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WO2018135404A1 - Disque de réception d'échantillon et détecteur de fluorescence - Google Patents

Disque de réception d'échantillon et détecteur de fluorescence Download PDF

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Publication number
WO2018135404A1
WO2018135404A1 PCT/JP2018/000620 JP2018000620W WO2018135404A1 WO 2018135404 A1 WO2018135404 A1 WO 2018135404A1 JP 2018000620 W JP2018000620 W JP 2018000620W WO 2018135404 A1 WO2018135404 A1 WO 2018135404A1
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WO
WIPO (PCT)
Prior art keywords
signal
track
sample storage
disk
sample
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Application number
PCT/JP2018/000620
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English (en)
Japanese (ja)
Inventor
謙司 永冨
靖裕 間宮
正教 石橋
Original Assignee
パナソニック株式会社
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Publication of WO2018135404A1 publication Critical patent/WO2018135404A1/fr

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    • 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
    • G01N21/01—Arrangements or apparatus for facilitating the optical investigation
    • G01N21/03—Cuvette constructions
    • 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
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N37/00—Details not covered by any other group of this subclass

Definitions

  • the present invention relates to a sample storage disk for storing a sample prepared by fluorescently staining a subject such as a cell and a fluorescence detection device used therewith.
  • Detecting cells infected with pathogenic bacteria or cells having a predetermined form from a large number of cells is particularly important in the medical field such as clinical sites.
  • a technique for performing such cell detection quickly and easily for example, a technique described in Patent Document 1 is introduced.
  • a fluorescently labeled antigen to be detected is fixed to a track on a disc using the principle of a sandwich method using an antigen-antibody reaction. Thereafter, the track is scanned with laser light serving as excitation light to generate fluorescence from the antigen to be detected, and the antigen to be detected is detected and counted.
  • address information in the radial direction and the track direction can be obtained from a disk by recording an address signal in advance in a track portion that is not connected to a flow path into which a sample flows.
  • address information in the radial direction and the track direction can be obtained from a disk by recording an address signal in advance in a track portion that is not connected to a flow path into which a sample flows.
  • the sample storage disk includes a substrate, a track formed on the upper surface of the substrate so as to turn around the center of the disk, and a sample storage unit disposed on the upper side of the track to store the sample.
  • the track is configured to be scanned in the scanning direction.
  • the track includes a plurality of track portions that are arranged in the radial direction away from the center of the disk and straddle the sample storage portion. Synchronization adjustment signals are recorded on the upstream side and the downstream side of the sample container in the scanning direction of each of the plurality of track portions.
  • This sample storage disk can generate a fluorescent image satisfactorily even when rotation unevenness occurs.
  • FIG. 1A is a plan view of a sample storage disk according to the embodiment.
  • FIG. 1B is an enlarged sectional view taken along line 1B-1B of the sample storage disk shown in FIG. 1A.
  • FIG. 2 is an enlarged view of the sample storage disk according to the embodiment.
  • FIG. 3A is a plan view schematically showing an area of the sample storage disk according to the embodiment.
  • FIG. 3B is a plan view schematically showing a zone of the sample storage disk according to the embodiment.
  • FIG. 4 is a diagram showing a groove and a land of the sample storage disk according to the embodiment developed in a straight line.
  • FIG. 5A is a diagram showing a format of each field of the sample storage disk according to the embodiment.
  • FIG. 5B is a diagram schematically illustrating an angle range of each field of the sample storage disk according to the embodiment.
  • FIG. 6A is a diagram showing a signal format of each field of the sample storage disk according to the embodiment.
  • FIG. 6B is a schematic enlarged view of the sample storage disk according to the embodiment.
  • FIG. 7 is a configuration diagram of a fluorescence detection pickup of the fluorescence detection apparatus according to the embodiment.
  • FIG. 8 is a configuration diagram of a signal calculation circuit of the fluorescence detection device according to the embodiment.
  • FIG. 9 is a configuration diagram of the fluorescence detection apparatus according to the embodiment.
  • FIG. 10A is a flowchart illustrating a fluorescence signal extraction process of the fluorescence detection device according to the embodiment.
  • FIG. 10B is a flowchart illustrating a cut-out signal invalidation process of the fluorescence detection device according to the embodiment.
  • FIG. 11 is a diagram for explaining a fluorescence signal cut-out process of the fluorescence detection device according to the embodiment.
  • FIG. 12 is a diagram schematically illustrating a method of generating a fluorescence image of the fluorescence detection device according to the embodiment.
  • FIG. 13A is a diagram illustrating a setting example of the extraction range of the fluorescence detection device according to the embodiment.
  • FIG. 13B is a diagram illustrating a setting example of the extraction range of the fluorescence detection device according to the embodiment.
  • FIG. 14 is a diagram illustrating an operation according to the first modification of the fluorescence detection device according to the embodiment.
  • FIG. 15 is a view schematically showing the structure of another semipermeable membrane according to the embodiment.
  • FIG. 1A is a plan view of a sample storage disk 100 according to the embodiment.
  • FIG. 1B is an enlarged cross-sectional view taken along line 1B-1B of the sample storage disk 100 shown in FIG. 1A, and shows a partially enlarged cross section of the sample storage disk 100 on a plane passing through the disk center Pc.
  • the sample storage disk 100 is used, for example, to detect red blood cells infected with malaria parasites.
  • the sample storage disk 100 has a disk shape similar to that of an optical disk such as a CD or a DVD, and a circular opening 101a is formed at the disk center Pc.
  • the sample storage disk 100 includes a substrate 102 and a substrate 101 bonded to the upper surface 102p of the substrate 102.
  • the substrate 101 constitutes a sample storage unit 101b.
  • the substrates 101 and 102 are made of a resin material.
  • the substrate 102 is made of a material that can transmit light.
  • FIG. 1A By bonding the substrate 101 to the substrate 102, nine sample accommodating portions 101b are formed as shown in FIG. 1A.
  • the sample storage portions 101b are arranged at regular intervals in the disc circumferential direction Dc with the disc center Pc as the center. Further, two boundaries (ends) aligned in the disk circumferential direction Dc of one sample storage portion 101b extend radially from the disk center Pc so as to be away from the disk center Pc.
  • the nine sample storage portions 101b extend over an angular range Wa centered on the disc center Pc.
  • the sample storage unit 101b is a space having a predetermined height. When viewed from above, the sample container 101b has a trapezoidal shape with rounded corners.
  • the nine sample storage portions 101b have the same shape and are arranged at the same position in the disk radial direction Dr perpendicular to the disk circumferential direction Dc from the disk center Pc.
  • Two holes 101c extending to the upper surface 101p are formed on the inner peripheral side facing the disc center Pc of the sample storage unit 101b.
  • the sample storage portion 101b is filled with the sample 100Sa from the one hole 101c.
  • Sample 100Sa is prepared such that the malaria parasite in red blood cells RC is labeled with a fluorescent dye.
  • the two holes 101c are closed with a lid.
  • the sample 100Sa prepared from nine types of specimens is filled in the sample storage unit 101b.
  • FIG. 1B schematically shows the red blood cells RC stored in the sample storage unit 101b.
  • the track 102c is composed of a series of grooves 111 that spirally turn around the disk center Pc. Groove 111 is formed in the track area 102a indicated by hatching in FIG. 1A from the outermost circumference that is the outermost edge farthest from the disk center Pc of the track area 102a to the innermost circumference that is the outermost edge closest to the disk center Pc.
  • the substrate 102 is formed by injection molding by the same process as that for CD and DVD.
  • the semi-transmissive film 102d is formed by a sputtering process.
  • the semi-transmissive film 102d reflects a part of the laser light incident from the lower surface 102q of the substrate 102 through the lower surface 102dq of the semi-transmissive film 102d, and transmits the remainder of the laser light to the semi-transmissive film 102d, thereby forming the semi-transmissive film 102d.
  • the sample is guided to the sample container 101b through the upper surface 102dp.
  • the semi-transmissive film 102d transmits the fluorescence generated in the sample storage portion 101b to the substrate 102.
