconductor laser 8, the first lens 9, the spot size adjusting lens 10, the first aperture 11 and the prism 15 are the same as the first semiconductor laser 8, the first lens 9, and the like shown in FIG. This is exactly the same as the spot size adjusting lens 10, the first aperture 11, and the prism 15. Therefore, the same numbers are assigned and detailed description is omitted.
  • the bandpass filter 17, the second lens 18, the second aperture 19 and the detector 20 are the same as the bandpass filter 17, the third lens 18, the second aperture 19 and the first aperture shown in FIG. 1 in the first embodiment. It is exactly the same as the detector 20. Therefore, the same numbers are assigned and detailed description is omitted.
  • the central portion including the optical axis is a convex lens portion 2a having a function of a normal convex lens (light is deflected only by refraction). Then, the excitation light emitted from the semiconductor laser 8 and reflected by the prism 15 passes through the convex lens portion 2a and is converged toward the sample 30. Further, light having a small radiation angle out of scattered light or fluorescence emitted from the sample 30 passes through the convex lens portion 2a and is condensed toward the detector 20.
  • the periphery of the convex lens portion 2a in the objective lens 2 is a truncated cone-shaped cylindrical body 2b that opens downward. Then, of the light emitted from the sample 6, the fluorescence having a large radiation angle that does not enter the convex lens portion 2a is incident on the cylindrical body 2b from the upper end surface of the cylindrical body 2b, and the cylindrical body 2b. Are totally reflected by the outer peripheral surface of the light source, deflected toward the optical axis, and emitted toward the detector 20 from the inner peripheral surface and the lower end surface of the cylindrical body 2b.
  • the band-pass filter 17 is disposed, for example, in the rotary folder 31 and can be replaced with a filter of another wavelength according to the wavelength of the fluorescence.
  • the optical module 5 is fixed, the glass stage 29 on which the sample 30 is placed is scanned in a two-dimensional direction, and a distribution image of scattered light or fluorescence intensity is read. It has become.
  • the glass stage 29 is relatively moved relative to the optical module 5 in the first scanning direction (long-side direction) and in the second scanning direction (short-side direction) orthogonal to the first scanning direction.
  • a scanning locus of a condensing point of the excitation light is drawn on the sample 30 by the objective lens 2.
  • the scanning trajectory includes a plurality of linear scanning lines reciprocating in the first scanning direction (long side direction) at intervals, and the end points and starting points of the adjacent scanning lines in the second scanning direction (short side direction). ) Are connected continuously and repeatedly.
  • the plurality of scanning lines extending in the first scanning direction (long side direction) correspond to the track in the first embodiment. Therefore, the two-dimensional coordinate system of the scanning line number and the scanning position obtained in the present embodiment can be replaced with the two-dimensional coordinate system of the track number and the rotation angle ( ⁇ ) in the first embodiment.
  • the PC 40 that has received the signal from the detector 20 uses the two-dimensional coordinate system of the scanning line number and the scanning position to determine the track number and the rotation angle ( ⁇ ) in the first embodiment.
  • the determination of the microparticle existence area and the count of the microparticles performed on the two-dimensional coordinate system are performed.
  • the determination of the microparticle existence area and the count of the microparticles by the disk rotation type microparticle detection apparatus are applied. It is possible to prevent double counting of the same microparticles and to accurately count the number of microparticles.
  • microparticle determination process described above can be performed without ending the detection of the microparticle existence area for all scanning lines, and the microparticle existence area information after the comparison of the microparticle existence areas is as follows. Since data can be deleted sequentially, the amount of data to be saved can be greatly reduced.
  • the sample 30 is set on the glass stage 29.
  • the sample 30 is a transparent and rectangular disk. It does not matter even if it is enclosed inside.
  • the microparticle detection apparatus of the present invention is An irradiation optical system 1 that irradiates the specimens 7 and 30 including fine particles with the light emitted from the light sources 8 and 12; Optical detection optical systems 3 and 4 for detecting light emitted from the microparticles in the specimens 7 and 30 by the irradiation of the light; A detection unit that detects the microparticles based on the intensity of light from the microparticles detected by the light detection optical systems 3 and 4; The irradiation optical system 1 and the light detection optical systems 3 and 4 relatively two-dimensionally scan the specimens 7 and 30 in the first scanning direction and the second scanning direction that intersects the first scanning direction.
  • the detection unit is Presence of the minute particles for each scanning line based on the intensity of light from the minute particles when the irradiation optical system 1 and the light detection optical systems 3 and 4 are scanned in the first scanning direction. Determine the area, By comparing the existing areas of the microparticles in the scanning lines adjacent to each other, it is determined whether or not the set of the existing areas in the two scanning lines is caused by the same microparticle. It is characterized by.
  • the set of the existing regions is the same by a simple method of comparing the existing regions of the microparticles in the adjacent scanning lines. It is determined whether or not it is caused by fine particles.
  • the comparison itself may be a very simple process. And the comparison regarding all the scanning lines can be performed by sequentially performing the comparison with respect to the adjacent scanning lines.
  • the comparison process can be performed simultaneously with the detection of the region where the microparticles exist in each scanning line, and the microparticles can be counted efficiently.
  • the detection unit determines that the set of the existing regions in both the scan lines is caused by the same microparticle, the detection unit is adjacent to one of the scan lines and the scan line. By comparing the existing area of the microparticles in the new scanning line, it is determined whether the set of the existing areas in the new adjacent scanning line is caused by the same microparticle. Yes.
  • the scanning lines adjacent to each other for performing the comparison process are sequentially updated. Therefore, as long as there is data on the existence area in the scanning lines adjacent to each other to be compared, it is possible to determine the overlap of the fine particles with respect to all the scanning lines.
  • the detection unit When the positions in the first scanning direction overlap each other with respect to the existence area of the microparticles in the scanning lines adjacent to each other, the detection unit has the same combination of the existence areas in both the scanning lines. It is determined that the region is a region where microparticles exist.
  • microparticle existence areas in the scanning lines adjacent to each other overlap each other in the first scanning direction, it is determined that the microparticle existence areas are the same microparticle existence area. ing.
  • the detection unit In the case where the existence area determined to be caused by the same minute particle is present in the scanning lines adjacent to each other, the detection unit detects the same minute particle based on the existence area caused by the same minute particle. Position information is set.
  • the same microparticles in addition to the same microparticles straddling the scan lines adjacent to each other, based on the data of the existence area in the scan lines adjacent to each other to be compared, the same microparticles It becomes possible to set the position information of the particles.
  • the detection unit In the case where the existence area determined to be caused by the same minute particle is present in the scanning lines adjacent to each other, the detection unit detects the same minute particle based on the existence area caused by the same minute particle. Shape information or size information is set.
  • the same microparticles in addition to the same microparticles straddling the scan lines adjacent to each other, based on the data of the existence area in the scan lines adjacent to each other to be compared, the same microparticles It is also possible to set particle shape information or size information.

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Abstract

This microparticle detection device is provided with an irradiation optical system (1) which irradiates light from light sources (8, 10) onto a sample (7), light detection optical systems (3, 4) which detect light from microparticles in the sample (7), and a detection unit (40) which detects the microparticles on the basis of the intensity of light detected by the light detection optical systems (3, 4). The irradiation optical system (1) and the light detection optical systems (3, 4) scan the sample (7) relatively in two directions, a first scanning direction and a second scanning direction intersecting the first scanning direction. For each scanning line, the detection unit (40) determines microparticle regions in which microparticles are present on the basis of the intensity of light from the microparticles during scanning in the first scanning direction by the irradiation optical system (1) and the light detection optical system (3, 4), compares the microparticle regions on mutually adjacent scanning lines, and determines whether or not pairs of the microparticle regions on both scanning lines are due to the same microparticles.

Description

微小粒子検出装置Fine particle detector
　この発明は、微小粒子検出装置に関する。 The present invention relates to a microparticle detection apparatus.
　従来より、微小粒子検出装置として、液体中あるいはメンブレンやスライドガラス上に展開された微小粒子に光を照射し、上記微小粒子から発生する蛍光あるいは散乱光を検出して、粒子の計数あるいは性状検査を行うものがある。ここで、上記微小粒子としては、無機粒子、微生物、細胞、血液中の赤血球、白血球、血小板、血管内皮細胞、上記組織の微小細胞片等が含まれる。そして、上記微小粒子は、液体中にある場合には微小粒子懸濁液となる。 Conventionally, as a microparticle detection device, light is irradiated to microparticles developed in a liquid or on a membrane or slide glass, and fluorescence or scattered light generated from the microparticles is detected, and particle counting or property inspection is performed. There is something to do. Here, the fine particles include inorganic particles, microorganisms, cells, erythrocytes in blood, leukocytes, platelets, vascular endothelial cells, fine cell fragments of the tissue, and the like. The microparticles become a microparticle suspension when in the liquid.
　上記微小粒子の検出方法としては、フローサイトメーターとイメージサイトメーターとの２つの方式が知られている。 As a method for detecting the fine particles, two methods of a flow cytometer and an image cytometer are known.
　上記フローサイトメーターにおいては、上記微小粒子の懸濁液を毛細管にシース液と共に流す。そして、上記毛細管の一部にレーザー光を照射し、上記微小粒子に光が当たった時に生ずる散乱光あるいは蛍光を検出することによって、粒子の種類や粒子の大きさを分類する。例えば、特定の粒子と結合する蛍光試薬で粒子を標識することによって、蛍光を発する粒子の数を計数して上記特定の粒子のみを計数することができる。このフローサイトメーターでは、サブミクロンから１０μｍ程度の粒子の検出が可能であり、高精度な検出が可能である。 In the flow cytometer, the fine particle suspension is flowed into the capillary together with the sheath liquid. Then, the type of particle and the size of the particle are classified by irradiating a part of the capillary with laser light and detecting scattered light or fluorescence generated when the fine particle is irradiated with light. For example, by labeling particles with a fluorescent reagent that binds to specific particles, the number of fluorescent particles can be counted to count only the specific particles. With this flow cytometer, particles of about submicron to 10 μm can be detected, and highly accurate detection is possible.
　しかしながら、上記サブミクロンの粒子まで測定可能なフローサイトメーターでは、装置が大型であって高価なシステムとなっている。 However, in the flow cytometer capable of measuring even the sub-micron particles, the apparatus is large and expensive.
　一方、上記イメージサイトメーターにおいては、粒子の画像を測定し、画像の情報から粒子の種類や大きさを判定する。画像化方法として、顕微鏡とデジタルカメラとによる撮像や、光学ヘッドを二次元的にスキャンしながら散乱光あるいは蛍光を検出し、画像化する方法がある。 On the other hand, in the above image cytometer, the image of the particle is measured, and the type and size of the particle are determined from the image information. Imaging methods include imaging with a microscope and a digital camera, and methods of detecting scattered light or fluorescence while scanning an optical head two-dimensionally and imaging.
　上記顕微鏡とデジタルカメラとによる撮像の場合には、１μｍ以上の粒子では高精度な画像測定が可能であるが、サブミクロンの粒子を測定する場合には、高倍率な対物レンズを有する顕微鏡と高感度な(つまり、低ノイズでダイナミックレンジの幅が広い)デジタルカメラとが必要になるので、非常に高価なシステムとなる。また、サブミクロン粒子の場合、光の波長と粒子のサイズとが同等になるので回折限界によって結像性能が低下し、粒子サイズの正確な判定が困難になる。 In the case of imaging with the above-mentioned microscope and digital camera, high-precision image measurement is possible with particles of 1 μm or more, but when measuring submicron particles, a microscope with a high-magnification objective lens and a high-power lens are used. A sensitive digital camera (that is, low noise and wide dynamic range) is required, which makes the system very expensive. In the case of submicron particles, the wavelength of light and the size of the particles are equivalent, so that the imaging performance is degraded due to the diffraction limit, making it difficult to accurately determine the particle size.
　さらに、上記顕微鏡として蛍光顕微鏡システムを用いれば、容易に粒子を検出することが可能である。ところが、同様に、サブミクロン粒子の場合に光の波長と粒子のサイズとが同等になるので、粒子サイズの正確な判定ができない。また、微小粒子からの蛍光は微弱なため、高感度なデジタルカメラが必要となる。 Furthermore, if a fluorescence microscope system is used as the microscope, particles can be easily detected. However, similarly, in the case of submicron particles, the wavelength of light is equal to the size of the particles, and thus the particle size cannot be accurately determined. Further, since the fluorescence from the minute particles is weak, a highly sensitive digital camera is required.
　これに対して、光学ヘッドをスキャンしながら散乱光あるいは蛍光を検出するシステムにおいては、光学ヘッドからレーザー光を粒子に集光して照射し、粒子から発生する散乱光あるいは蛍光を検出しながら、上記光学ヘッドを二次元的に走査して画像化する。 On the other hand, in a system that detects scattered light or fluorescence while scanning the optical head, the laser light from the optical head is focused on the particle and irradiated, and while detecting scattered light or fluorescence generated from the particle, The optical head is scanned two-dimensionally to form an image.
　このように、上記光学ヘッドを上記サンプルに対して相対的にスキャンしながら光を検出するシステムにおいては、サブミクロンの粒子を検出する場合、レーザー光の照射スポット径は、粒子サイズと同等かそれ以上に大きくなる。そのために、二次元スキャンの結果得られる画像は、粒子の一つ一つが解像された画像になっていないので、画像から直接粒子の大きさを計測することは困難である。ところが、上記レーザー光の照射スポットが粒子サイズよりも大きくても、粒子サイズによって粒子から生じる散乱光強度が異なるので、散乱光の強さから粒子径を判定することは可能である。その理由は、粒子径と散乱光強度とに相関があるためである。 Thus, in a system that detects light while scanning the optical head relative to the sample, when detecting submicron particles, the laser spot diameter is equal to or smaller than the particle size. More than that. For this reason, the image obtained as a result of the two-dimensional scan is not an image in which each particle is resolved, and it is difficult to directly measure the size of the particle from the image. However, even if the laser beam irradiation spot is larger than the particle size, the intensity of the scattered light generated from the particle varies depending on the particle size, so that the particle diameter can be determined from the intensity of the scattered light. The reason is that there is a correlation between the particle diameter and the scattered light intensity.
　その場合、散乱光を高感度に検出する検出器(低ノイズで広ダイナミックレンジな検出器)とレーザー光源とが必要になるが、高倍率な対物レンズを有する顕微鏡と高感度なデジタルカメラとを用いるシステムに比較して、安価なシステム構成が可能である。 In that case, a detector that detects scattered light with high sensitivity (a detector with low noise and a wide dynamic range) and a laser light source are required, but a microscope with a high-magnification objective lens and a high-sensitivity digital camera are required. Compared to the system to be used, an inexpensive system configuration is possible.
　しかしながら、上記従来の光学ヘッドをサンプルに対して相対的にスキャンしながら光を検出するシステムにおいては、以下のような問題がある。 However, the system that detects light while scanning the conventional optical head relative to the sample has the following problems.
　すなわち、上記二次元スキャンを行ってサンプルからの散乱光を検出するシステムにおいては、検出の感度や分解能を高めるためには照射スポットを集光させて小さくする必要がある。ＬＥＤ(Light Emitting Diode：発光ダイオード)やランプ光源では十分に集光できないため、レーザー光源を用いる必要がある。上記レーザー光源の場合、半導体レーザーが小型で安価であり、有利である。ところが、波長帯によっては、高価になる場合や、上記半導体レーザーと比較して体積が大きい固体レーザーしか得られない場合がある。そのために、波長帯によっては高価なシステムになる場合がある。 That is, in the system that detects the scattered light from the sample by performing the above-described two-dimensional scan, it is necessary to condense and reduce the irradiation spot in order to increase the sensitivity and resolution of detection. It is necessary to use a laser light source because light cannot be sufficiently collected by an LED (Light Emitting Diode) or a lamp light source. In the case of the laser light source, a semiconductor laser is advantageous because it is small and inexpensive. However, depending on the wavelength band, it may be expensive or only a solid laser having a volume larger than that of the semiconductor laser may be obtained. Therefore, depending on the wavelength band, there may be an expensive system.
　粒子画像を取得する場合には、検出対象物がμｍオーダーで小さいために、サンプル上の蛍光や散乱光の情報を狭ピッチで取得する必要がある。さらに、装置としての現実的な測定時間(数分程度)を考えると、サンプリング周波数を高くせざるを得ない。上述のような二次元スキャン方式に対して、ディスク回転方式は、大幅に測定時間を短縮できるものの、依然として測定時間が長くなることが課題となる。また、画像化を行うのは一般にパソコン等のモニターであるため、大量の取得データを高速で上記パソコン等に送信する必要もある。 When acquiring a particle image, it is necessary to acquire information on fluorescence and scattered light on the sample at a narrow pitch because the detection target is small on the order of μm. Furthermore, considering a realistic measurement time (about several minutes) as an apparatus, the sampling frequency must be increased. In contrast to the two-dimensional scanning method as described above, the disk rotation method can greatly reduce the measurement time, but the measurement time is still long. Further, since it is generally a monitor such as a personal computer that performs imaging, it is necessary to transmit a large amount of acquired data to the personal computer or the like at high speed.
　粒子画像の取得と並行して粒子数をカウントする場合には、処理データはさらに膨大なものになる。 If the number of particles is counted in parallel with the acquisition of the particle image, the processing data will be enormous.
　上記粒子カウントを行う際には、散乱光または蛍光を読み取って二値化処理を行う。その際に、バックグラウンドやノイズと検出対象粒子とを、散乱光または蛍光の輝度が閾値よりも高いか低いかによって判別し、輝度が閾値よりも高い箇所をカウントすることによって上記粒子カウントを行うのである。 When performing the above particle count, binarization processing is performed by reading scattered light or fluorescence. At that time, the background and noise and the detection target particle are discriminated based on whether the brightness of scattered light or fluorescence is higher or lower than the threshold value, and the particle count is performed by counting the places where the brightness is higher than the threshold value. It is.
　尚、上記ディスク回転方式によって粒子画像を取得する場合には、別トラックで同一粒子を重複して検出するために、データ量が増大する。このように、上記ディスク回転方式によって粒子カウントを行う方法として、特開２００４‐３２５０９１号公報(特許文献１)に開示されたセルカウント方法がある。 In addition, when acquiring a particle image by the said disk rotation system, since the same particle is detected in another track | truck repeatedly, data amount increases. As described above, there is a cell counting method disclosed in Japanese Patent Application Laid-Open No. 2004-325091 (Patent Document 1) as a method of performing particle counting by the disk rotation method.
　上記特許文献１に開示された従来のセルカウント方法においては、読み取った散乱光または蛍光の二値化画像に対して、粒子検出ウインドウとサイズ判別ウインドウとを有する走査ウインドウを用いた走査を行って、セルのサイズ別にセルの個数をカウントするようにしている。 In the conventional cell counting method disclosed in Patent Document 1, the scanned scattered light or fluorescent binary image is scanned using a scanning window having a particle detection window and a size discrimination window. The number of cells is counted for each cell size.
　したがって、上記従来のセルカウント方法によれば、粒子検出ウインドウとサイズ判別ウインドウに分けて、同一粒子を重複してカウントしないようにすることによって、粒子のサイズ毎にカウントすることが可能になる。 Therefore, according to the conventional cell counting method, it is possible to count for each particle size by dividing into a particle detection window and a size determination window so that the same particles are not counted repeatedly.
　その反面、一旦、全体のマップデータを取得する必要があるためデータ量が増大する。さらに、上記走査ウインドウを作用させて粒子検出およびサイズ判別を行うので演算処理の負担が大きくなってしまう。そのために、必要なデータ量やデータ処理の負荷が大きくなるという問題がある。 On the other hand, the amount of data increases because it is necessary to obtain the entire map data once. Furthermore, since the particle size is detected and the size is discriminated by operating the scanning window, the burden of calculation processing becomes large. Therefore, there is a problem that a necessary data amount and a data processing load increase.
特開２００４‐３２５０９１号公報JP 2004-325091 A
　そこで、この発明の課題は、微小粒子を含む検体から微小粒子を検出してカウントを行う際におけるデータ量を削減できる微小粒子検出装置を提供することにある。 Therefore, an object of the present invention is to provide a microparticle detection apparatus capable of reducing the amount of data when counting is performed by detecting microparticles from a specimen containing microparticles.
　上記課題を解決するため、この発明の微小粒子検出装置は、
　光源から出射された光を、微小粒子を含む検体に対して照射する照射光学系と、
　上記光の照射によって上記検体中の上記微小粒子から発せられた光を検出する光検出光学系と、
　上記光検出光学系によって検出された上記微小粒子からの光の強度に基づいて、上記微小粒子を検出する検出部と
を備え、
　上記照射光学系および上記光検出光学系は、第１走査方向とこの第１走査方向に交差する第２走査方向とに、上記検体を相対的に二次元走査するようになっており、
　上記検出部は、
　上記照射光学系および上記光検出光学系が上記第１走査方向に走査した場合の上記微小粒子からの光の強度に基づいて、各走査ライン毎に、上記微小粒子が存在する存在領域を判定し、
　互いに隣接する上記走査ラインにおける上記微小粒子の存在領域を比較することによって、上記両走査ラインに在る上記存在領域の組みが同一微小粒子に起因するか否かを判定する
ようになっている
ことを特徴としている。 In order to solve the above problems, a microparticle detection apparatus according to the present invention includes:
An irradiation optical system for irradiating a specimen containing fine particles with light emitted from a light source;
A light detection optical system for detecting light emitted from the microparticles in the specimen by the light irradiation;
A detection unit that detects the microparticles based on the intensity of light from the microparticles detected by the photodetection optical system;
The irradiation optical system and the light detection optical system are configured to relatively two-dimensionally scan the specimen in a first scanning direction and a second scanning direction that intersects the first scanning direction,
The detection unit is
Based on the intensity of light from the microparticles when the irradiation optical system and the light detection optical system scan in the first scanning direction, an existence region where the microparticles are present is determined for each scanning line. ,
By comparing the existing areas of the microparticles in the scanning lines adjacent to each other, it is determined whether or not the set of the existing areas in the two scanning lines is caused by the same microparticle. It is characterized by.
　ここで、上記「微小粒子が存在する存在領域」とは、各走査ラインにおいて、上記微小粒子からの光の強度が閾値を超えている区間の始点と終点に挟まれた領域のことを言う。 Here, the “existing region where the microparticles exist” refers to a region sandwiched between the start point and the end point of the section where the intensity of light from the microparticles exceeds the threshold in each scanning line.
　また、一実施の形態の微小粒子検出装置では、
　上記検出部は、上記両走査ラインに在る上記存在領域の組みが同一微小粒子に起因すると判定した場合には、上記両走査ラインの何れか一方の走査ラインと、当該の走査ラインに隣接する新たな走査ラインとにおける、上記微小粒子の存在領域を比較することによって、上記新たな隣接走査ラインに在る上記存在領域の組みが同一微小粒子に起因するか否かを判定するようになっている。 Moreover, in the microparticle detection apparatus of one embodiment,
When the detection unit determines that the set of the existing regions in both the scan lines is caused by the same microparticle, the detection unit is adjacent to one of the scan lines and the scan line. By comparing the existing area of the microparticles in the new scanning line, it is determined whether the set of the existing areas in the new adjacent scanning line is caused by the same microparticle. Yes.
　また、一実施の形態の微小粒子検出装置では、
　上記検出部は、互いに隣接する上記走査ラインにおける上記微小粒子の存在領域に関して、上記第１走査方向の位置が互いに重なっている場合には、上記両走査ラインに在る上記存在領域の組みは同一の微小粒子の存在領域であると判定するようになっている。 Moreover, in the microparticle detection apparatus of one embodiment,
When the positions in the first scanning direction overlap each other with respect to the existence area of the microparticles in the scanning lines adjacent to each other, the detection unit has the same combination of the existence areas in both the scanning lines. It is determined that the region is a region where microparticles exist.
　また、一実施の形態の微小粒子検出装置では、
　上記検出部は、互いに隣接する上記走査ラインに、同一微小粒子に起因すると判定された上記存在領域が在る場合には、上記同一微小粒子に起因する上記存在領域に基づいて上記同一微小粒子の位置情報を設定するようになっている。 Moreover, in the microparticle detection apparatus of one embodiment,
In the case where the existence area determined to be caused by the same minute particle is present in the scanning lines adjacent to each other, the detection unit detects the same minute particle based on the existence area caused by the same minute particle. Position information is set.
　また、一実施の形態の微小粒子検出装置では、
　上記検出部は、互いに隣接する上記走査ラインに、同一微小粒子に起因すると判定された上記存在領域が在る場合には、上記同一微小粒子に起因する上記存在領域に基づいて上記同一微小粒子の形状情報またはサイズ情報を設定するようになっている。 Moreover, in the microparticle detection apparatus of one embodiment,
In the case where the existence area determined to be caused by the same minute particle is present in the scanning lines adjacent to each other, the detection unit detects the same minute particle based on the existence area caused by the same minute particle. Shape information or size information is set.
　以上より明らかなように、この発明の微小粒子検出装置は、
　上記第１走査方向に走査した場合の各走査ラインに関し、互いに隣接する上記走査ラインにおける上記微小粒子の存在領域を比較するという単純な方法によって、上記存在領域の組みが同一微小粒子に起因するか否かを判定するようにしている。 As is clear from the above, the microparticle detection apparatus of the present invention is
For each scanning line when scanned in the first scanning direction, whether the group of the existing regions is caused by the same minute particles by a simple method of comparing the existing regions of the minute particles in the adjacent scanning lines. It is determined whether or not.
　したがって、上記判定には、比較の対象となる上記互いに隣接する走査ラインにおける上記存在領域のデータさえあればよく、全体のマップデータは必ずしも必要ではない。また、比較自体も非常に簡単な処理でよい。そして、上記比較を、隣接する上記走査ラインに対して順次行うことによって、全走査ラインに関する比較行うことができる。 Therefore, for the determination, it is only necessary to have the data of the existing area in the scanning lines adjacent to each other to be compared, and the entire map data is not necessarily required. Further, the comparison itself may be a very simple process. And the comparison regarding all the scanning lines can be performed by sequentially performing the comparison with respect to the adjacent scanning lines.
　すなわち、この発明によれば、上述の比較処理時のデータ量や演算負荷を大幅に軽減することができる。さらに、その上に、各走査ラインにおける上記微小粒子の存在領域の検出と同時に上記比較処理を行うことができ、効率的に微小粒子のカウントを行うことが可能になる。 That is, according to the present invention, it is possible to greatly reduce the amount of data and the calculation load during the comparison process described above. In addition, the comparison process can be performed simultaneously with the detection of the region where the microparticles exist in each scanning line, and the microparticles can be counted efficiently.