  • the reflectivity of the semi-transmissive film 102d is about 5% to 20% so that more laser light can be guided to the sample storage portion 101b and more fluorescence can be transmitted to the base portion 102b of the substrate 102. Is set.
  • the sample storage disk 100 is divided into nine areas A0 to A8 in the disk circumferential direction Dc. Each area includes one sample container 101b. As will be described later, one track portion Ta in each area of the track 102c constitutes one unit of information recording area. Various signals are recorded in portions of the track portion Ta that do not overlap the sample storage portion 101b when viewed from above. In the present embodiment, these signals are recorded by a pit string composed of one or more bits.
  • FIG. 2 is an enlarged view of the semi-transmissive film 102d located on the upper surface 102p of the substrate 102, and schematically shows the structure of the groove 111, the land 112, and the pit 113.
  • FIG. 2 shows only the semipermeable membrane 102d.
  • the upper side is the substrate 102 side. That is, in FIG. 2, the upper surface 102dp of the semi-permeable membrane 102d faces downward and the lower surface 102dq faces upward.
  • a groove 111 is formed on the substrate 102 (semi-transmissive film 102d).
  • the lands 112 connected to the grooves 111 and between the grooves 111 constitute an upper surface 102 p of the substrate 102.
  • a pit 113 is formed in a groove 111 corresponding to a portion of the track portion Ta that does not overlap the sample accommodating portion 101b, and a predetermined signal is recorded.
  • the groove 111 includes pits 113 and spaces 113s. In the space 113s, the pit 113 is not formed, and the groove 111 extends monotonously. The format of the signal to be recorded will be described later. No signal is recorded on the land 112 between adjacent grooves 111. Further, the groove 111 and the land 112 extend spirally around the disc center Pc without meandering.
  • the beam spot B1 of the laser beam applied to the lower surface 102q of the substrate 102 moves relatively along the groove 111 and scans the track 102c in the scanning direction Ds.
  • the beam spot B1 scans the groove 111 (track 102c) from the outermost peripheral part farthest from the disk center Pc of the groove 111 toward the innermost peripheral part closest to the disk center Pc.
  • the laser beam forming the beam spot B1 hits the lower surface 102dq of the semi-transmissive film 102d, a part of the laser beam is reflected by the lower surface 102q as described above and becomes reflected light.
  • the intensity of the reflected light from the groove 111 decreases.
  • the reflected light is modulated by the pits 113 and the intensity of the reflected light changes.
  • the photodetector receives the modulated reflected light and outputs a detection signal that changes according to the intensity of the reflected light. By demodulating the detection signal, various information recorded in the pit 113 is reproduced.
  • the diameter of the beam spot B1 is substantially the same as the pitch of the tracks 102c (grooves 111) extending spirally in the disc radial direction Dr, that is, the track pitch that is the interval between the track portions Ta adjacent to each other in the disc radial direction Dr. In the embodiment, the track pitch is about 0.3 ⁇ m to 2.0 ⁇ m.
  • FIG. 3A is a plan view of the sample storage disk 100, schematically showing areas A0 to A8 arranged in the disk circumferential direction Dc.
  • FIG. 3B is a plan view of the sample storage disk 100.
  • the track region 102a of the sample storage disk 100 is divided into a plurality of zones Z0 to Zn arranged in the disk radial direction Dr.
  • FIG. 3B schematically shows a plurality of zones Z0 to Zn.
  • Areas A0 to A8 shown in FIG. 3A and zones Z0 to Zn shown in FIG. 3B are logically assigned to the sample storage disk 100 in order to set a signal format to be described later on the track 102c in relation to the sample storage unit 101b.
  • the areas A0 to A8 and the zones Z0 to Zn are not partitioned by physical structures such as barriers and grooves.
  • the sample storage disk 100 is divided into a plurality of areas at a predetermined constant angular interval with respect to the disc center Pc. It is divided into A0 to A8.
  • a track portion included in each area is a track portion Ta shown in FIG. 1A.
  • the track area 102a shown in FIG. 1A is detected between the outer area 102e farthest from the disk center Pc, the inner area 102f closest to the disk center Pc, and the outer area 102e and the inner area 102f in the disk radial direction Dr. It is divided into an area 102g.
  • the outer area 102e is a lead-in area
  • the inner area 102f is a lead-out area and an appearance identification area.
  • the groove 111 of the lead-in area (outer area 102e), various pieces of information necessary for scanning the sample storage disk 100 are recorded as bit strings.
  • a signal indicating the lead-out area is recorded by a pit string.
  • the appearance identification area (inner area 102f) is provided with a structure for visually displaying the type and the like of the sample storage disk 100 by making the groove 111 discontinuous.
  • the appearance identification area is set on the inner peripheral side near the disc center Pc in the lead-out area.
  • the detection region 102g of the sample storage disk 100 is divided into a plurality of zones Z0 to Zn in the disk radial direction Dr.
  • the sample storage disk 100 is divided into, for example, 75 zones.
  • the number of track portions Ta arranged in the disk radial direction Dr included in each zone in the track 102c is the same.
  • signals are recorded on the plurality of track portions Ta at a constant angular velocity.
  • the track 102c (groove 111) of one zone is scanned by the beam spot B1 at the same angular velocity with respect to the disc center Pc.
  • the angular velocity of each zone is set so that the track portion Ta of the track 102c (groove 111) at the center position of the zone in the disc radial direction Dr is scanned by the beam spot B1 at the same linear velocity.
  • FIG. 4 shows the groove 111 and the land 112 of each zone developed in a straight line.
  • the groove 111 and the land 112 for one round are shown by one straight line.
  • the lengths of the grooves 111 and lands 112 shown in FIG. 4 are not physical lengths, but are standardized so that the length of one round is the same in all the grooves 111 and lands 112 for convenience. Has been.
  • the detection area 102g is divided into a plurality of zones Z0 to Zn in the disk radial direction Dr.
  • Each zone includes a plurality of tracks 102c (grooves 111) arranged in the disk radial direction Dr.
  • track numbers T0 to Tm from the outer peripheral side are given to the track portion Ta of the track 102c in one zone.
  • the number of track portions Ta of the track 102c included in one zone is, for example, 800.
  • FIG. 5A shows the formats of the fields F1 to F8 set in the track portion Ta (groove 111) in each of the areas A0 to A8.
  • FIG. 5B schematically shows the angle range of the fields F1 to F8.
  • fields F1 to F8 are set in each of a plurality of track portions Ta (groove 111) in one area Ax.
  • the fields F2, F5, and F6 no signal is recorded with the bit 113 (see FIG. 2), and only the groove 111 (G) including only the monotonically extending space 113s (see FIG. 2) is formed.
  • the field F5 overlaps the sample storage portion 101b in the entire length. That is, both ends of the field F5 coincide with two boundaries (ends) arranged in the disk circumferential direction Dc of the sample storage unit 101b. No signal is recorded on the track portion that overlaps the sample storage portion 101b when viewed from above, and only the groove 111 that extends monotonously and flatly is formed.
  • the track portion Ta starts at the start end SP and ends at the end EP along the scanning direction Ds.
  • the start ends SP of all track portions Ta in the same area are aligned in one disk radial direction Dr, that is, located on a straight line Lr1 extending from the disk center Pc in the disk radial direction Dr1.
  • the end points EP of all track portions Ta in the same area are aligned in one disc radial direction Dr, that is, located on a straight line Lr2 extending from the disc center Pc in the disc radial direction Dr2.
  • the start ends of the fields F5 in all the track portions Ta in the same area are aligned in the disc radial direction Dr, and the end ends of the field F5 are aligned in another disc radial direction Dr.
  • the start ends of the field F1 in all the track portions Ta in the same area are aligned in another disk radial direction Dr, and the ends of the field F1 are aligned in another disk radial direction Dr.
  • the start ends of the field F3 in all the track portions Ta in the same area are aligned in another disk radial direction Dr, and the ends of the field F3 are aligned in another disk radial direction Dr.
  • the start ends of the fields F4 in all track portions Ta in the same area are aligned in another disk radial direction Dr, and the ends of the field F4 are aligned in another disk radial direction Dr.