この発明の微小粒子検出装置における概略構成を示す図である。It is a figure which shows schematic structure in the microparticle detection apparatus of this invention. ディスク上における各トラックと微小粒子との関係を示す模式図である。It is a schematic diagram which shows the relationship between each track | truck and fine particle on a disk. 微小粒子の位置をトラック番号と回転角度の二次元座標で表した図である。It is the figure which represented the position of the microparticle by the two-dimensional coordinate of the track number and the rotation angle. 図１とは異なる微小粒子検出装置の概略構成を示す図である。It is a figure which shows schematic structure of the microparticle detection apparatus different from FIG. 図４における光学モジュールの断面概略図である。It is a cross-sectional schematic diagram of the optical module in FIG.
　以下、この発明を図示の実施の形態により詳細に説明する。 Hereinafter, the present invention will be described in detail with reference to embodiments shown in the drawings.
　・第１実施の形態
　図１は、本実施の形態の微小粒子検出装置の概略構成を示す図である。この微小粒子検出装置は、検体が注入されたディスクと、ディスクを回転させる回転駆動系と、散乱光または蛍光を検出する光検出光学系と、上記検出光学系を半径方向に駆動させる駆動機構と、上記光検出光学系からの信号を受けて上記微小粒子を検出する検出部とから、概略構成されている。 -1st Embodiment FIG. 1: is a figure which shows schematic structure of the microparticle detection apparatus of this Embodiment. The microparticle detection apparatus includes a disk into which a specimen is injected, a rotation drive system that rotates the disk, a light detection optical system that detects scattered light or fluorescence, and a drive mechanism that drives the detection optical system in a radial direction. And a detection unit that receives the signal from the optical detection optical system and detects the fine particles.
　図１において、１は光源装置、２は対物レンズ、３は第１検出装置、４は第２検出装置である。光源装置１と対物レンズ２と第１検出装置３と第２検出装置４は、枠体内に収納されて光学モジュール５を構成している。そして、光学モジュール５の上方には対物レンズ２に対向して円形のディスク６が配置され、ディスク６内には例えば蛍光物質によって標識された微小粒子が分布する懸濁液やゲル支持体やメンブレン等の転写支持体がサンプル(上記検体)７として封入されている。 In FIG. 1, 1 is a light source device, 2 is an objective lens, 3 is a first detection device, and 4 is a second detection device. The light source device 1, the objective lens 2, the first detection device 3, and the second detection device 4 are housed in a frame to constitute an optical module 5. A circular disk 6 is disposed above the optical module 5 so as to face the objective lens 2. In the disk 6, for example, a suspension, a gel support, or a membrane in which fine particles labeled with a fluorescent substance are distributed. Or the like is encapsulated as a sample (the specimen) 7.
　上記光学モジュール５の光源装置１には、光源としての第１半導体レーザー８が設けられており、第１半導体レーザー８の光軸上には、第１レンズ９,スポットサイズ調整レンズ１０および第１アパーチャ１１をこの順で配置している。さらに、第１半導体レーザー８に加えて、第１半導体レーザー８から出射されるレーザー光の第１波長とは異なる、第２波長のレーザー光を出射する第２半導体レーザー１２を配置している。さらに、第２半導体レーザー１２からのレーザー光を平行光化する第２レンズ１３を配置している。そして、第１半導体レーザー８の光軸と第２半導体レーザー１２の光軸との交差位置には、第１波長のレーザー光を透過する一方、第２波長のレーザー光を反射する第１ダイクロイックミラー１４を配置している。ここで、第１半導体レーザー８,第１レンズ９,スポットサイズ調整レンズ１０,第１アパーチャ１１,第２半導体レーザー１２,第２レンズ１３および第１ダイクロイックミラー１４は、一つのケース内に収納されて上記照射光学系の一例である光源装置１を構成している。 The light source device 1 of the optical module 5 is provided with a first semiconductor laser 8 as a light source. On the optical axis of the first semiconductor laser 8, a first lens 9, a spot size adjusting lens 10, and a first The apertures 11 are arranged in this order. Further, in addition to the first semiconductor laser 8, a second semiconductor laser 12 that emits laser light having a second wavelength different from the first wavelength of laser light emitted from the first semiconductor laser 8 is disposed. Further, a second lens 13 for collimating the laser beam from the second semiconductor laser 12 is disposed. A first dichroic mirror that transmits the laser light having the first wavelength and reflects the laser light having the second wavelength is transmitted to the intersection between the optical axis of the first semiconductor laser 8 and the optical axis of the second semiconductor laser 12. 14 is arranged. Here, the first semiconductor laser 8, the first lens 9, the spot size adjusting lens 10, the first aperture 11, the second semiconductor laser 12, the second lens 13 and the first dichroic mirror 14 are accommodated in one case. The light source device 1 which is an example of the irradiation optical system is configured.
　さらに、上記第１半導体レーザー８の光軸上には、第１ダイクロイックミラー１４を透過した光を、対物レンズ２側に向かうように反射させるプリズム１５を配置している。また、プリズム１５によって反射された光と対物レンズ２の光軸との交差位置には、プリズム１５からの光を対物レンズ２に入射するように反射させる第２ダイクロイックミラー１６を配置している。ここで、第２ダイクロイックミラー１６は、サンプル７からの蛍光を透過する一方、散乱光を反射する。 Furthermore, on the optical axis of the first semiconductor laser 8, a prism 15 that reflects the light transmitted through the first dichroic mirror 14 toward the objective lens 2 is disposed. Further, a second dichroic mirror 16 that reflects the light from the prism 15 so as to enter the objective lens 2 is disposed at the intersection of the light reflected by the prism 15 and the optical axis of the objective lens 2. Here, the second dichroic mirror 16 transmits the fluorescence from the sample 7 and reflects the scattered light.
　尚、この発明で言うところの「散乱光」とは、第１半導体レーザー８または第２半導体レーザー１２から出射された光が、サンプル７の照射箇所から周囲に等方的に散乱された光であり、出射光と同じ波長の光である。これに対し、「蛍光」とは、第１半導体レーザー８または第２半導体レーザー１２から出射された光がサンプル７を照射して微小粒子を標識している蛍光物質を励起し、サンプル７の照射箇所から周囲に等方的に散乱された蛍光であり、出射光とは異なる波長の光である。 The “scattered light” referred to in the present invention is light in which the light emitted from the first semiconductor laser 8 or the second semiconductor laser 12 is isotropically scattered from the irradiated position of the sample 7 to the surroundings. Yes, the light has the same wavelength as the emitted light. On the other hand, “fluorescence” means that the light emitted from the first semiconductor laser 8 or the second semiconductor laser 12 irradiates the sample 7 to excite the fluorescent substance labeling the microparticles, and irradiates the sample 7. It is fluorescence scattered isotropically from a location to the surroundings, and is light having a wavelength different from that of outgoing light.
　ここで、詳述はしないが、上記対物レンズ２はレンズホルダ(図示せず)に格納されており、ステッピングモータ等の駆動部(図示せず)によって光軸方向に移動されて、焦点位置を変更可能になっている。また、スポットサイズ調整レンズ１０はレンズホルダ(図示せず)に格納されており、駆動部(図示せず)によって光軸方向に移動されて、スポットサイズを調整可能になっている。 Here, although not described in detail, the objective lens 2 is stored in a lens holder (not shown), and is moved in the optical axis direction by a drive unit (not shown) such as a stepping motor, so that the focal position is adjusted. It can be changed. The spot size adjustment lens 10 is stored in a lens holder (not shown), and is moved in the optical axis direction by a drive unit (not shown) so that the spot size can be adjusted.
　また、図１において、上記対物レンズ２の光軸上における第２ダイクロイックミラー１６の下方には、第２ダイクロイックミラー１６側から順に、対物レンズ２によって集光されて平行光に変換されたサンプル７からの光(蛍光とは異なる波長の光)を減光するバンドパスフィルタ１７、バンドパスフィルタ１７を通過した蛍光を集光する第３レンズ１８、および、第３レンズ１８を通過した蛍光の迷光をカットする第２アパーチャ１９が配置されている。さらに、対物レンズ２の光軸上における第２アパーチャ１９の下方には、第２アパーチャ１９を通過した蛍光を検出する光電子増倍管(ＰＭＴ)等の検出素子を含む第１検出器２０が配置されている。ここで、第２アパーチャ１９と第１検出器２０とは、一つのケース内に収納されて上記光検出光学系の一例である第１検出装置３を構成している。 Further, in FIG. 1, a sample 7 that is condensed by the objective lens 2 and converted into parallel light is sequentially from the second dichroic mirror 16 side below the second dichroic mirror 16 on the optical axis of the objective lens 2. Bandpass filter 17 that attenuates light (light having a wavelength different from that of fluorescence), a third lens 18 that collects fluorescence that has passed through bandpass filter 17, and stray light of fluorescence that has passed through third lens 18 The 2nd aperture 19 which cuts is arranged. Furthermore, a first detector 20 including a detection element such as a photomultiplier tube (PMT) that detects fluorescence that has passed through the second aperture 19 is disposed below the second aperture 19 on the optical axis of the objective lens 2. Has been. Here, the second aperture 19 and the first detector 20 are housed in one case and constitute a first detection device 3 which is an example of the light detection optical system.
　上記プリズム１５と第２ダイクロイックミラー１６とを結ぶ光軸上におけるプリズム１５の図１中の左方には、プリズム１５側から順に、対物レンズ２により集光されて平行光に変換されたサンプル７からの散乱光を減光するＮＤ(減光)フィルタ２１、ＮＤフィルタ２１を通過した散乱光を集光する第４レンズ２２、および、第４レンズ２２を通過した散乱光の迷光をカットする第３アパーチャ２３が配置されている。さらに、第３アパーチャ２３の左方には、第３アパーチャ２３を通過した散乱光を検出する上記ＰＭＴ等の検出素子を含む第２検出器２４が配置されている。ここで、第３アパーチャ２３および第２検出器２４は、一つのケース内に収納されて上記光検出光学系の一例である第２検出装置４を構成している。尚、ダイクロイックミラーを追加して、上記検出装置を３つ以上としても良い。 On the left side of the prism 15 in FIG. 1 on the optical axis connecting the prism 15 and the second dichroic mirror 16, the sample 7 condensed by the objective lens 2 and converted into parallel light in order from the prism 15 side. ND (attenuating) filter 21 that attenuates scattered light from the ND filter, a fourth lens 22 that condenses the scattered light that has passed through the ND filter 21, and a first lens that cuts stray light from the scattered light that has passed through the fourth lens 22. Three apertures 23 are arranged. Further, on the left side of the third aperture 23, a second detector 24 including a detection element such as the PMT for detecting scattered light that has passed through the third aperture 23 is disposed. Here, the 3rd aperture 23 and the 2nd detector 24 are stored in one case, and constitute the 2nd detection device 4 which is an example of the above-mentioned optical detection optical system. Note that three or more detection devices may be added by adding dichroic mirrors.
　尚、上記構成においては、上記第１半導体レーザー８および第２半導体レーザー１２の複数の光源を搭載しているが、必ずしも複数の光源が必要ではない。 In addition, in the said structure, although the several light source of the said 1st semiconductor laser 8 and the 2nd semiconductor laser 12 is mounted, a several light source is not necessarily required.
　上記ディスク６は透明に且つ円形に構成されており、中心軸２５に固定された円形の皿状のホルダ２６に収容されて、中心軸２５に対して固定されている。中心軸２５は、上記回転駆動系の一例としてのスピンドルモータ２７で回転可能になっている。これに対し、光学モジュール５は、ディスク６が成す円板の半径方向に、上記駆動機構によって段階的に移動可能になっている。尚、光学モジュール５の上記駆動機構については特に限定するものではない。例えば、光学モジュール５の枠体を、ステッピングモータ等で上記半径方向に往復動されるタイミングベルト等により、上記半径方向に配設されたガイドレールで案内されて、移行可能に構成する。 The disk 6 is configured to be transparent and circular, and is accommodated in a circular dish-shaped holder 26 fixed to the central shaft 25 and fixed to the central shaft 25. The central shaft 25 can be rotated by a spindle motor 27 as an example of the rotational drive system. On the other hand, the optical module 5 can be moved stepwise by the drive mechanism in the radial direction of the disk formed by the disk 6. The drive mechanism of the optical module 5 is not particularly limited. For example, the frame of the optical module 5 is configured to be movable by being guided by the guide rail disposed in the radial direction by a timing belt or the like reciprocated in the radial direction by a stepping motor or the like.
　上記ディスク６は、上述したように、円形に形成されている。そして、ディスク６が一回転する毎に、光学モジュール５は１ステップ移動するように設定するのである。但し、円板状のディスク６について、上述の動作をディスク６を連続回転させながら行うには、ディスク６に一定幅を有して半径方向に延在する帯状の非検出領域を設定し、対物レンズ２からの励起光のスポットが上記非検出領域を移動する間に光学モジュール５を１ステップ移動させる必要がある。 The disk 6 is formed in a circular shape as described above. The optical module 5 is set to move one step each time the disk 6 makes one revolution. However, for the disc-like disc 6, in order to perform the above-described operation while the disc 6 is continuously rotated, a band-like non-detection area having a certain width and extending in the radial direction is set on the disc 6 and the objective is set. It is necessary to move the optical module 5 by one step while the spot of the excitation light from the lens 2 moves in the non-detection region.