  • the start ends of the fields F6 in all the track portions Ta in the same area are aligned in another disk radial direction Dr, and the ends of the field F6 are aligned in another disk radial direction Dr.
  • the start ends of the field F8 in all the track portions Ta in the same area are aligned in another disk radial direction Dr, and the ends of the field F8 are aligned in another disk radial direction Dr.
  • FIG. 6A shows a signal format of fields F1 to F8 of a certain track portion Ta1 among a plurality of track portions Ta.
  • Each of the plurality of track portions Ta has the same configuration as the track portion Ta1.
  • a hatched portion indicates a region where the pit 113 is formed in the groove 111, and a white portion where the hatched hatch is not illustrated indicates a region of only the groove 111.
  • the time length 1T indicates the time length of the minimum pit when the groove 111 is scanned at a constant angular velocity as described above.
  • a signal En composed of pits and spaces alternately repeated 10 times is recorded. Both the pit and the space of the signal En have a time length 2T that is twice the time length 1T.
  • the signal En recorded in the field F1 indicates the start point SP (see FIG. 5A) of the track portion Ta1 of one area, and the signal En recorded in the field F8 indicates the end point EP of the track portion Ta1 of one area. .
  • Field F4 starts with a space having a time length 8T which is eight times the time length 1T, a pit having a time length 1T and a space having a time length 1T which are alternately repeated four times after the space.
  • a signal V3 is recorded.
  • the start signal V3 is a synchronization adjustment signal for correcting the synchronization shift of the fluorescence signal when the fluorescence image is generated. The synchronization adjustment using the start signal V3 will be described later.
  • the field F3 includes two header areas HE1 and HE2.
  • an identification signal for identifying the header area HE1 an address signal indicating the position of the track portion Ta1, and an error correction signal for performing error detection or error correction on the address signal are recorded. These signals have a fixed bit length.
  • the address signal includes the track number of the track portion Ta1 of the track numbers T0 to Tm shown in FIG. 4, the zone number indicating the zone including the track portion Ta of the zones Z0 to Zn, and the areas A0 to A8. And the area number of the area including the track portion Ta1.
  • signals similar to those in the header area HE1 are recorded.
  • the field F7 includes two footer areas FT1 and FT2.
  • an identification signal Id8 In the footer area FT1, an identification signal Id8, an address signal, and an error correction signal are recorded. These signals have a fixed bit length.
  • the address signal includes the track number of the track portion Ta1 of the track numbers T0 to Tm shown in FIG. 4, the zone number indicating the zone including the track portion Ta1 of the zones Z0 to Zn, and the areas A0 to A8. And an area number indicating an area including the track portion Ta1.
  • a signal similar to that in the footer area FT1 is recorded.
  • the identification signals Id8 in the footer areas FT1 and FT2 are different from the identification signals in the header areas HE1 and HE2.
  • the address signals in the footer areas FT1 and FT2 are the same as the address signals in the header areas HE1 and HE2.
  • digital signals (bit signals) having values of 1 and 0 are recorded by pits and spaces.
  • FIG. 6A further shows the signal format of the identification signal Id8 in the footer areas FT1 and FT2.
  • the identification signal Id8 of the footer areas FT1 and FT2 is also a synchronization adjustment signal for correcting the synchronization shift of the fluorescence signal when the fluorescence image is generated.
  • the identification signal Id8 of the footer areas FT1 and FT2 includes a pit b5 and a space s5 that are alternately repeated four times, and a pit b1 and a space s1 that are alternately repeated 20 times.
  • the pit b5 and the space s5 have a time length 5T that is five times the time length 1T.
  • the pit b1 and the space s1 have a time length of 1T.
  • the identification signal Id8 and the start signal V3 the synchronization deviation of the fluorescence signal is corrected.
  • the synchronization adjustment using the identification signal Id8 in the footer area FT1 and the start signal V3 in the field F4, both of which are synchronization adjustment signals, will be described later.
  • each field other than the fields F3 and F7 in which the address signal is recorded has a space having the same time length as the pits having the same time length arranged in the same arrangement.
  • the same signal is recorded.
  • the pits and spaces formed in these fields are aligned in the disc radial direction Dr in all track portions Ta in the same zone and the same area.
  • the pits and spaces formed in the fields F1 and F8 are aligned in the disc radial direction in the track portion Ta included in all the zones of the same area.
  • the pits and spaces formed in the fields F3 and F7 are shifted in the circumferential direction between the track portions Ta because the lengths of the pits and spaces change according to the contents of the address signal.
  • FIG. 6B is a schematic enlarged view of the sample storage disk 100, and shows a field F4 in a track portion Ta of track numbers T11 to T15. As shown in FIG.
  • a space Sc1 having a time length 8T, a pit Pt1 having a time length 1T, and a space Sc2 having a time length 1T along the scanning direction Ds A pit Pt2 having a time length 1T, a space Sc3 having a time length 1T, a pit Pt3 having a time length 1T, a space Sc4 having a time length 1T, a pit Pt4 having a time length 1T, and a time length 1T Space Sc5 is arranged in this order. As shown in FIG.
  • both ends of the space Sc1 of the field F4 in all the track portions Ta of each zone including the track numbers T11 to T15 are aligned in the disk radial directions Dr11 and Dr12 that are separated from the disk center Pc. And located on straight lines Lr11 and Lr12 extending in the disk radial direction Dr11 and Dr12 from the disk center Pc, respectively.
  • Both ends of the pit Pt1 of the field F4 of all the track portions Ta of each zone of each area including the track numbers T11 to T15 are aligned in the disk radial directions Dr12 and Dr13 away from the disk center Pc, and the disk diameter from the disk center Pc. They are located on straight lines Lr12 and Lr13 extending in directions Dr12 and Dr13, respectively.
  • Both ends of the space Sc2 of the field F4 of all track portions Ta of each zone of each area including the track numbers T11 to T15 are aligned in the disk radial directions Dr13 and Dr14 away from the disk center Pc, and the disk diameter from the disk center Pc. They are located on straight lines Lr13 and Lr14 extending in directions Dr13 and Dr14, respectively. Both ends of the pit Pt2 of the field F4 of all track portions Ta of each zone of each area including the track numbers T11 to T15 are aligned in the disk radial directions Dr14 and Dr15 away from the disk center Pc, respectively. They are located on straight lines Lr14 and Lr15 extending in directions Dr14 and Dr15, respectively.
  • Both ends of the space Sc3 of the field F4 of all track portions Ta of each zone of each area including the track numbers T11 to T15 are aligned in the disk radial directions Dr15 and Dr16 away from the disk center Pc, and the disk diameter from the disk center Pc. They are located on straight lines Lr15 and Lr16 extending in the directions Dr15 and Dr16, respectively.
  • Both ends of the pit Pt3 of the field F4 of all track portions Ta of each zone of each area including the track numbers T11 to T15 are aligned in the disc radial directions Dr16 and Dr17, respectively, away from the disc center Pc. They are located on straight lines Lr16 and Lr17 extending in directions Dr16 and Dr17, respectively.
  • Both ends of the space Sc4 of the field F4 of all the track portions Ta of each zone of each area including the track numbers T11 to T15 are aligned in the disk radial directions Dr17 and Dr18 away from the disk center Pc. They are located on straight lines Lr17 and Lr18 extending in the directions Dr17 and Dr18, respectively. Both ends of the pit Pt4 of the field F4 of all track portions Ta of each zone of each area including the track numbers T11 to T15 are aligned in the disc radial directions Dr18 and Dr19, respectively, away from the disc center Pc. They are located on straight lines Lr18 and Lr19 extending in directions Dr18 and Dr19, respectively.
  • Both ends of the space Sc5 of the field F4 of all the track portions Ta of each zone of each area including the track numbers T11 to T15 are aligned in the disk radial directions Dr19 and Dr110 that are separated from the disk center Pc, and the disk diameter from the disk center Pc. They are located on straight lines Lr19 and Lr110 extending in directions Dr19 and Dr110, respectively.
  • the end of the start signal V3 recorded in the field F4 is aligned in the disc radial direction Dr in the track portion Ta included in all the zones of the same area. That is, the end of the start signal V3 recorded in the field F4 is aligned with the direction DV3 from the disc center Pc in the track portion Ta included in all the zones of the same area.