　尚、得られた蛍光画像を解析する際に支障がなければ、ディスク６を連続回転させながら、光学モジュール５を連続的に移動させて、螺旋状に走査するようにしても構わない。 If there is no problem in analyzing the obtained fluorescent image, the optical module 5 may be continuously moved while the disk 6 is continuously rotated to scan in a spiral manner.
　上記構成において、光検出を行う際には、上記ディスク６を回転させながら光学モジュール５をディスク６の半径方向に移動させて、サンプル７内の微小粒子からの蛍光または散乱光の検出を行う。 In the above configuration, when detecting light, the optical module 5 is moved in the radial direction of the disk 6 while rotating the disk 6 to detect fluorescence or scattered light from the microparticles in the sample 7.
　以下、一例として、上記散乱光を検出する散乱光検出の場合を例に挙げて説明する。 Hereinafter, as an example, the case of scattered light detection for detecting the scattered light will be described as an example.
　上記光源装置１の第１半導体レーザー８および第２半導体レーザー１２のうち、第２ダイクロイックミラー１６で反射される波長のレーザー光を出射する方の半導体レーザー、例えば第１半導体レーザー８から第１波長のレーザー光を出射させる。 Of the first semiconductor laser 8 and the second semiconductor laser 12 of the light source device 1, a semiconductor laser emitting a laser beam having a wavelength reflected by the second dichroic mirror 16, for example, the first wavelength from the first semiconductor laser 8. The laser beam is emitted.
　上記第１半導体レーザー８から出射された励起光(レーザー光)は、第１レンズ９,スポットサイズ調整レンズ１０および第１アパーチャ１１で収束され、第１ダイクロイックミラー１４を透過する。次いで、プリズム１５および第２ダイクロイックミラー１６によって反射され、対物レンズ２およびディスク６を通過して、サンプル７における下面上の一点に集光される。その場合、プリズム１５の長手方向(水平方向)の長さは短く、上記長手方向に直交する方向の幅は狭くなっており、第１半導体レーザー８からの励起光は対物レンズ２の光軸付近(励起光透過部)のみを通過するようになっている。こうして、サンプル７中の微小粒子に集束光が照射されると、上記集束光が照射された部分から周囲に等方的に散乱された散乱光が生ずる。 The excitation light (laser light) emitted from the first semiconductor laser 8 is converged by the first lens 9, the spot size adjusting lens 10 and the first aperture 11, and passes through the first dichroic mirror 14. Next, the light is reflected by the prism 15 and the second dichroic mirror 16, passes through the objective lens 2 and the disk 6, and is collected at one point on the lower surface of the sample 7. In that case, the length of the prism 15 in the longitudinal direction (horizontal direction) is short, the width in the direction orthogonal to the longitudinal direction is narrow, and the excitation light from the first semiconductor laser 8 is near the optical axis of the objective lens 2. It passes through only (excitation light transmission part). Thus, when the focused light is irradiated to the fine particles in the sample 7, scattered light isotropically scattered from the portion irradiated with the focused light to the surroundings.
　上記散乱光は、上記サンプル７における上記集束光が照射された部分から周囲に等方的に出射される。そして、出射された散乱光のうちのディスク６を透過して対物レンズ２に入射した成分が、対物レンズ２を通過し、第２ダイクロイックミラー１６によって反射され、ＮＤフィルタ２１,第４レンズ２２および第３アパーチャ２３を通過して、第２検出器２４によって検出される。そして、第２検出器２４で検出された信号は、内蔵されるＡＤ変換器等によってＡＤ変換等の処理が施された後に、上記検出部の一例であるＰＣ(パーソナルコンピュータ)４０等へ送出される。こうして、サンプル７上の各測定点での蛍光強度の分布が内部メモリ等に記録される。また、検出信号に基づいて粒子カウントを行った場合には、粒子カウントデータが上記内部メモリ等に記録される。 The scattered light is isotropically emitted from the portion of the sample 7 irradiated with the focused light to the surroundings. The component of the emitted scattered light that has passed through the disk 6 and entered the objective lens 2 passes through the objective lens 2 and is reflected by the second dichroic mirror 16, and the ND filter 21, the fourth lens 22, and the like. It passes through the third aperture 23 and is detected by the second detector 24. The signal detected by the second detector 24 is subjected to processing such as AD conversion by a built-in AD converter and the like, and then sent to a PC (personal computer) 40, which is an example of the detection unit. The Thus, the fluorescence intensity distribution at each measurement point on the sample 7 is recorded in the internal memory or the like. Further, when the particle count is performed based on the detection signal, the particle count data is recorded in the internal memory or the like.
　上記第３アパーチャ２３は、空間的な迷光をカットするために配置されている。また、共焦点アパーチャとしても機能しており、サンプル７が存在する面以外からの不必要な反射光や迷光を除去する。例えば、ディスク６の面やレンズ面で発生した反射光は対物レンズ２の焦点位置からずれているので、対物レンズ２の後段に続く光学系によって第３アパーチャ２３の位置で広がった光となり、効率よく第３アパーチャ２３を透過することができない。 The third aperture 23 is arranged to cut spatial stray light. It also functions as a confocal aperture and removes unnecessary reflected light and stray light from areas other than the surface where the sample 7 exists. For example, since the reflected light generated on the surface of the disk 6 or the lens surface is deviated from the focal position of the objective lens 2, it becomes light spread at the position of the third aperture 23 by the optical system following the objective lens 2. It cannot pass through the third aperture 23 well.
　こうして、上記ディスク６を回転させながら上述のような検出を行うことによって、各測定点での散乱光強度が上記ＰＣ４０の内部メモリ等に記録される。 Thus, by performing the above-described detection while rotating the disk 6, the scattered light intensity at each measurement point is recorded in the internal memory of the PC 40 or the like.
　尚、上述の説明は、上記第１半導体レーザー８からの第１波長のレーザー光による散乱光検出の場合を説明したが、第２半導体レーザー１２からの第２波長のレーザー光による場合も、第１ダイクロイックミラー１４で反射される以外は、全く同様である。また、上記蛍光を検出する蛍光検出の場合も、第２ダイクロイックミラー１６を透過する波長のレーザー光を出射する方の半導体レーザーを用い、第２ダイクロイックミラー１６を透過した蛍光を第１検出装置３で検出する以外は、全く同様である。 In the above description, the scattered light detection by the first wavelength laser light from the first semiconductor laser 8 has been described. However, the second wavelength laser light from the second semiconductor laser 12 can also be detected by the first wavelength. Except for being reflected by the one dichroic mirror 14, the same is true. In the case of fluorescence detection for detecting the fluorescence, the semiconductor laser that emits laser light having a wavelength that passes through the second dichroic mirror 16 is used, and the fluorescence that has passed through the second dichroic mirror 16 is detected by the first detection device 3. It is exactly the same except for detecting at.
　以下、上記光強度データ取得のシーケンスについて説明する。 Hereinafter, the above-described light intensity data acquisition sequence will be described.
　上述したように、上記サンプル７である微小粒子を含む検体は、ディスク６に封入されている。そして、ディスク６を回転させながら光学モジュール５によって上記光強度のデータを取得する。ここで、ディスク６上における光学モジュール５によって読み取り可能な一周分の上記データが記録されている帯状の部分のことをトラックと言う。ここで、上記トラックは、上記走査ラインの一例である。また、円周方向は上記第１走査方向の一例であり、半径方向は上記第２走査方向の一例である。 As described above, the specimen including the microparticles as the sample 7 is sealed in the disk 6. Then, the optical intensity data is acquired by the optical module 5 while the disk 6 is rotated. Here, the belt-like portion on the disk 6 where the above-mentioned data for one round which can be read by the optical module 5 is recorded is called a track. Here, the track is an example of the scan line. The circumferential direction is an example of the first scanning direction, and the radial direction is an example of the second scanning direction.
　上記ディスク６が一回転する毎に、ディスク６の半径方向に光学モジュール５を移動させて、上記光強度のデータを読み取るトラックを移動させる。または、ディスク６が一回転する間に光学モジュール５を徐々に移動させて、渦巻状に上記光強度のデータの取得を行っても良い。 Each time the disk 6 makes one revolution, the optical module 5 is moved in the radial direction of the disk 6 to move the track for reading the light intensity data. Alternatively, the optical module 5 may be gradually moved while the disk 6 rotates once, and the data of the light intensity may be acquired in a spiral shape.
　ここで、上記ホルダ２６の外周面２６aには、周方向に一定の角度のピッチで反射面を配列したエンコーダリング(図示せず)を設けている。さらに、ホルダ２６の外周面２６aに対向する位置には、一対の発光素子と受光素子とが搭載されたヘッド２８を配置している。こうして、ヘッド２８からのエンコーダ信号によって中心軸２５の回転変位、つまりディスク６の回転変位が検出可能になっている。したがって、ヘッド２８からのエンコーダ信号に同期して、タンジェンシャル方向の一定角度毎に上記散乱光強度のデータを取得することが可能になっている。 Here, the outer peripheral surface 26a of the holder 26 is provided with an encoder ring (not shown) in which reflecting surfaces are arranged at a constant angular pitch in the circumferential direction. Furthermore, a head 28 on which a pair of light emitting elements and light receiving elements are mounted is disposed at a position facing the outer peripheral surface 26 a of the holder 26. Thus, the rotational displacement of the central shaft 25, that is, the rotational displacement of the disk 6 can be detected by the encoder signal from the head 28. Therefore, in synchronization with the encoder signal from the head 28, it is possible to acquire the scattered light intensity data at every fixed angle in the tangential direction.
　尚、上記エンコーダ信号は、上記エンコーダリングおよびヘッド２８に限らず、スピンドルモータ２７に付属するロータリエンコーダによって生成するようにしても差し支えない。 The encoder signal is not limited to the encoder ring and the head 28, and may be generated by a rotary encoder attached to the spindle motor 27.
　上記光学モジュール５は、上述したように、散乱光と蛍光を同時に検出可能になっている。そこで、例えば、散乱光は画像化して、粒子の大きさを判定するのに用いる。また、特定種類の微小粒子のみを蛍光物質で染色することによって、サンプル７からの蛍光は上記特定種類の微小粒子からのみ発光する。そこで、サンプル７からの蛍光は、特定粒子の数をカウントするのに用いる。 The optical module 5 can simultaneously detect scattered light and fluorescence as described above. Thus, for example, the scattered light is imaged and used to determine the size of the particles. Further, by staining only a specific type of microparticles with a fluorescent material, the fluorescence from the sample 7 is emitted only from the specific type of microparticles. Therefore, the fluorescence from the sample 7 is used to count the number of specific particles.
　さらに、粒子の位置情報(座標)も記録し、蛍光と散乱光とを異なる検出器で検出することも可能である。その場合には、上記両検出器による検出画像において座標が一致する点を探すことにより、ある粒子の種類(蛍光)と大きさ(散乱光)との両方を判定することができる。この方法は、大きさが任意の閾値以上の蛍光粒子をカウントから除きたい場合等に適用できる。 Furthermore, it is also possible to record particle position information (coordinates) and detect fluorescence and scattered light with different detectors. In that case, both the type (fluorescence) and size (scattered light) of a certain particle can be determined by searching for a point where the coordinates match in the detection images obtained by the two detectors. This method can be applied, for example, when it is desired to exclude fluorescent particles having a size equal to or larger than an arbitrary threshold from the count.
　このように、上記光学モジュール５を散乱光と蛍光とを同時に検出可能にするメリットとして、光学モジュール５の小型化による低コストで、且つ省スペース化や、同時検出による測定時間の短縮等が挙げられる。 As described above, advantages of enabling the optical module 5 to detect scattered light and fluorescence at the same time include low cost due to downsizing of the optical module 5, space saving, and shortening of measurement time due to simultaneous detection. It is done.
　以下、上記散乱光や蛍光による粒子カウント方法の詳細について説明する。 Hereinafter, the details of the particle counting method using the scattered light and fluorescence will be described.
　前提として、上記光学モジュール５によってサンプル７に光を照射し、サンプル７に含まれる微小粒子から発せられる散乱光や蛍光を検出することによって、サンプル７に含まれる微小粒子を検出する。 As a premise, the optical module 5 irradiates the sample 7 with light, and detects the scattered light and fluorescence emitted from the microparticles included in the sample 7, thereby detecting the microparticles included in the sample 7.
　上記サンプル７が封入されたディスク６は、ホルダ２６に収容されて中心軸２５に対して固定されている。 The disk 6 enclosing the sample 7 is accommodated in the holder 26 and fixed to the central shaft 25.
　上記光学モジュール５によるスキャンニングの手順は、以下の通りである。
（１）上述のごとく上記ホルダ２６内にディスク６を収容して固定した状態で、ディスク６を回転初期位置に設置する。
（２）上記中心軸２５をスピンドルモータ２７で回転駆動することによって、ディスク６を回転させる。回転角度θは上記エンコーダリングおよびヘッド２８によって検出する。
（３）上記光学モジュール５を、上記ディスク６の径方向に１トラック分だけ移動させながら、１回転(１トラック)毎にディスク６上を走査させることによって、ディスク６上の微小粒子から発せられる散乱光や蛍光を検出する。
（４）検出され散乱光や蛍光の強度から、各トラック毎に微小粒子が存在する存在領域を判定する。 The scanning procedure by the optical module 5 is as follows.
(1) As described above, the disk 6 is placed at the initial rotation position while the disk 6 is received and fixed in the holder 26.
(2) The disk 6 is rotated by rotationally driving the central shaft 25 with a spindle motor 27. The rotation angle θ is detected by the encoder ring and the head 28.
(3) The optical module 5 is emitted from the fine particles on the disk 6 by scanning the disk 6 every rotation (one track) while moving the optical module 5 by one track in the radial direction of the disk 6. Detects scattered light and fluorescence.
(4) From the detected intensity of scattered light or fluorescence, the existence area where the microparticles exist for each track is determined.
　上記微小粒子の存在領域の判定における具体的な手順は、下記の通りである。 The specific procedure for determining the region where the fine particles are present is as follows.
　上記トラック毎に微小粒子を検出する場合に問題となるのは、複数のトラックに跨がっている同一の微小粒子を検出する場合である。この問題は、微小粒子が大きい、トラック間のピッチが小さい、励起光の照射スポット径が大きい等の場合に生ずる。このような場合には、同一の微小粒子を別のトラックでもカウントしてしまうダブルカウントが生じ、一つの大きな微粒子を複数の小さな微粒子として誤ってカウントしてしまうため、検出および計数が正確にできないことになる。 When a minute particle is detected for each track, a problem arises when the same minute particle straddling a plurality of tracks is detected. This problem occurs when the fine particles are large, the pitch between tracks is small, or the excitation light irradiation spot diameter is large. In such a case, double counting occurs in which the same minute particles are counted even in different tracks, and one large particle is erroneously counted as a plurality of small particles, so that detection and counting cannot be performed accurately. It will be.
　図２は、上記ディスク６上における各トラックと微小粒子ａ,ｂとの関係を示す模式図である。ここで、図２(a)は、ディスク６の全体を示す模式図である。図２から分かるように、複数のトラックに跨がる微小粒子としては、微小粒子ａのように、形状が略円形であってその直径自体が複数のトラックに跨がるものと、微小粒子ｂのように、形状が細長い楕円形であって小幅はトラック間と略同じであるがトラックに対して斜めに配置されているため複数のトラックに跨がるものとがある。尚、図２(b)は、微小粒子ａを含む領域の拡大模式図である。また、図２(c)は、微小粒子ｂを含む領域の拡大模式図である。 FIG. 2 is a schematic diagram showing the relationship between each track on the disk 6 and the fine particles a and b. Here, FIG. 2A is a schematic diagram showing the entire disk 6. As can be seen from FIG. 2, the microparticles straddling a plurality of tracks are those having a substantially circular shape and having a diameter itself straddling the plurality of tracks, such as a microparticle a, As described above, the shape is an elongated ellipse, and the small width is substantially the same as that between the tracks. FIG. 2B is an enlarged schematic view of a region including the microparticles a. FIG. 2 (c) is an enlarged schematic view of a region including the fine particles b.
　そこで、本実施の形態においては、微小粒子ａの場合であっても、微小粒子ｂの場合であっても、正確な検出および計数を可能にする方法を提案するものである。 Therefore, in the present embodiment, a method is proposed that enables accurate detection and counting in the case of the fine particles a and the fine particles b.
　以下、上記微小粒子ａの場合と微小粒子ｂの場合とに分けて、微小粒子の存在領域の判定について説明する。 Hereinafter, the determination of the presence region of the microparticle will be described separately for the case of the microparticle a and the case of the microparticle b.
　（微小粒子ａについて）
　(５）上記微小粒子ａおよび微小粒子ｂの判定は、上記第１検出器２０または第２検出器２４による検出光強度を、閾値と比較することによって行われる。尚、閾値の設定方法については、特に限定するものではない。例えば、図２(b)において、トラックｎで、タンジェンシャル方向θ1からθ2の間で閾値よりも強い強度の光が検出されたとする。その場合には、上記ＰＣ４０は、θ1からθ2の間の角度領域に微小粒子が存在すると判定する。そして、上記ＰＣ４０の内部メモリ等に、始点と終点との角度(θ1,θ2)を記録しておく。 (About microparticles a)
(5) The determination of the fine particles a and the fine particles b is performed by comparing the light intensity detected by the first detector 20 or the second detector 24 with a threshold value. The threshold setting method is not particularly limited. For example, in FIG. 2B, it is assumed that light having an intensity stronger than the threshold is detected in the track n between the tangential directions θ1 and θ2. In that case, the PC 40 determines that there are fine particles in the angle region between θ1 and θ2. The angles (θ1, θ2) between the start point and the end point are recorded in the internal memory of the PC 40 or the like.
　(６）上記ディスク６の検出位置を、トラックｎから径方向に１トラック分移動した状態で、同様にしてトラック(ｎ＋１)の測定を行う。 (6) The track (n + 1) is measured in the same manner with the detection position of the disk 6 moved from the track n by one track in the radial direction.
　(７）例えば、図２(b)において、トラック(ｎ＋１)で、タンジェンシャル方向のθ3からθ4の間で閾値よりも強い強度の光が検出されたとする。その場合には、上記ＰＣ４０は、θ3からθ4の間の角度領域に微小粒子が存在すると判定する。そして、上記ＰＣ４０の内部メモリ等に、始点と終点との角度(θ3,θ4)を記録しておく。 (7) For example, in FIG. 2B, it is assumed that light having an intensity stronger than the threshold is detected between θ3 and θ4 in the tangential direction in the track (n + 1). In that case, the PC 40 determines that there are fine particles in the angle region between θ3 and θ4. Then, the angles (θ3, θ4) between the start point and the end point are recorded in the internal memory of the PC 40 or the like.
　(８）次に、互いに隣接するトラックであるトラックｎとトラック(ｎ＋１)とで夫々検出された微小粒子の存在領域(角度領域)を比較する。ここで、微小粒子の存在位置の測定データ(上記始点と終点との角度データ)は、トラック番号と回転角度(θ)との二次元座標系に置き換えることができる。図３は、図２(b)および図２(c)において得られた微小粒子の存在位置の測定データを、トラック番号と回転角度(θ)との二次元座標系に置き換えた図である。以下においては、上記微小粒子の存在領域の比較について、分かり易く説明するために、図３を用いる。 (8) Next, the presence areas (angle areas) of the microparticles detected in the tracks n and (n + 1), which are adjacent to each other, are compared. Here, the measurement data of the position where the microparticles are present (the angle data between the start point and the end point) can be replaced with a two-dimensional coordinate system of the track number and the rotation angle (θ). FIG. 3 is a diagram in which the measurement data of the positions of the microparticles obtained in FIGS. 2B and 2C are replaced with a two-dimensional coordinate system of the track number and the rotation angle (θ). In the following, FIG. 3 is used in order to easily understand the comparison of the region where the microparticles exist.
　(９）図３において、トラックｎとトラック(ｎ＋１)との間で、検出された微小粒子のタンジェンシャル方向の存在領域を比較する。図３から分かるように、θ3からθ2の角度領域においてトラックｎとトラック(ｎ＋１)との両トラックにおいて微小粒子が検出されており、重複領域となっている。この場合には、トラックｎとトラック(ｎ＋１)とで夫々検出された微小粒子は重複領域ありとして同一の微小粒子と見なす。 (9) In FIG. 3, the existence area of the detected microparticles in the tangential direction is compared between the track n and the track (n + 1). As can be seen from FIG. 3, minute particles are detected in both the track n and the track (n + 1) in the angle region from θ3 to θ2, and this is an overlapping region. In this case, the microparticles detected in the track n and the track (n + 1) are regarded as the same microparticles with an overlapping region.
　(１０）さらに、上記トラック(ｎ＋１)とトラック(ｎ＋２)とで夫々検出された微小粒子の存在領域を比較する。その際に、上記ＰＣ４０の内部メモリ等に記憶されているトラックｎの角度(θ1,θ2)は、削除してもよい。 (10) Further, the presence areas of the microparticles detected in the track (n + 1) and the track (n + 2) are compared. At this time, the angle (θ1, θ2) of the track n stored in the internal memory or the like of the PC 40 may be deleted.
　図３において、トラック(ｎ＋１)ではθ3からθ4の角度領域で微小粒子が検出されているのに対して、トラック(ｎ＋２)では対応するθ3からθ4の角度領域で微粒子が検出されてはいない。そこで、トラックｎ～トラック(ｎ＋１)において同一と見なされた微小粒子を、一つの微小粒子ａとしてカウントする。 In FIG. 3, fine particles are detected in the angle region from θ3 to θ4 in the track (n + 1), whereas fine particles are not detected in the corresponding angle region from θ3 to θ4 in the track (n + 2). Therefore, the minute particles regarded as the same in the track n to the track (n + 1) are counted as one minute particle a.
　(１１）以下、同様にして、上記ディスク６の検出位置を１トラック分移動して微小粒子が存在する領域を判定し、隣接するトラック同士に同一微小粒子と判断可能な対応する微小粒子の有無を判定する。そして、同一微小粒子と判断可能な対応する微小粒子が存在しない場合には、それまでに同一と見なされた微小粒子を一つの微小粒子としてカウントするのである。 (11) Hereinafter, similarly, the detection position of the disk 6 is moved by one track to determine the area where the microparticles exist, and the presence or absence of corresponding microparticles that can be determined as the same microparticles between adjacent tracks. Determine. When there is no corresponding microparticle that can be determined as the same microparticle, the microparticles that have been regarded as the same are counted as one microparticle.
　（微小粒子ｂについて）
　上述の微小粒子ａの場合と同様に、隣接するトラック同士において、夫々検出された微小粒子の存在領域を比較する。そして、存在領域に重複がある場合には同一の微小粒子に関する存在領域であると判定する。 (About microparticles b)
As in the case of the fine particle a described above, the detected regions of the fine particles are compared between adjacent tracks. And when there exists duplication in an existing area, it determines with it being an existing area regarding the same microparticle.
　図３において、互いに隣接するトラックであるトラックｎとトラック(ｎ＋１)とで夫々検出された微小粒子の存在領域を比較する。θ7からθ8の角度領域で各トラックで検出された微小粒子の存在領域が重複している。したがって、これらのトラックに重複している存在領域に関する微小粒子を同一の微小粒子であると判定する。 In FIG. 3, the microparticle existing areas detected in track n and track (n + 1), which are adjacent to each other, are compared. In the angle region from θ7 to θ8, the microparticle existence regions detected in each track overlap. Therefore, it is determined that the microparticles related to the existing region overlapping these tracks are the same microparticles.
　次に、互いに隣接するトラック(ｎ＋１)とトラック(ｎ＋２)とで夫々検出された微小粒子の存在領域を比較する。θ9からθ10の角度領域で夫々のトラックで検出された微小粒子の存在領域が重複している。このような場合は、先ほど同一の微小粒子であると判定されたトラックｎとトラック(ｎ＋１)との微小粒子に加えて、さらにトラック(ｎ＋２)で検出された微小粒子も含めて、トラックｎからトラック(ｎ＋２)までの総ての微小粒子を同一の微小粒子ｂであると判定する。 Next, the presence areas of the microparticles detected in the track (n + 1) and the track (n + 2) adjacent to each other are compared. In the angle region from θ9 to θ10, the existing regions of the microparticles detected in each track overlap. In such a case, in addition to the microparticles of the track n and the track (n + 1) that have been determined to be the same microparticles earlier, the microparticles detected in the track (n + 2) are further included from the track n. All the fine particles up to the track (n + 2) are determined to be the same fine particle b.
　また、上記トラック(ｎ＋２)とトラック(ｎ＋３)とで夫々検出された微小粒子の存在領域を比較する。そうすると、トラック(ｎ＋２)ではθ9からθ10の角度領域で微小粒子を検出しているのに対し、トラック(ｎ＋３)では対応するθ9からθ10の角度領域で微小粒子が検出されてはいない。そこで、その時点で、トラックｎからトラック(ｎ＋２)までにおいて検出された微小粒子を一つの微小粒子として粒子数をカウントするのである。 Also, the presence areas of the microparticles detected in the track (n + 2) and the track (n + 3) are compared. Then, while the fine particles are detected in the angle region from θ9 to θ10 in the track (n + 2), the fine particles are not detected in the corresponding angle region from θ9 to θ10 in the track (n + 3). Therefore, at that time, the number of particles is counted with the minute particles detected from track n to track (n + 2) as one minute particle.
　以上のごとく、上記ディスク６の検出位置を１トラック分移動する毎に、微小粒子が存在する領域を判定する。そして、互いに隣接するトラック毎に順次微小粒子の存在領域を比較し、隣接するトラック同士に同一微小粒子と判断可能な対応する微小粒子が無い場合には、その時点で一つの微小粒子として粒子数をカウントする。 As described above, every time the detection position of the disk 6 is moved by one track, the region where the fine particles are present is determined. Then, the area of the microparticles is sequentially compared for each adjacent track, and if there is no corresponding microparticle that can be determined as the same microparticle between adjacent tracks, the number of particles as one microparticle at that time Count.