  • the end of the identification signal Id8 in the footer area FT1 is aligned in the disk radial direction in the track portion Ta included in all the zones of the same area. That is, the end of the identification signal Id8 in the footer region FT1 is aligned in the direction DFT1 from the disc center Pc in the track portion Ta included in all the zones of the same area.
  • FIG. 7 is a configuration diagram of a fluorescence detection pickup 200 for reading fluorescence from the sample storage disk 100.
  • fluorescence is detected from the sample storage portion 101b of the sample storage disk 100 using the fluorescence detection pickup 200.
  • fluorescence is detected from the sample storage disk 100 in order to determine whether the red blood cells RC are infected with malaria parasites.
  • the sample 100Sa is prepared so that the malaria parasite in the red blood cells RC is labeled with a fluorescent dye.
  • the fluorescent dye emits fluorescence having a wavelength of about 450 to 540 nm when irradiated with light having a wavelength of 405 nm, for example.
  • the sample 100Sa thus prepared is filled into the nine sample storage portions 101b of the sample storage disk 100 for each specimen. Thereafter, the opening 101a (see FIG. 1A) of the sample storage disk 100 is fixed to the turntable 230 supported by the spindle motor 220.
  • the fluorescence detection pickup 200 includes a semiconductor laser 201, a half-wave plate 202, a polarizing beam splitter (PBS) 203, a collimator lens 204, a quarter-wave plate 205, an objective lens 206, and an objective lens actuator. 207, a dichroic prism 208, an anamorphic lens 209, a photodetector 210, and a fluorescence detector 211.
  • PBS polarizing beam splitter
  • the semiconductor laser 201 emits laser light having a wavelength of about 405 nm.
  • the polarization direction of the laser light emitted from the semiconductor laser 201 is adjusted by the half-wave plate 202 so as to be S-polarized with respect to the PBS 203.
  • the laser beam is reflected by the PBS 203 and enters the collimator lens 204.
  • the PBS 203 has polarization dependency of characteristics such as reflection and transmission only with respect to light having a wavelength of about 405 nm of the laser light emitted from the semiconductor laser 201, and the polarization dependence of the above characteristics for light with a wavelength of about 450 to 540 nm. Does not have sex.
  • the collimator lens 204 converts laser light incident from the PBS 203 side into parallel light.
  • the quarter-wave plate 205 converts the laser light incident from the collimator lens 204 side into circularly polarized light, and the polarization direction when the laser light incident from the objective lens 206 side is incident from the collimator lens 204 side.
  • the laser light reflected by the semi-transmissive film 102d of the sample storage disk 100 passes through the PBS 203 as it is and is not reflected toward the fluorescence detector 211.
  • the objective lens 206 converges the laser light incident from the 1 ⁇ 4 wavelength plate 205 side on the semi-transmissive film 102d of the sample storage disk 100.
  • the objective lens actuator 207 is displaced by driving the objective lens 206 in the focus direction and the tracking direction so that the laser beam converges on the groove 111 of the sample storage disk 100 by a servo circuit 50 (see FIG. 9) described later.
  • the dichroic prism 208 transmits light having a wavelength of about 405 nm of the laser light emitted from the semiconductor laser 201, and emits light having a wavelength other than the wavelength of the laser light emitted from the semiconductor laser 201, for example, light having a wavelength of about 450 to 540 nm. It is configured to reflect. As a result, the fluorescence incident from the PBS 203 side is reflected by the dichroic prism 208, and the laser light incident from the PBS 203 side passes through the dichroic prism 208.
  • the anamorphic lens 209 introduces astigmatism into the laser light transmitted through the dichroic prism 208.
  • the laser light that has passed through the anamorphic lens 209 enters the photodetector 210.
  • the photodetector 210 has a four-divided sensor for receiving laser light on the light receiving surface.
  • the detection signal output from the photodetector 210 is processed by a signal arithmetic circuit 300 (see FIG. 8) described later.
  • the fluorescence reflected by the dichroic prism 208 is guided to the light receiving surface of the fluorescence detector 211 while being converged by the collimator lens 204.
  • the fluorescence detector 211 has a sensor provided on the light receiving surface for receiving fluorescence.
  • a fluorescence signal that is a detection signal of the fluorescence detector 211 is amplified by a signal amplification circuit.
  • a barrier or the like for preventing the laser light emitted from the semiconductor laser 201 from entering the fluorescence detector 211 is appropriately used. It is preferable to arrange in the optical system.
  • FIG. 8 is a configuration diagram of the signal arithmetic circuit 300.
  • FIG. 9 is a configuration diagram of the fluorescence detection apparatus 1 according to the first embodiment.
  • the sample storage disk 100 and the fluorescence detection apparatus 1 constitute a fluorescence detection system.
  • the light detector 210 has a quadrant sensor for receiving laser light as described above.
  • the upper left sensor 2101, the upper right sensor 2102, the lower right sensor 2103, and the lower left sensor 2104 of the quadrant sensor output detection signals S1, S2, S3, and S4, respectively, based on the beam spot of the received laser beam.
  • the signal arithmetic circuit 300 processes the detection signals S1 to S4 to generate a focus error signal FE, a tracking error signal TE, and a reproduction RF signal RF.
  • the focus error signal FE and the tracking error signal TE are generated according to the astigmatism method and the one-beam push-pull method used in existing optical disc apparatuses.
  • the signal operation circuit 300 includes adders 301 to 304 and 307 and subtractors 305 and 306.
  • the adder 301 outputs a signal (S1 + S3) obtained by adding the detection signals S1 and S3 to the subtractor 305
  • the adder 302 outputs a signal (S2 + S4) obtained by adding the detection signals S2 and S4 to the subtractor 305.
  • the adder 303 outputs a signal (S1 + S4) obtained by adding the detection signals S1 and S4 to the subtractor 306 and the adder 307
  • the adder 304 adds the signal (S2 + S3) obtained by adding the detection signals S2 and S3 to the subtractor 306. And output to the adder 307.
  • the subtracter 305 subtracts the output signal of the adder 302 from the output signal of the adder 301 and outputs a focus error signal FE.
  • the subtracter 306 subtracts the output signal of the adder 304 from the output signal of the adder 303 and outputs a tracking error signal TE.
  • the adder 307 adds the output signals of the adders 303 and 304 and outputs a reproduction RF signal RF (SUM signal).
  • the focal position of the objective lens 206 when the focal position of the objective lens 206 is positioned on the semi-transmissive film 102d of the sample storage disk 100, the beam spot on the four-divided sensors 2101 to 2104 of the photodetector 210 becomes a minimum circle of confusion, and a focus error signal.
  • the value of FE becomes 0.
  • the focal position of the objective lens 206 is positioned at the center position in the disk radial direction Dr of the track 102c (groove 111) of the sample storage disk 100, the beam spots on the quadrant sensors 2101 to 2104 of the photodetector 210.
  • the left side of the two sensors 2101 and 2104 are equal in size to the right side of the two sensors 2102 and 2103, and the value of the tracking error signal TE is zero.
  • the objective lens actuator 207 shown in FIG. 7 has the objective lens 206 mounted on the substrate of the sample storage disk 100 so that both the focus error signal FE and the tracking error signal TE become zero under the control of the servo circuit 50 shown in FIG.
  • the pickup 200 is driven so as to be displaced in a focus direction perpendicular to the upper surface 102p of the 102 and a tracking direction parallel to the upper surface 102p of the substrate 102.
  • the fluorescence detection apparatus 1 includes a signal processing circuit 10, an image processing unit 20, an input / output unit 30, a controller 40, and a servo. A circuit 50 and a thread motor 240 are further provided. A signal operation circuit 300 shown in FIG. 8 is provided in the fluorescence detection pickup 200.
  • the signal processing circuit 10 processes the fluorescence signal FL and the reproduction RF signal RF output from the fluorescence detection pickup 200.
  • the fluorescence signal FL is output from the fluorescence detector 211 shown in FIG. 7, and the reproduction RF signal RF is output from the adder 307 shown in FIG.