　こうすることによって、同一微小粒子のダブルカウントを防止して、微小粒子の数を正確にカウントすることができる。また、上述の同一微小粒子判定処理は、隣接するトラック同士の微小粒子の存在領域情報があれば行うことができるため、全てのトラックに関する微小粒子の存在領域の検出を終了しなくとも行うことができる。また、上記微小粒子の存在領域の比較後における微小粒子の存在領域情報は順次削除してもよいため、保存するテータ量を大幅に少なくできる。 By doing so, it is possible to accurately count the number of microparticles by preventing double counting of the same microparticles. In addition, since the same minute particle determination process described above can be performed if there is information on the existence area of minute particles between adjacent tracks, the detection of the existence area of minute particles for all tracks can be performed without ending. it can. In addition, since the microparticle existence area information after the comparison of the microparticle existence area may be sequentially deleted, the amount of data to be saved can be greatly reduced.
　したがって、上述の同一微小粒子判定処理時のデータ量や演算負荷を大幅に軽減することができる。さらに、その上に、各トラックの微小粒子の存在領域の検出と同時に同一微小粒子の判定を行うことができるため、効率的に微小粒子のカウントを行うことができるである。 Therefore, it is possible to greatly reduce the amount of data and the calculation load during the same minute particle determination process described above. Furthermore, since the same minute particle can be determined simultaneously with the detection of the region where the minute particle exists in each track, the minute particle can be counted efficiently.
　尚、本実施の形態においては、上記微小粒子が分布するサンプル７は、透明且つ円形のディスク６に封入されており、ディスク６を中心軸２５に固定されたホルダ２６に収容することによっ回転可能にしている。 In the present embodiment, the sample 7 in which the fine particles are distributed is enclosed in a transparent and circular disk 6, and is rotated by accommodating the disk 6 in a holder 26 fixed to the central shaft 25. It is possible.
　しかしながら、この発明はこれに限定されるものではなく、上記中心軸２５に固定されたガラスステージの表面に、サンプル７を直接固定するようにしても差し支えない。 However, the present invention is not limited to this, and the sample 7 may be directly fixed to the surface of the glass stage fixed to the central shaft 25.
　・第２実施の形態
　本実施の形態における微小粒子検出装置の構成は、上記第１実施の形態において図１に示す微小粒子検出装置の概略構成と同様である。したがって、本実施の形態における微小粒子検出装置の詳細な説明は省略し、以下の説明においては適宜図１～図３を用いる。 -2nd Embodiment The structure of the microparticle detection apparatus in this Embodiment is the same as that of the schematic structure of the microparticle detection apparatus shown in FIG. 1 in the said 1st Embodiment. Therefore, a detailed description of the microparticle detection apparatus in this embodiment is omitted, and FIGS. 1 to 3 are used as appropriate in the following description.
　本実施の形態においては、上記微小粒子検出装置の上記ＰＣ４０で、微小粒子数のカウントに加えて、微小粒子の位置情報を取得するようにしている。 In this embodiment, in addition to counting the number of microparticles, the PC40 of the microparticle detection apparatus acquires the position information of the microparticles.
　以下、上記微小粒子数のカウントと共に微小粒子の位置情報を取得する場合の動作手順について説明する。 Hereinafter, the operation procedure in the case of acquiring the position information of the fine particles together with the count of the fine particles will be described.
　上記第１実施の形態の場合と同様にして、各トラックにおいて微小粒子を検出する。 In the same manner as in the first embodiment, fine particles are detected in each track.
　その場合における上記第１実施の形態との違いは、各トラックにおける微小粒子の存在領域を上記ＰＣ４０の内部メモリ等に記録するだけでなく、各微小粒子の代表座標情報をも記録することである。ここで、上記代表座標とは、微小粒子の存在領域における上記始点または上記終点の図３に示す回転角‐トラック番号座標上の座標、または、上記始点と上記終点との中間点の座標等である。尚、上記存在領域における上記始点から上記終点までの全ての座標を記録しても良い。その場合には、扱うデータ量は大きくなるが、微小粒子のより詳細な情報を得ることができる。 The difference from the first embodiment in that case is that not only the microparticle existing area in each track is recorded in the internal memory of the PC 40 but also the representative coordinate information of each microparticle. . Here, the representative coordinates are the coordinates on the rotation angle-track number coordinates shown in FIG. 3 of the start point or the end point in the microparticle existence region, or the coordinates of the intermediate point between the start point and the end point. is there. Note that all coordinates from the start point to the end point in the existence area may be recorded. In that case, the amount of data to be handled is increased, but more detailed information on the fine particles can be obtained.
　次に、上記第１実施の形態の場合と同様にして、互いに隣接するトラック同士で、夫々のトラックで検出された微小粒子の存在領域を比較して、同一の微小粒子であるか否かを判定する。 Next, in the same way as in the case of the first embodiment, by comparing the existing regions of the microparticles detected in the tracks between adjacent tracks, it is determined whether or not they are the same microparticles. judge.
　上記比較によって同一の微小粒子であると判定された場合には、同一であると判定された微小粒子に対して一つの上記代表座標が対応するように、上記代表座標を設定する。その理由は、同一微小粒子であると判定された複数の存在領域情報(角度領域情報)には、夫々固有の代表座標情報がある。したがって、そのままでは同一であると判定された微小粒子に複数の代表座標情報が対応することになってしまうことを回避するためである。 When it is determined by the comparison that they are the same microparticles, the representative coordinates are set so that one representative coordinate corresponds to the microparticles determined to be the same. The reason is that each of the plurality of existence area information (angle area information) determined to be the same minute particle has unique representative coordinate information. Therefore, it is for avoiding that several representative coordinate information will respond | correspond to the microparticle determined to be the same as it is.
　具体的な例としては、トラックｎとトラック(ｎ＋１)との比較によって、夫々のトラックで検出された微小粒子の存在領域情報が同一微小粒子に基づくと判定された場合は、トラックｎの存在領域情報の代表座標を、上記同一と見なされた微小粒子の代表座標であると設定する。そして、トラック(ｎ＋１)で検出された存在領域情報の代表座標は削除するのである。 As a specific example, when it is determined by comparing the track n and the track (n + 1) that the microparticle existence area information detected in each track is based on the same microparticle, the existence area of the track n The representative coordinates of the information are set to be the representative coordinates of the microparticles regarded as the same. Then, the representative coordinates of the existing area information detected in the track (n + 1) are deleted.
　こうすることによって、同一と見なされた微小粒子に対して一つの代表座標が対応するように関連付けることができるのである。 In this way, one representative coordinate can be associated with a minute particle that is regarded as the same.
　そして、上記第１実施の形態の場合と同様に、互いに隣接するトラック同士の比較を順次行っていき、隣接するトラックに対応する微小粒子が無くなった時点で、一つの微小粒子としてカウントすると共に、当該微小粒子の代表座標を設定し、上記ＰＣ４０の内部メモリ等に記録する。 Then, as in the case of the first embodiment, the adjacent tracks are sequentially compared, and when there are no more microparticles corresponding to the adjacent tracks, they are counted as one microparticle, The representative coordinates of the fine particles are set and recorded in the internal memory of the PC 40 or the like.
　・第３実施の形態
　本実施の形態における微小粒子検出装置の構成は、上記第１実施の形態において図１に示す微小粒子検出装置の概略構成と同様である。したがって、本実施の形態における微小粒子検出装置の詳細な説明は省略し、以下の説明においては適宜図１～図３を用いる。 Third Embodiment The configuration of the microparticle detection device in the present embodiment is the same as the schematic configuration of the microparticle detection device shown in FIG. 1 in the first embodiment. Therefore, a detailed description of the microparticle detection apparatus in this embodiment is omitted, and FIGS. 1 to 3 are used as appropriate in the following description.
　本実施の形態においては、上記微小粒子検出装置の上記ＰＣ４０で、微小粒子数のカウントに加えて、微小粒子の形状やサイズの情報を取得するようにしている。 In the present embodiment, in addition to counting the number of microparticles, the PC40 of the microparticle detection apparatus acquires information on the shape and size of the microparticles.
　以下、上記微小粒子数のカウントと共に微小粒子の形状やサイズの情報を取得する場合の動作手順について説明する。 Hereinafter, an operation procedure in the case of acquiring the shape and size information of the fine particles together with the count of the fine particles will be described.
　上記第１実施の形態の場合と同様にして、各トラックにおいて微小粒子を検出する。 In the same manner as in the first embodiment, fine particles are detected in each track.
　上記第１実施の形態の場合と同様にして、互いに隣接するトラック同士において、検出された微小粒子の存在領域が比較される。その場合における上記第１実施の形態との違いは、各トラックにおける微小粒子の存在領域を上記ＰＣ４０の内部メモリ等に記録するだけでなく、各微小粒子の形状やサイズをも判定して記録することである。 In the same manner as in the case of the first embodiment, the detected microparticle existence regions are compared between adjacent tracks. In this case, the difference from the first embodiment is that not only the microparticle existing area in each track is recorded in the internal memory of the PC 40 but also the shape and size of each microparticle is determined and recorded. That is.
　上記第１実施の形態の場合と同様にして、互いに隣接するトラック同士で、夫々のトラックで検出された微小粒子の存在領域を比較して、同一の微小粒子であるか否かを判定する。そして、上記比較によって同一の微小粒子であると判定された場合には、夫々のトラックで検出した微小粒子の存在領域を足し合わせた領域を、同一微小粒子の存在領域であると設定するのである。 In the same manner as in the case of the first embodiment, the adjacent regions of the microparticles detected in the respective tracks are compared with each other to determine whether or not they are the same microparticles. Then, if it is determined by the above comparison that they are the same microparticles, the area obtained by adding the microparticle existence areas detected in the respective tracks is set as the same microparticle existence area. .
　具体的な例としては、図２において、トラックｎとトラック(ｎ＋１)との比較により、夫々のトラックで検出された微小粒子の存在領域情報が同一微小粒子に基づくと判定された場合は、トラックｎの存在領域情報の存在領域とトラック(ｎ＋１)の存在領域情報の存在領域とを、ディスク６の半径方向に足し合わせた領域を、上記同一と見なされた微小粒子の存在領域であると設定する。 As a specific example, in FIG. 2, when it is determined by comparing the track n and the track (n + 1) that the microparticle existence area information detected in each track is based on the same microparticle, The area obtained by adding the existence area of the existence area information of n and the existence area of the existence area information of the track (n + 1) in the radial direction of the disk 6 is set as the existence area of the microparticles regarded as the same. To do.
　こうすることによって、同一と見なされた微小粒子全体の存在領域を設定することができるのである。 By doing this, it is possible to set the existence region of the entire microparticles that are regarded as the same.
　そして、上記第１実施の形態の場合と同様に、互いに隣接するトラック同士の比較を順次行っていき、隣接するトラックに対応する微小粒子が無くなった時点で、一つの微小粒子としてカウントすると共に、当該微小粒子の形状やサイズに関する情報を設定し、上記ＰＣ４０の内部メモリ等に記録する。 Then, as in the case of the first embodiment, the adjacent tracks are sequentially compared, and when there are no more microparticles corresponding to the adjacent tracks, they are counted as one microparticle, Information on the shape and size of the microparticles is set and recorded in the internal memory of the PC 40 or the like.
　ここで、上記形状に関する情報とは、同一と見なされた微小粒子全体の存在領域の形状の情報である。また、上記サイズに関する情報とは、上記形状に関する情報を上記内部メモリ等に記録するとデータ量が大きくなるため、上記微小粒子全体の存在領域の面積等、微小粒子全体の存在領域から得られるサイズ情報を記録するような場合での上記サイズ情報を意味している。 Here, the information on the shape is information on the shape of the entire region of the microparticles regarded as the same. Also, the size information is the size information obtained from the entire area of the microparticles, such as the area of the entire area of the microparticles, because the amount of data increases when the information about the shape is recorded in the internal memory or the like. Means the above-mentioned size information in the case of recording.
　・第４実施の形態
　本実施の形態は、上記第１実施の形態における微小粒子数のカウント方法を、サンプルまたは光学モジュールを互いに直交する二方向への二次元走査する二次元スキャン方式に適用したものに関する。 Fourth Embodiment In this embodiment, the method for counting the number of fine particles in the first embodiment is applied to a two-dimensional scanning method in which a sample or an optical module is two-dimensionally scanned in two directions orthogonal to each other. About things.
　図４および図５は、一例として、Ｙ方向に上記サンプルを移動させながらＸ方向にスキャンする微小粒子検出装置を示す。図４は全体を示す斜視図であり、図５は上記光学モジュールの断面概略図である。 4 and 5 show, as an example, a microparticle detection apparatus that scans in the X direction while moving the sample in the Y direction. 4 is a perspective view showing the whole, and FIG. 5 is a schematic sectional view of the optical module.
　図４において、１は光源装置であり、２は対物レンズであり、３は検出装置である。光源装置１と対物レンズ２と検出装置３とは、枠体内に収納されて上記光学モジュール５を構成している。そして、光学モジュール５の上方には対物レンズ２に対向してガラスステージ２９が配置され、ガラスステージ２９上には例えば蛍光物質によって標識された微小粒子が分布する懸濁液やゲル支持体やメンブレン等の転写支持体がサンプル(上記検体)３０としてセットされている。 In FIG. 4, 1 is a light source device, 2 is an objective lens, and 3 is a detection device. The light source device 1, the objective lens 2, and the detection device 3 are housed in a frame to constitute the optical module 5. A glass stage 29 is disposed above the optical module 5 so as to face the objective lens 2. On the glass stage 29, for example, a suspension, a gel support, or a membrane in which fine particles labeled with a fluorescent substance are distributed. A transfer support such as the above is set as a sample (the specimen) 30.