  • the signal processing circuit 10 includes a signal detection unit 11, a signal reproduction unit 12, a cutout unit 13, and a superposition unit 14.
  • the signal detection unit 11 and the signal reproduction unit 12 constitute a signal acquisition unit 11a.
  • the signal detection unit 11 processes the reproduction RF signal RF input from the fluorescence detection pickup 200, detects various signals shown in FIG. 6A, and sends the detected signals to the signal reproduction unit 12, the cutout unit 13, and the controller 40. Output.
  • the signal reproduction unit 12 reproduces the signals of the fields F3 and F7 input from the signal detection unit 11, that is, the signals of the header areas HE1 and HE2 and the footer areas FT1 and FT2, and acquires an address signal.
  • the signal reproducing unit 12 outputs the acquired address signal to the superimposing unit 14.
  • the cutout unit 13 converts each sample value obtained by sampling the fluorescence signal FL input from the fluorescence detection pickup 200 with a sampling clock of a predetermined period into a digital signal and outputs the digital signal to the superposition unit 14 as the fluorescence signal FLd. To do.
  • the cutout unit 13 starts sampling of the fluorescent signal FL in response to detection of the start signal V3 (see FIG. 5A) by the signal detection unit 11, and the signal detection unit 11 detects the identification signal Id8 of the footer region FT1. In response to this, the sampling of the fluorescence signal FL is terminated.
  • the sample storage disk 100 is rotated at a different angular velocity for each zone. Accordingly, the time during which the track portion Ta is scanned with the laser light varies from zone to zone. For this reason, when a fluorescence signal is cut out with a timing signal having the same period for each zone, the number of cut out signal groups differs for each zone.
  • the sampling clock cycle of the fluorescence signal FL in the cutout unit 13 is adjusted so that the same number of signal groups are cut out from the track portion Ta of each zone. Thereby, in each zone, the fluorescence signal FL is cut out at substantially the same angular interval.
  • the superimposing unit 14 adds the address signal input from the signal reproducing unit 12 to the fluorescent signal FLd acquired by the cutout unit 13 and outputs it to the image processing unit 20.
  • the image processing unit 20 connects the input fluorescent signal FLd based on the address signal, and generates a fluorescent image for each of the areas A0 to A8. Further, the image processing unit 20 performs image processing on the fluorescent image, counts the number of fluorescent bright spots in the fluorescent image, and calculates the infection rate of malaria in the red blood cells RC. These fluorescent images, count values, infection rates, and the like are output from the image processing unit 20 to the input / output unit 30 as needed.
  • the image processing unit 20 includes a programmable arithmetic processing circuit and a memory 20m.
  • the input / output unit 30 includes input devices such as a keyboard, a mouse, and a touch panel, and output devices such as a monitor and a speaker.
  • An instruction for starting fluorescence detection is input via the input / output unit 30.
  • the fluorescence image, the number of bright spots, the infection rate of malaria, and the like are displayed on the input / output unit 30.
  • the controller 40 includes a processing circuit such as a CPU (Central Processing Unit) and a memory such as a ROM (Read Only Memory) and a RAM (Random Access Memory), and controls each unit according to a program stored in the memory.
  • a processing circuit such as a CPU (Central Processing Unit) and a memory such as a ROM (Read Only Memory) and a RAM (Random Access Memory), and controls each unit according to a program stored in the memory.
  • the servo circuit 50 controls the objective lens actuator 207 based on the focus error signal FE and the tracking error signal TE generated by the signal calculation circuit 300 in FIG.
  • the servo circuit 50 controls the spindle motor 220 so that the zones Z0 to Zn shown in FIG. 3B are scanned by the beam spot B1 at the angular velocity set in each zone.
  • the servo circuit 50 is a thread for sending the fluorescence detection pickup 200 in the disk radial direction Dr of the sample storage disk 100 so that the beam spot B1 can be scanned from the outermost position to the innermost position of the track 102c.
  • the motor 240 is controlled.
  • the servo circuit 50, the fluorescence detection pickup 200, the spindle motor 220, and the sled motor 240 constitute a scanning unit 1200 that irradiates the sample storage disk 100 with light and scans the sample storage disk 100 in the scanning direction Ds.
  • FIG. 10A is a flowchart showing a cutting process for cutting out a fluorescent signal.
  • step S11 When the signal detection unit 11 detects the start signal V3 (see FIG. 5A) (step S11: YES), the extraction unit 13 starts extraction of the fluorescent signal FL, that is, sampling (step S12). Thereafter, when the signal detection unit 11 detects the identification signal Id8 (see FIG. 6A) of the footer region FT1 (step S13: YES), the extraction unit 13 ends extraction of the fluorescent signal FL, that is, sampling (step S14).
  • FIG. 10B is a flowchart showing the invalidation processing of the cut out signal, that is, the sampled signal.
  • the controller 40 acquires the address signal reproduced from the header areas HE1 and HE2 and the address signal reproduced from the footer areas FT1 and FT2 while scanning one track portion Ta (steps S21 and S22).
  • the controller 40 determines whether or not the address signal reproduced from the header areas HE1 and HE2 matches the address signal reproduced from the footer areas FT1 and FT2 (step S23). If the two address signals do not match (step S23: NO), the controller 40 invalidates the fluorescent signal FL cut out from the track portion Ta (step S24), and scans the track portion Ta again with laser light.
  • a process of cutting out, that is, sampling, the fluorescence signal is executed (step S25).
  • the controller 40 outputs the fluorescence signal FL cut out from the track portion Ta as the fluorescence signal FLd without invalidating it, and ends the process.
  • the laser beam spot B1 may have moved out of the groove 111 and moved to another groove while scanning the groove 111 overlapping the sample storage unit 101b. .
  • the fluorescence signal cut out in the meantime is acquired across two track portions, and is not a fluorescence signal acquired from one track portion.
  • the beam spot B1 of the laser beam is detached from the groove 111 and moved to another groove while the process shown in FIG. 10A is executed and the groove 111 overlapping the sample storage unit 101b is scanned.
  • the fluorescence signal acquired during that time is invalidated and the fluorescence signal is cut out again. Thereby, a fluorescence signal is appropriately acquired from one track portion Ta.
  • FIG. 11 is a diagram for explaining the fluorescence signal cut-out process.
  • FIG. 11 schematically shows the sampling clock Sck and signals cut out from one group of track portions Ta (track numbers T0 to Tm) included in the same area in the same zone.
  • k signal groups SP1 to SPk are obtained from one track portion Ta.
  • red blood cells RC infected with malaria are present in the sample 100Sa when the signal SPn is scanned while the track portion Ta of the track number T1 is scanned with laser light.
  • the higher the sample value of the signal the higher the hatching density.
  • the sampling value of the signal SPn of the track number T1 is higher than the other signals SP1 to SPn ⁇ 1, SPn + 1 to SPk of the track number T1 and the signals SP1 to SPk of the other tracks (track numbers T0, T2 to Tm).
  • the sample values of the signals around the signal SPn (the signals SPn ⁇ 1 and SPn + 1 of the track number T1 and the signals SPn of the track numbers T0 and T2) are higher than other signals of those signals.
  • the sample values of signals around the signal SPn (the signals SPn ⁇ 1 and Pn + 1 of the track numbers T0 and T2) are higher than those of other signals.
  • the image processing unit 20 in FIG. 9 scans a signal group including signals respectively obtained from a plurality of track portions Ta included in the same area in the same zone based on the signal input from the superimposing unit 14 and the address signal.
  • a fluorescent image showing one sample storage unit 101b is generated by arranging in the order of track numbers and track numbers. In the generation of the fluorescence image, the synchronization shift between the signal groups is corrected. Details of the method for generating the fluorescence image will be described later.
  • the image processing unit 20 analyzes the fluorescence image thus generated, counts the number of fluorescent bright spots, that is, the number of red blood cells RC infected with malaria, and based on the number, malaria of red blood cells RC contained in the sample 100Sa. Calculate the infection rate.
  • the image processing unit 20 outputs the acquired number and infection rate to the input / output unit 30 together with the fluorescence image. As a result, the fluorescence image, the number of detected malaria, the malaria infection rate, and the like are displayed on the input / output unit 30.