　ここで、上記ガラスステージ２９は矩形を成しており、長辺方向の第１走査方向と、第１走査方向に直交する短辺方向の第２走査方向との、二次元方向に走査するように構成されている。その場合における走査方法については、特に限定するものではない。要は、ガラスステージ２９を上記第１走査方向に往復動作をさせる第１動作部と、上記第２走査方向に往復動作をさせる第２動作部とを備えていれば良いのである。あるいは、光学モジュール５側を二次元方向に走査してもよい。 Here, the glass stage 29 has a rectangular shape, and scans in a two-dimensional direction of a first scanning direction in the long side direction and a second scanning direction in the short side direction orthogonal to the first scanning direction. It is configured. The scanning method in that case is not particularly limited. In short, it is only necessary to include a first operation unit that reciprocates the glass stage 29 in the first scanning direction and a second operation unit that reciprocates in the second scanning direction. Alternatively, the optical module 5 side may be scanned in a two-dimensional direction.
　図５において、半導体レーザー８,第１レンズ９,スポットサイズ調整レンズ１０,第１アパーチャ１１およびプリズム１５は、上記第１実施の形態において図１に示す第１半導体レーザー８,第１レンズ９,スポットサイズ調整レンズ１０,第１アパーチャ１１およびプリズム１５と、全く同様である。そこで、同じ番号を付けて詳細な説明は省略する。また、バンドパスフィルタ１７,第２レンズ１８,第２アパーチャ１９および検出器２０は、上記第１実施の形態において図１に示すバンドパスフィルタ１７,第３レンズ１８,第２アパーチャ１９および第１検出器２０と、全く同様である。そこで、同じ番号を付けて詳細な説明は省略する。 In FIG. 5, the semiconductor laser 8, the first lens 9, the spot size adjusting lens 10, the first aperture 11 and the prism 15 are the same as the first semiconductor laser 8, the first lens 9, and the like shown in FIG. This is exactly the same as the spot size adjusting lens 10, the first aperture 11, and the prism 15. Therefore, the same numbers are assigned and detailed description is omitted. Further, the bandpass filter 17, the second lens 18, the second aperture 19 and the detector 20 are the same as the bandpass filter 17, the third lens 18, the second aperture 19 and the first aperture shown in FIG. 1 in the first embodiment. It is exactly the same as the detector 20. Therefore, the same numbers are assigned and detailed description is omitted.
　本実施の形態における対物レンズ２は、光軸を含む中央部分が、通常の凸レンズの機能(屈折のみで光を偏向)を有する凸レンズ部２aとなっている。そして、半導体レーザー８から出射されてプリズム１５で反射された励起光は、この凸レンズ部２aの部分を通過してサンプル３０に向かって収束される。また、サンプル３０から出射された散乱光または蛍光のうちの放射角度が小さい光は、この凸レンズ部２aの部分を通過して検出器２０に向かって集光される。 In the objective lens 2 in the present embodiment, the central portion including the optical axis is a convex lens portion 2a having a function of a normal convex lens (light is deflected only by refraction). Then, the excitation light emitted from the semiconductor laser 8 and reflected by the prism 15 passes through the convex lens portion 2a and is converged toward the sample 30. Further, light having a small radiation angle out of scattered light or fluorescence emitted from the sample 30 passes through the convex lens portion 2a and is condensed toward the detector 20.
　上記対物レンズ２における凸レンズ部２aの周囲は、下方に向かって開いた円錐台形の筒状体２bとなっている。そして、サンプル６から出射された光のうちの、凸レンズ部２aに入りきらないような放射角度の大きい蛍光は、筒状体２bの上端面から筒状体２b内に入射し、筒状体２bの外周面で全反射されて光軸側に偏向され、筒状体２bの内周面および下端面から検出器２０に向かって出射される。 The periphery of the convex lens portion 2a in the objective lens 2 is a truncated cone-shaped cylindrical body 2b that opens downward. Then, of the light emitted from the sample 6, the fluorescence having a large radiation angle that does not enter the convex lens portion 2a is incident on the cylindrical body 2b from the upper end surface of the cylindrical body 2b, and the cylindrical body 2b. Are totally reflected by the outer peripheral surface of the light source, deflected toward the optical axis, and emitted toward the detector 20 from the inner peripheral surface and the lower end surface of the cylindrical body 2b.
　また、上記バンドパスフィルタ１７は、図４に示すように、例えば回転フォルダ３１に配置されて、蛍光の波長に応じて他の波長のフィルタと交換可能になっている。 Further, as shown in FIG. 4, the band-pass filter 17 is disposed, for example, in the rotary folder 31 and can be replaced with a filter of another wavelength according to the wavelength of the fluorescence.
　上述したように、本実施の形態においては、上記光学モジュール５を固定して、サンプル３０を載せたガラスステージ２９を二次元方向に走査させて、散乱光または蛍光の強度の分布画像を読取る構成になっている。 As described above, in the present embodiment, the optical module 5 is fixed, the glass stage 29 on which the sample 30 is placed is scanned in a two-dimensional direction, and a distribution image of scattered light or fluorescence intensity is read. It has become.
　このように、上記光学モジュール５に対してガラスステージ２９を相対的に第１走査方向(長辺方向)とこの第１走査方向に直交する第２走査方向(短辺方向)とに交互に二次元走査した場合に、サンプル３０上には、対物レンズ２によって励起光の集光点の走査軌跡が描かれる。この走査軌跡は、間隔を空けて第１走査方向(長辺方向)に往復動する複数の直線状の走査ラインと、互いに隣接する走査ラインの終点と始点とを第２走査方向(短辺方向)につなぐ接続ラインとが、連続して繰り返して構成されている。 In this manner, the glass stage 29 is relatively moved relative to the optical module 5 in the first scanning direction (long-side direction) and in the second scanning direction (short-side direction) orthogonal to the first scanning direction. When the dimension scanning is performed, a scanning locus of a condensing point of the excitation light is drawn on the sample 30 by the objective lens 2. The scanning trajectory includes a plurality of linear scanning lines reciprocating in the first scanning direction (long side direction) at intervals, and the end points and starting points of the adjacent scanning lines in the second scanning direction (short side direction). ) Are connected continuously and repeatedly.
　その場合、上記第１走査方向(長辺方向)に延在する複数の走査ラインは、上記第１実施の形態における上記トラックに相当する。そこで、本実施の形態において得られる走査ライン番号と走査位置との二次元座標系を、上記第１実施の形態におけるトラック番号と回転角度(θ)との二次元座標系に置き換えることができる。 In this case, the plurality of scanning lines extending in the first scanning direction (long side direction) correspond to the track in the first embodiment. Therefore, the two-dimensional coordinate system of the scanning line number and the scanning position obtained in the present embodiment can be replaced with the two-dimensional coordinate system of the track number and the rotation angle (θ) in the first embodiment.
　したがって、上記検出器２０からの信号を受けたＰＣ４０は、上記走査ライン番号と走査位置との二次元座標系を用いて、上記第１実施の形態において上記トラック番号と回転角度(θ)との二次元座標系に対して行った微小粒子の存在領域の判定と微小粒子数のカウントとを行うのである。 Therefore, the PC 40 that has received the signal from the detector 20 uses the two-dimensional coordinate system of the scanning line number and the scanning position to determine the track number and the rotation angle (θ) in the first embodiment. The determination of the microparticle existence area and the count of the microparticles performed on the two-dimensional coordinate system are performed.
　以上のように、本実施の形態によれば、二次元スキャン方式の微小粒子検出装置においても、ディスク回転方式の微小粒子検出装置による微小粒子の存在領域の判定と微小粒子数のカウントとを適用でき、同一微小粒子のダブルカウントを防止して、微小粒子の数を正確にカウントすることができるのである。 As described above, according to the present embodiment, even in the two-dimensional scan type microparticle detection apparatus, the determination of the microparticle existence area and the count of the microparticles by the disk rotation type microparticle detection apparatus are applied. It is possible to prevent double counting of the same microparticles and to accurately count the number of microparticles.
　また、上述の同一微小粒子判定処理は、全ての走査ラインに関する微小粒子の存在領域の検出を終了しなくとも行うことができ、上記微小粒子の存在領域の比較後における微小粒子の存在領域情報は順次削除できるので、保存するテータ量を大幅に少なくできる。 The same microparticle determination process described above can be performed without ending the detection of the microparticle existence area for all scanning lines, and the microparticle existence area information after the comparison of the microparticle existence areas is as follows. Since data can be deleted sequentially, the amount of data to be saved can be greatly reduced.
　したがって、上述の同一微小粒子判定処理時のデータ量や演算負荷を大幅に軽減することができる。 Therefore, it is possible to greatly reduce the amount of data and the calculation load during the same minute particle determination process described above.
　尚、本実施の形態においては、上記ガラスステージ２９上にサンプル３０をセットするようにしているが、上記第１実施の形態の場合のように、サンプル３０を透明に且つ矩形に構成されたディスク内に封入するようにしても差し支えない。 In this embodiment, the sample 30 is set on the glass stage 29. However, as in the case of the first embodiment, the sample 30 is a transparent and rectangular disk. It does not matter even if it is enclosed inside.
　上述したように、この発明の微小粒子検出装置は、
　光源８,１２から出射された光を、微小粒子を含む検体７,３０に対して照射する照射光学系１と、
　上記光の照射によって上記検体７,３０中の上記微小粒子から発せられた光を検出する光検出光学系３,４と、
　上記光検出光学系３,４によって検出された上記微小粒子からの光の強度に基づいて、上記微小粒子を検出する検出部と
を備え、
　上記照射光学系１および上記光検出光学系３,４は、第１走査方向とこの第１走査方向に交差する第２走査方向とに、上記検体７,３０を相対的に二次元走査するようになっており、
　上記検出部は、
　上記照射光学系１および上記光検出光学系３,４が上記第１走査方向に走査した場合の上記微小粒子からの光の強度に基づいて、各走査ライン毎に、上記微小粒子が存在する存在領域を判定し、
　互いに隣接する上記走査ラインにおける上記微小粒子の存在領域を比較することによって、上記両走査ラインに在る上記存在領域の組みが同一微小粒子に起因するか否かを判定する
ようになっていることを特徴としている。 As described above, the microparticle detection apparatus of the present invention is
An irradiation optical system 1 that irradiates the specimens 7 and 30 including fine particles with the light emitted from the light sources 8 and 12;
Optical detection optical systems 3 and 4 for detecting light emitted from the microparticles in the specimens 7 and 30 by the irradiation of the light;
A detection unit that detects the microparticles based on the intensity of light from the microparticles detected by the light detection optical systems 3 and 4;
The irradiation optical system 1 and the light detection optical systems 3 and 4 relatively two-dimensionally scan the specimens 7 and 30 in the first scanning direction and the second scanning direction that intersects the first scanning direction. And
The detection unit is
Presence of the minute particles for each scanning line based on the intensity of light from the minute particles when the irradiation optical system 1 and the light detection optical systems 3 and 4 are scanned in the first scanning direction. Determine the area,
By comparing the existing areas of the microparticles in the scanning lines adjacent to each other, it is determined whether or not the set of the existing areas in the two scanning lines is caused by the same microparticle. It is characterized by.
　上記構成によれば、上記第１走査方向に走査した場合の各走査ラインに関し、互いに隣接する上記走査ラインにおける上記微小粒子の存在領域を比較するという単純な方法によって、上記存在領域の組みが同一微小粒子に起因するか否かを判定するようにしている。 According to the above configuration, for each scanning line when scanned in the first scanning direction, the set of the existing regions is the same by a simple method of comparing the existing regions of the microparticles in the adjacent scanning lines. It is determined whether or not it is caused by fine particles.
　したがって、上記判定には、比較の対象となる上記互いに隣接する走査ラインにおける上記存在領域のデータさえあればよく、全体のマップデータは必ずしも必要ではない。また、比較自体も非常に簡単な処理でよい。そして、上記比較を、隣接する上記走査ラインに対して順次行うことによって、全走査ラインに関する比較行うことができる。 Therefore, for the determination, it is only necessary to have the data of the existing area in the scanning lines adjacent to each other to be compared, and the entire map data is not necessarily required. Further, the comparison itself may be a very simple process. And the comparison regarding all the scanning lines can be performed by sequentially performing the comparison with respect to the adjacent scanning lines.
　すなわち、この発明によれば、上述の比較処理時のデータ量や演算負荷を大幅に軽減することができる。さらに、その上に、各走査ラインにおける上記微小粒子の存在領域の検出と同時に上記比較処理を行うことができ、効率的に微小粒子のカウントを行うことが可能になる。 That is, according to the present invention, it is possible to greatly reduce the amount of data and the calculation load during the comparison process described above. In addition, the comparison process can be performed simultaneously with the detection of the region where the microparticles exist in each scanning line, and the microparticles can be counted efficiently.
　また、一実施の形態の微小粒子検出装置では、
　上記検出部は、上記両走査ラインに在る上記存在領域の組みが同一微小粒子に起因すると判定した場合には、上記両走査ラインの何れか一方の走査ラインと、当該の走査ラインに隣接する新たな走査ラインとにおける、上記微小粒子の存在領域を比較することによって、上記新たな隣接走査ラインに在る上記存在領域の組みが同一微小粒子に起因するか否かを判定するようになっている。 Moreover, in the microparticle detection apparatus of one embodiment,
When the detection unit determines that the set of the existing regions in both the scan lines is caused by the same microparticle, the detection unit is adjacent to one of the scan lines and the scan line. By comparing the existing area of the microparticles in the new scanning line, it is determined whether the set of the existing areas in the new adjacent scanning line is caused by the same microparticle. Yes.