  • the fluorescence signal is sampled according to the sampling clock Sck having a certain period, and the signals SP1 to SPk are acquired.
  • the sample storage disk 100 is rotated at a constant angular velocity for each zone as described above.
  • the angular velocity may be uneven.
  • the rotation unevenness causes a distortion in the scanning direction in the fluorescent signal of each track portion Ta, and as a result, the accuracy of the fluorescent image may be lowered.
  • the fluorescence detection apparatus 1 has the start signal V3 recorded in the field F4 that is the upstream synchronization adjustment signal in the scanning direction Ds and the footer region FT1 that is the downstream synchronization adjustment signal in the scanning direction Ds.
  • the fluorescent signal is synchronously corrected using the identification signal Id8.
  • FIG. 12 schematically shows a method of generating a fluorescence image while performing synchronization correction in the fluorescence detection device 1 according to the present embodiment.
  • FIG. 12 shows a signal obtained by cutting out the fluorescence signal acquired from one zone of one area by the cutting unit 13 in a state where it is stored in the memory area 120m of the memory 20m and developed.
  • FIG. 12 shows a fluorescence image extracted by extracting a signal within a predetermined range from the signal developed in the memory area 220m and saving it in the memory area 220m of the memory 20m.
  • FIG. 12 shows the memory area 120m by the track portion Ta, the upstream and downstream directions in the scanning direction Ds, and the radial direction Dr indicating the outer peripheral side of the sample storage disk 100.
  • Tk,..., Tm and the signals acquired from the track portion Ta developed on the memory area 120m shown in FIG. 12 are partially extracted to generate a fluorescent image and are shown in FIG.
  • the memory area 220m shown in FIG. For example, a signal obtained from the track portion Ta with the track number T0 and stored in the memory area 120m is extracted in the extraction ranges R0-1, R0-2,..., R0-10 and stored in the memory area 220m. Signals obtained from the track portion Ta of the track number Tk and stored in the memory area 120m are extracted in the extraction ranges Rk-1, Rk-2,..., Rk-10 and stored in the memory area 220m.
  • ten extraction ranges are set for a signal group of one track portion Ta, but the number of extraction ranges is not limited to this.
  • the same number (10 in this case) of extraction ranges as the extraction ranges of the track numbers T0 and Tk are set in the signal group of other track portions Ta.
  • a signal obtained by sampling the fluorescence signal FL is present in the extraction range.
  • the number of signals extracted by sampling fluorescent signals FL included in a plurality of extraction ranges in one track portion is the same.
  • the signal group is cut from each track portion Ta during the cut-out period from the detection of the signal V3 recorded in the field F4 to the detection of the identification signal Id8 in the footer area FT1. Is issued.
  • This cut-out period changes according to the rotation unevenness in the sample storage disk 100. That is, the faster the rotation speed of the sample storage disk 100, the shorter the cutout period.
  • the cutout period Ck in the track part Ta of the track number Tk is shorter than the cutout period C0 in the track part Ta of the track number T0.
  • a synchronization shift occurs between the signal groups acquired from Ta.
  • the Sck cycle of the sampling clock for sampling the fluorescent signal FL is constant as described above, the number of signals acquired from the track portion Ta of the track number T0 is acquired from the track portion Ta of the track number Tk. More than the number of signals.
  • the extraction range for the signal group acquired from each track portion Ta is changed according to the length of the extraction period so as to eliminate this synchronization shift. For example, as shown in the memory area 120m of FIG. 12, the extraction range for the signal group with the track number Tk is shifted to the upstream side in the scanning direction Ds from the extraction range for the signal group with the track number T0.
  • the shift amount and the shift direction for shifting the extraction range for the signal group of the track portion Ta of the other target track number are determined based on the signal group of the track portion Ta of the track number T0.
  • the extraction range is shifted upstream in the scanning direction Ds, and the extraction periods C0, Ck
  • the shift amount of the extraction range is determined according to the ratio.
  • the extraction range in which the signal group of the track part Ta with the track number Tk is extracted is the scanning direction Ds. Shifted upstream.
  • the extraction range is shifted upstream in the scanning direction Ds.
  • the signal portion of the extraction range set in the signal group of each track number is developed in the memory area 220m for forming the fluorescence image.
  • FIG. 12 shows lines L0, Lk, and Lm corresponding to the track portions of track numbers T0, Tk, and Tm, respectively.
  • signals extracted from the signal group of each track portion are mapped to each line in the same order as the track number.
  • a region corresponding to each of the plurality of lines in the fluorescent image memory region 220m is divided into ten divided regions Wp.
  • a signal included in the extraction range among the signal group obtained from the track portion Ta of the track number Ti corresponding to the line Li is stored and mapped.
  • the fluorescence image is generated by mapping the signal in which the synchronization error is corrected to the fluorescence image memory area 220m. Thereby, a fluorescence image without distortion can be obtained.
  • the position on the memory of each signal sampled and extracted from the fluorescence signal FL is referred to as a pixel, and the upstream side in the scanning direction Ds from the downstream side in each line on the memory. Defines the order of the pixels.
  • extraction ranges R0-1 to R0-10 are set for the signal group of the reference track number T0.
  • ten extraction ranges R0-1 to R0-10 are set for the P1th to P11th pixels.
  • the signal may be deteriorated due to the influence of the boundary in the circumferential direction Dc of the sample container 101b.
  • the range of pixels from P1 to P11 is assumed to be able to ensure the signal quality without being influenced by the vicinity of the boundary of the sample storage unit 101b even if the sample storage disk 100 is unevenly rotated. The range is set.
  • the image processing unit 20 sets the extraction ranges Ri-1 to Ri-10 for the signal group of each track portion Ta of the track number Ti (i: integer of 2 to m).
  • the image processing unit 20 uses the following expression (1) based on the extraction period Ci of the track number Ti and the extraction period C0 of the track number T0 to extract the extraction range R0-1 to R0 of the track number T0.
  • the shift value Dp for shifting the extraction range Ri-1 to Ri-10 of the track number Ti is set for -10.
  • Dp ⁇ (Ci / C0) ⁇ 1 ⁇ ⁇ VCp (1)
  • Dp is a shift value of the p-th (p: 1 to 10) extraction range Ri-p from the upstream side.
  • VCp is the position of the center pixel in the scanning direction Ds of the p-th extraction range Ri-p from the upstream side of the track number T0.
  • the shift value Dp calculated by the equation (1) is positive when the cutout period Ci is longer than the cutout period C0, and is negative when the cutout period Ci is shorter than the cutout period C0.
  • the shift value Dp is positive
  • the p-th extraction range R1-p of the track number T1 is shifted by the absolute value of the shift value Dp upstream from the p-th extraction range R0-p of the track number T0.
  • the shift value Dp is negative, the p-th extraction range R1-p of the track number T1 is shifted to the shift position by the absolute value of the shift value Dp on the downstream side with respect to the p-th extraction range R0-p of the track number T0. Is set.
  • the shift value Dp is zero.
  • the p-th extraction range Ri-p of the track number Ti is set at the same position in the scanning direction Ds with respect to the p-th extraction range R0-p of the track number T0.
  • FIG. 13A and 13B show the first extraction range Ri-1 of the track number Ti, respectively.
  • FIG. 13A shows the extraction range Ri-1 when the cutout period Ci is shorter than the cutout period C0
  • FIG. 13B shows the extraction range Ri-1 when the cutout period Ci is longer than the cutout period C0.
  • the extraction range Ri-1 is the first extraction of the track number T0.
  • a position shifted by the absolute value of the shift value D1 is set upstream of the range R0-1.
  • the first shift value D1 of the track number Ti is positive, and therefore the extraction range Ri-1 is 1 of the track number T0.
  • a position shifted by the absolute value of the shift value D1 is set downstream from the first extraction range R0-1.
  • the setting of the extraction range and the generation of the fluorescence image are performed by the image processing unit 20 shown in FIG.
  • the image processing unit 20 develops a signal group cut out from the fluorescence signal in the internal memory 20m, and sets an extraction range for the signal group according to the above-described method.