　この実施の形態によれば、上記比較処理を行う互いに隣接する上記走査ラインを順次更新するようにしている。したがって、比較の対象となる上記互いに隣接する上記走査ラインにおける上記存在領域のデータさえあれば、全走査ラインに関する上記微小粒子の重複を判定することが可能になる。 According to this embodiment, the scanning lines adjacent to each other for performing the comparison process are sequentially updated. Therefore, as long as there is data on the existence area in the scanning lines adjacent to each other to be compared, it is possible to determine the overlap of the fine particles with respect to all the scanning lines.
　また、一実施の形態の微小粒子検出装置では、
　上記検出部は、互いに隣接する上記走査ラインにおける上記微小粒子の存在領域に関して、上記第１走査方向の位置が互いに重なっている場合には、上記両走査ラインに在る上記存在領域の組みは同一の微小粒子の存在領域であると判定するようになっている。 Moreover, in the microparticle detection apparatus of one embodiment,
When the positions in the first scanning direction overlap each other with respect to the existence area of the microparticles in the scanning lines adjacent to each other, the detection unit has the same combination of the existence areas in both the scanning lines. It is determined that the region is a region where microparticles exist.
　この実施の形態によれば、互いに隣接する上記走査ラインにおける上記微小粒子の存在領域が、上記第１走査方向で互いに重なっている場合に、同一の微小粒子の存在領域であると判定するようにしている。 According to this embodiment, when the microparticle existence areas in the scanning lines adjacent to each other overlap each other in the first scanning direction, it is determined that the microparticle existence areas are the same microparticle existence area. ing.
　したがって、比較の対象となる上記互いに隣接する上記走査ラインにおける上記存在領域のデータに基づいて、互いに隣接する上記走査ラインに跨がる同一微小粒子を的確に判定することができる。 Therefore, it is possible to accurately determine the same microparticles straddling the scan lines adjacent to each other based on the data of the existence area in the scan lines adjacent to each other to be compared.
　また、一実施の形態の微小粒子検出装置では、
　上記検出部は、互いに隣接する上記走査ラインに、同一微小粒子に起因すると判定された上記存在領域が在る場合には、上記同一微小粒子に起因する上記存在領域に基づいて上記同一微小粒子の位置情報を設定するようになっている。 Moreover, in the microparticle detection apparatus of one embodiment,
In the case where the existence area determined to be caused by the same minute particle is present in the scanning lines adjacent to each other, the detection unit detects the same minute particle based on the existence area caused by the same minute particle. Position information is set.
　この実施の形態によれば、比較の対象となる上記互いに隣接する上記走査ラインにおける上記存在領域のデータに基づいて、互いに隣接する上記走査ラインに跨がる同一微小粒子に加えて、上記同一微小粒子の位置情報を設定することが可能になる。 According to this embodiment, in addition to the same microparticles straddling the scan lines adjacent to each other, based on the data of the existence area in the scan lines adjacent to each other to be compared, the same microparticles It becomes possible to set the position information of the particles.
　また、一実施の形態の微小粒子検出装置では、
　上記検出部は、互いに隣接する上記走査ラインに、同一微小粒子に起因すると判定された上記存在領域が在る場合には、上記同一微小粒子に起因する上記存在領域に基づいて上記同一微小粒子の形状情報またはサイズ情報を設定するようになっている。 Moreover, in the microparticle detection apparatus of one embodiment,
In the case where the existence area determined to be caused by the same minute particle is present in the scanning lines adjacent to each other, the detection unit detects the same minute particle based on the existence area caused by the same minute particle. Shape information or size information is set.
　この実施の形態によれば、比較の対象となる上記互いに隣接する上記走査ラインにおける上記存在領域のデータに基づいて、互いに隣接する上記走査ラインに跨がる同一微小粒子に加えて、上記同一微小粒子の形状情報またはサイズ情報をも設定することが可能になる。 According to this embodiment, in addition to the same microparticles straddling the scan lines adjacent to each other, based on the data of the existence area in the scan lines adjacent to each other to be compared, the same microparticles It is also possible to set particle shape information or size information.
　１…光源装置、
　２…対物レンズ、
　３,４…検出装置、
　５…光学モジュール、
　６…ディスク、
　７,３０…サンプル、
　８,１２…半導体レーザー、
　９,１３,１８,２２…レンズ、
１０…スポットサイズ調整レンズ、
１１,１９,２３…アパーチャ、
１４,１６…ダイクロイックミラー、
１５…プリズム、
１７…バンドパスフィルタ、
２０,２４…検出器、
２１…ＮＤフィルタ、
２５…中心軸、
２６…ホルダ、
２７…スピンドルモータ、
２８…ヘッド、
２９…ガラスステージ、
４０…ＰＣ。 1 ... light source device,
2 ... Objective lens,
3, 4 ... detection device,
5 ... Optical module,
6 ... disc,
7,30 ... sample,
8,12 ... Semiconductor laser,
9, 13, 18, 22 ... lens,
10 ... Spot size adjustment lens,
11, 19, 23 ... Aperture,
14,16 ... Dichroic mirror,
15 ... Prism,
17 ... band pass filter,
20, 24 ... detector,
21: ND filter,
25 ... central axis,
26 ... Holder,
27 ... Spindle motor,
28 ... Head,
29 ... Glass stage,
40 ... PC.

Claims (5)

  1. 　光源（８，１２）から出射された光を、微小粒子（ａ，ｂ）を含む検体（７，３０）に対して照射する照射光学系（１）と、
    　上記光の照射によって上記検体中の上記微小粒子（ａ，ｂ）から発せられた光を検出する光検出光学系（３，４）と、
    　上記光検出光学系（３，４）によって検出された上記微小粒子（ａ，ｂ）からの光の強度に基づいて、上記微小粒子を検出する検出部（４０）と
    を備え、
    　上記照射光学系（１）および上記光検出光学系（３，４）は、第１走査方向とこの第１走査方向に交差する第２走査方向とに、上記検体（７，３０）を相対的に二次元走査するようになっており、
    　上記検出部（４０）は、
    　上記照射光学系（１）および上記光検出光学系（３，４）が上記第１走査方向に走査した場合の上記微小粒子（ａ，ｂ）からの光の強度に基づいて、各走査ライン毎に、上記微小粒子（ａ，ｂ）が存在する存在領域を判定し、
    　互いに隣接する上記走査ラインにおける上記微小粒子（ａ，ｂ）の存在領域を比較することによって、上記両走査ラインに在る上記存在領域の組みが同一微小粒子に起因するか否かを判定する
    ようになっている
    ことを特徴とする微小粒子検出装置。     An irradiation optical system (1) for irradiating the specimen (7, 30) containing the fine particles (a, b) with the light emitted from the light source (8, 12);
    A light detection optical system (3, 4) for detecting light emitted from the microparticles (a, b) in the specimen by the light irradiation;
    A detection unit (40) for detecting the microparticles based on the intensity of light from the microparticles (a, b) detected by the photodetection optical system (3,4),
    The irradiation optical system (1) and the light detection optical system (3,4) relatively move the sample (7, 30) in the first scanning direction and the second scanning direction intersecting the first scanning direction. 2D scanning,
    The detection unit (40)
    Based on the intensity of light from the fine particles (a, b) when the irradiation optical system (1) and the light detection optical system (3,4) scan in the first scanning direction, And determining the existence region where the microparticles (a, b) are present,
    By comparing the existing areas of the microparticles (a, b) in the scanning lines adjacent to each other, it is determined whether or not a set of the existing areas in the two scanning lines is caused by the same microparticle. A microparticle detection device characterized by the above.
  2. 　請求項１に記載の微小粒子検出装置において、
    　上記検出部（４０）は、上記両走査ラインに在る上記存在領域の組みが同一微小粒子に起因すると判定した場合には、上記両走査ラインの何れか一方の走査ラインと、当該の走査ラインに隣接する新たな走査ラインとにおける、上記微小粒子の存在領域を比較することによって、上記新たな隣接走査ラインに在る上記存在領域の組みが同一微小粒子に起因するか否かを判定するようになっている
    ことを特徴とする微小粒子検出装置。     The fine particle detection apparatus according to claim 1,
    When the detection unit (40) determines that the set of the existing regions present in both the scan lines is caused by the same minute particle, the scan line of either of the two scan lines and the scan line By comparing the existing area of the microparticles in a new scanning line adjacent to the same, it is determined whether or not the set of the existing areas in the new adjacent scanning line is caused by the same microparticle. A microparticle detection device characterized by the above.
  3. 　請求項１または請求項２に記載の微小粒子検出装置において、
    　上記検出部（４０）は、互いに隣接する上記走査ラインにおける上記微小粒子の存在領域に関して、上記第１走査方向の位置が互いに重なっている場合には、上記両走査ラインに在る上記存在領域の組みは同一の微小粒子の存在領域であると判定するようになっている
    ことを特徴とする微小粒子検出装置。     In the microparticle detection apparatus according to claim 1 or 2,
    When the positions in the first scanning direction are overlapped with each other with respect to the region where the microparticles exist in the scanning lines adjacent to each other, the detection unit (40) A microparticle detection apparatus characterized in that a set is determined to be an area where the same microparticles exist.
  4. 　請求項１から請求項３までの何れか一つに記載の微小粒子検出装置において、
    　上記検出部（４０）は、互いに隣接する上記走査ラインに、同一微小粒子に起因すると判定された上記存在領域が在る場合には、上記同一微小粒子に起因する上記存在領域に基づいて上記同一微小粒子の位置情報を設定するようになっている
    ことを特徴とする微小粒子検出装置。     In the microparticle detection apparatus according to any one of claims 1 to 3,
    The detection unit (40), when there is the existence area determined to be caused by the same microparticles in the scanning lines adjacent to each other, the detection unit (40) is configured based on the existence area caused by the same microparticles. A fine particle detection apparatus configured to set position information of fine particles.
  5. 　請求項１から請求項４までの何れか一つに記載の微小粒子検出装置において、
    　上記検出部（４０）は、互いに隣接する上記走査ラインに、同一微小粒子に起因すると判定された上記存在領域が在る場合には、上記同一微小粒子に起因する上記存在領域に基づいて上記同一微小粒子の形状情報またはサイズ情報を設定するようになっている
    ことを特徴とする微小粒子検出装置。     In the microparticle detection apparatus according to any one of claims 1 to 4,
    The detection unit (40), when there is the existence area determined to be caused by the same microparticles in the scanning lines adjacent to each other, the detection unit (40) is configured based on the existence area caused by the same microparticles. A microparticle detection apparatus configured to set shape information or size information of microparticles.
PCT/JP2015/065303 2014-08-11 2015-05-27 Microparticle detection device WO2016024430A1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113624649A (en) * 2021-08-05 2021-11-09 西安航空学院 Road aggregate needle flake content detection system and method based on machine vision

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP6834676B2 (en) * 2017-03-28 2021-02-24 株式会社Ｊｖｃケンウッド Analytical device and analytical method
JP2019100988A (en) * 2017-12-08 2019-06-24 株式会社島津製作所 Particle image analysis device, and particle image analysis method
ES2938687T3 (en) * 2019-04-03 2023-04-13 Mecwins S A Biomarker Optical Detection Procedure
JP7056773B2 (en) * 2021-02-02 2022-04-19 株式会社Ｊｖｃケンウッド Analytical equipment and analytical method

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5264991A (en) * 1975-11-26 1977-05-28 Nippon Regulator Kk Particle image measuring method
JPS5425760A (en) * 1977-07-28 1979-02-26 Resuka Kk Device of measuring maximum diameter of particle
JPS63261139A (en) * 1987-04-17 1988-10-27 Hamamatsu Photonics Kk Particle counter
JP2002310886A (en) * 2001-04-11 2002-10-23 Canon Inc Analyzing method and device by disc cytometry
JP2004325091A (en) * 2003-04-22 2004-11-18 Matsushita Electric Ind Co Ltd Cell count method
JP2005156538A (en) * 2003-10-30 2005-06-16 Matsushita Electric Ind Co Ltd Optical analysis device and particle counting method for it

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS5264991A (en) * 1975-11-26 1977-05-28 Nippon Regulator Kk Particle image measuring method
JPS5425760A (en) * 1977-07-28 1979-02-26 Resuka Kk Device of measuring maximum diameter of particle
JPS63261139A (en) * 1987-04-17 1988-10-27 Hamamatsu Photonics Kk Particle counter
JP2002310886A (en) * 2001-04-11 2002-10-23 Canon Inc Analyzing method and device by disc cytometry
JP2004325091A (en) * 2003-04-22 2004-11-18 Matsushita Electric Ind Co Ltd Cell count method
JP2005156538A (en) * 2003-10-30 2005-06-16 Matsushita Electric Ind Co Ltd Optical analysis device and particle counting method for it

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113624649A (en) * 2021-08-05 2021-11-09 西安航空学院 Road aggregate needle flake content detection system and method based on machine vision
CN113624649B (en) * 2021-08-05 2023-12-08 西安航空学院 System and method for detecting needle-shaped content of road aggregate based on machine vision

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