  • the image processing unit 20 acquires the extraction period of each track number by, for example, counting an internal fixed clock having a fixed frequency, and based on the acquired extraction period (number of clocks), the expression ( The operation of 1) is executed. Then, the image processing unit 20 sets an extraction range for the signal group of each track number based on the calculated shift value. And the signal of each extraction range is preserve
  • FIG. 12 shows a method of generating a fluorescence image from a signal group obtained by cutting out the fluorescence signal acquired from one zone of one area by the cutout unit 13.
  • a plurality of zones are set in the radial direction in a region corresponding to the sample storage unit 101b arranged in one area. Therefore, when acquiring the fluorescence image of the whole sample accommodating part 101b, the process which integrates the fluorescence image acquired about each zone in radial direction Dr is performed.
  • the image processing unit 20 integrates the fluorescence images acquired for each zone in the radial direction Dr, and generates a fluorescence image of the entire sample storage unit 101b.
  • 10 extraction ranges are set for the signal group obtained from the track portion Ta of one track number.
  • the number of extraction ranges set for a signal group with one track number is not limited to 10. In actual processing, a larger number of extraction ranges are set for a signal group. In this case, the number of divided areas Wp set in the fluorescent image memory area 220m is also changed according to the number of extraction ranges.
  • ⁇ Effect of embodiment> As an effective method for detecting cells infected with pathogenic bacteria or cells having a predetermined form, a method of obtaining a fluorescent image of the entire flow path by staining the cells to be detected with a fluorescent dye and storing them in the flow path Can be used.
  • the presence / absence and number of detection target cells can be acquired by analyzing the acquired fluorescence image, and based on this, the infection rate of pathogenic bacteria and the like can be acquired. It is also possible to display the acquired fluorescence image as appropriate and visually confirm the occurrence of fluorescence.
  • the sample storage disk 100 is disposed upstream and downstream of the sample storage portion 101b in the scanning direction Ds of the track 102c on each track portion Ta straddling the sample storage portion 101b.
  • the start signal V3 of the field F4 and the identification signal Id8 of the footer area FT1 that are synchronization adjustment signals are recorded on the side. For this reason, when rotation unevenness occurs in the sample storage disk 100, from the detection of the start signal V3 that is the upstream synchronization adjustment signal to the detection of the identification signal Id8 that is the downstream synchronization adjustment signal. It is possible to measure the extraction period, which is the time of.
  • the synchronization adjustment signals recorded on the upstream side in the plurality of track portions Ta of the sample storage portion 101b are recorded so as to be aligned in the disc radial direction Dr, that is, the direction DV3 away from the disc center Pc, and in the plurality of track portions Ta.
  • the synchronization adjustment signal recorded on the side is recorded so as to be aligned in the disc radial direction Dr, that is, the direction DFT1 away from the disc center Pc, so that the synchronization deviation of the fluorescent signal FL obtained from each track portion Ta can be detected.
  • the identification signal Id8 of the footer region FT1 that is the downstream synchronization adjustment signal is detected after the start signal V3 that is the upstream synchronization adjustment signal is detected.
  • the time until is longer.
  • the image processing unit 20 can correct the synchronization shift between the fluorescent signals according to the detected amount of synchronization shift. Therefore, a higher quality fluorescent image can be acquired.
  • the image processing unit 20 of the fluorescence detection apparatus 1 identifies the footer region FT1 that is the downstream synchronization adjustment signal after the start signal V3 of the field F4 that is the upstream synchronization adjustment signal is detected.
  • the cut-out period which is the time until the signal Id (is detected) is measured, and the synchronization deviation of the signal group corresponding to each track portion Ta cut out by the cut-out unit 13 is corrected based on the measured cut-out period.
  • the image processing unit 20 divides each of the lines L0 to Lm on the fluorescent image into a plurality of regions.
  • the divided areas Wp of the memory area 220m are allocated to the plurality of areas of the lines L0 to Lm, respectively.
  • the extraction range of the signal group that is stored and manually mapped in each of the divided areas Wp is set based on the measured extraction period.
  • the image processing unit 20 extracts the cutting period C0 measured in the track part Ta having the reference track number T0 and the tracks having the other track numbers T2 to Tm among the track parts Ta crossing the sample storage unit 101b. Based on the cutting periods C2 to Cm measured at the portion Ta, the shift value Dp is obtained by the equation (1).
  • the image processing unit 20 sets an extraction range for the signal group acquired from the track number T2 to Tm track portion Ta according to the obtained shift value Dp, and the signals included in each extraction range are divided into regions Wp corresponding to the extraction range. Save and map to. Thereby, the synchronization shift between signals arranged in each divided region Wp can be effectively eliminated. Therefore, a high-quality fluorescent image can be acquired.
  • the sample storage disk 100 is divided into areas A0 to A8 in the disk circumferential direction Dc. Two boundaries (ends) aligned in the disk circumferential direction Dc of each area extend radially from the disk center Pc in the disk radial direction Dr.
  • the sample storage portions 101b are arranged in the areas A0 to A8, respectively, and the track portion Ta included in each area straddles the sample storage portion 101b.
  • the track portions Ta included in each area are all scanned with the same time length. Therefore, the same signal format can be applied uniformly to all track portions Ta.
  • the angle ranges of the areas A0 to A8 centered on the disc center Pc in the disc circumferential direction Dc are set to be equal to each other. For this reason, fluorescence signals can be cut out from all areas A0 to A8 by the same process.
  • the sample storage disk 100 is divided into a plurality of zones Z0 to Zn in the disk radial direction Dr, and a signal is recorded at a constant angular velocity in the track portion Ta of each zone.
  • the angular velocities of the zones Z0 to Zn are set so that the linear velocities of the track portions Ta at the center position in the disk radial direction Dr of each zone are the same.
  • the difference between the linear velocity on the inner circumferential side and the linear velocity on the outer circumferential side of the sample storage disk 100 can be suppressed, For any zone, it is possible to stably extract the fluorescence signal and read the signal from the track portion Ta.
  • the sample storage disk 100 stores the substrate 100, the track 102c formed on the upper surface of the substrate 102 so as to turn around the disk center Pc, and the sample 100Sa disposed above the track 102c.
  • One or more sample storage portions 101b are provided.
  • the track 102c is configured to be scanned in the scanning direction Ds.
  • the track 102c includes a plurality of track portions Ta that straddle the sample storage portions 101b of the one or more sample storage portions 101b in a row in the disc radial direction Dr away from the disc center Pc.
  • a synchronization adjustment signal (V3) is recorded on the upstream side of each of the sample storage portions 101b in the scanning direction Ds of each of the plurality of track portions Ta.
  • a synchronization adjustment signal (Id8) is recorded on the downstream side of each sample storage portion 101b in the scanning direction Ds of each track portion Ta of the plurality of track portions Ta.
  • the synchronization adjustment signal (V3) may be aligned in the direction DV3 away from the disc center Pc.
  • the synchronization adjustment signal (Id8) may be aligned in the direction DFT1 away from the disk center Pc.
  • each sample storage portion 101b that intersect the disc circumferential direction Dc centered on the disc center Pc may extend radially from the disc center Pc.
  • the one or more sample storage units 101b may include a plurality of sample storage units 101b that are arranged on the upper side of the track 102c and store the sample 100Sa.
  • the track region 102a of the sample storage disk 100 on which the track 102c is formed may be divided into a plurality of areas A0 to A8 in the disk circumferential direction Dc at a plurality of boundaries extending radially from the disk center Pc.
  • a plurality of sample storage portions 101b are respectively disposed in the areas A0 to A8, and the portion of the track 102c included in each of the plurality of areas A0 to A8 covers the track portion Ta straddling the respective sample storage portions 101b.
  • the plurality of areas A0 to A8 may be arranged in an equal angular range around the disc center Pc.
  • the sample storage disk 100 may be divided into a plurality of zones Z0 to Zn along the disk radial direction Dr. In each of the plurality of zones Z0 to Zn, a signal may be recorded at a constant angular velocity on the plurality of track portions Ta.
  • the angular velocities of the respective zones may be set so that the linear velocities of the portions located in the center in the disc radial direction Dr of the plurality of zones Z0 to Zn of the track portion Ta are the same.
  • the fluorescence detection apparatus 1 is used together with a sample storage disk 100 that stores a sample 100Sa.
  • the scanning unit 1200 irradiates the sample storage disk 100 with light and scans the sample storage disk 100 with the light in the scanning direction Ds.
  • the photodetector 210 outputs a detection signal (S1 to S2) according to the light reflected by the sample storage disk 100.
  • the signal acquisition unit 11a operates based on the detection signals (S1 to S2).
  • the fluorescence detector 211 outputs a fluorescence signal FL corresponding to the fluorescence generated from the sample 100Sa accommodated in the sample accommodating portion 101b by the irradiated light.
  • the cutout unit 13 cuts out a plurality of signals FLd obtained by sampling the fluorescent signal FL, and outputs a plurality of signal groups respectively corresponding to a plurality of track portions Ta from the cut out signals FLd.
  • the image processing unit 20 generates a fluorescence image of the sample storage unit 101b based on the plurality of signal groups.
  • a plurality of synchronization adjustment signals (V3) and a plurality of synchronization adjustment signals (Id8) are recorded on the track 102c of the sample storage disk 100.
  • the track 102c includes a plurality of track portions Ta that straddle the sample storage portion 101b side by side in the disc radial direction Dr away from the disc center Pc.
  • Each synchronization adjustment signal (V3) of the plurality of synchronization adjustment signals (V3) is recorded on the upstream side of the sample accommodating portion 101b in the scanning direction Ds of each track portion Ta of the plurality of track portions Ta.
  • the respective synchronization adjustment signals (Id8) of the plurality of synchronization adjustment signals (Id8) are recorded on the downstream side of the sample storage portion 101b in the scanning direction Ds of each of the track portions Ta.
  • the image processing unit 20 arranges a plurality of signal groups respectively corresponding to the plurality of track portions Ta in order of the plurality of track portions Ta in the disk radial direction Dr.
  • the image processing unit 20 measures the time from when each of the synchronization adjustment signals (V3) is detected by the signal acquisition unit 11a to when each of the synchronization adjustment signals (Id8) is detected by the signal acquisition unit 11a. To do.
  • the image processing unit 20 generates a fluorescence image by correcting the synchronization deviation of the plurality of signal groups based on the measured time.
  • the plurality of synchronization adjustment signals (V3) may be recorded so as to be aligned in the direction DV3 from the disc center Pc.
  • a plurality of synchronization adjustment signals (Id8) may be recorded so as to be aligned in the direction DFT1 from the disk center Pc.
  • the cutout unit 13 may start sampling the fluorescence signal FL based on the signal acquisition unit 11a acquiring the respective synchronization adjustment signals (V3).
  • the cutout unit 13 may end the sampling of the fluorescence signal FL based on the signal acquisition unit 11a acquiring the respective synchronization adjustment signals (Id8).
  • the fluorescent image is composed of a plurality of lines L0 to Lm respectively corresponding to a plurality of track portions Ta.
  • the image processing unit 20 includes a memory 20m that stores a fluorescent image.
  • a line Li corresponding to each of the track portions Ta among the plurality of lines L0 to Lm may be divided into a plurality of regions.
  • the memory 20m may include a memory area 220m divided into a plurality of divided areas Wp that respectively correspond to the plurality of areas of the line Li.
  • the image processing unit 20 uses a plurality of extraction ranges Ri-1 to Ri assigned to the plurality of regions of the line Li, with the signal groups corresponding to the respective track portions Ta among the plurality of signal groups. Extracted at ⁇ 10 and mapped to a plurality of divided areas Wp of the memory area 220m.
  • the image processing unit 20 sets a plurality of extraction ranges Ri-1 to Ri-10 based on the measured time.
  • Other synchronization adjustment signals (V3) recorded on the upstream side of the sample storage portion 101b in the scanning direction Ds of the other track portions Ta among the plurality of track portions Ta may be included.
  • a plurality of synchronization adjustment signals (Id8) are transmitted to a certain synchronization adjustment signal (Id8) recorded on the downstream side of the sample storage unit 101b in the scanning direction Ds of a certain track portion Ta and the other track portions Ta.
  • synchronization adjustment signals (Id8) recorded on the downstream side of the sample storage unit 101b in the scanning direction Ds may be included.
  • the image processing unit 20 performs the first process from when a certain synchronization adjustment signal (V3) is detected by the signal acquisition unit 11a to when a certain synchronization adjustment signal (Id8) is detected by the signal acquisition unit 11a. Measure the time.
  • the image processing unit 20 calculates the second time from when the other synchronization adjustment signal (V3) is detected by the signal acquisition unit 11a to when the other synchronization adjustment signal (Id8) is detected by the signal acquisition unit 11a. taking measurement.
  • the image processing unit 20 sets a plurality of extraction ranges Ri-1 to Ri-10 in the other track portion Ta based on the first time and the second time.
  • the image processing unit 20 sets each extraction range based on the shift value Dp obtained by the calculation of Expression (1).
  • the image processing unit 20 obtains a correlation coefficient between signals between the extraction range set in this way and the extraction range adjacent to the extraction range in the radial direction Dr, and the correlation coefficient is the highest. Reset the extraction range to the position.
  • FIG. 14 shows a method for resetting the extraction range.
  • FIG. 14 schematically shows the relationship between the extraction range R0-1 of the track number T0 and the extraction range R1-1 of the track number T1.
  • FIG. 14 further shows a correlation coefficient between the signal group of the extraction range R1-1 and the signal group of the extraction range R0-1 when the extraction range R1-1 is shifted by one pixel.
  • the extraction range of the signal group obtained from a certain track portion is set as the reference extraction range.
  • the extraction range of the signal group of another track portion adjacent to the track portion in the radial direction Dr is set as the target extraction range according to the equation (1).
  • the order of the reference extraction ranges in the scanning direction Ds is the same as the order of the target extraction ranges in the scanning direction Ds.
  • the image processing unit 20 obtains a correlation coefficient between the signals of the plurality of candidate extraction ranges, which are extraction ranges obtained by shifting the target extraction range to the upstream side and the downstream side in the scanning direction Ds, and the signal of the reference extraction range.
  • the candidate extraction range in which the largest correlation coefficient among the obtained correlation coefficients is obtained is reset as the target extraction range.
  • the image processing unit 20 sets the first extraction range R1-1 that is the target extraction range from the upstream side by the calculation of Expression (1) for the signal group of the track number T1.
  • the correlation coefficient between the signal in the target extraction range R1-1 and the signal in the first extraction range R0-1 from the upstream side that is the reference extraction range of the track number T0 adjacent in the radial direction Dr is obtained.
  • the target extraction range R1-1 is shifted by one pixel upstream from the initial set position PD0 in the predetermined search range Q1, and the target extraction range R11 is shifted downstream from the initial set position PD0 in the predetermined search range Q2.
  • the correlation coefficient with the reference extraction range R01 is obtained while shifting one pixel at a time, that is, while shifting the target extraction range R11 one pixel at a time from the initial set position PD0 in the search range QT that combines the search ranges Q1 and Q2. Then, the position of the target extraction range R11 having the maximum correlation coefficient is searched.
  • the correlation coefficient continuously decreases in a range in which the target extraction range R11 is shifted 10 pixels from a certain position to the upstream side, and the target extraction range R11 is set to 10 downstream from the predetermined position.
  • the certain position is specified as the position where the correlation coefficient is maximized.
  • the position PDa shifted by ⁇ P upstream from the initial setting position PD0 is the position where the correlation coefficient is maximized.
  • the correlation coefficient at a certain position exceeds a predetermined threshold SH (for example, 0.9)
  • the target extraction range R11 at the certain position is from the upstream side with respect to the signal group of the track number T1. It is reset to the first extraction range R1-1.
  • the search range is expanded and the same search is performed again.
  • the extraction range R1-1 is shifted and reset by the correction amount from the position obtained by the equation (1) to the position reset by the search.
  • the image processing unit 20 performs the same search process on the other extraction ranges R1-2 to R1-10 of the track number T1, obtains correction amounts for the respective extraction ranges, and extracts the extraction ranges R1-2 to R1-10.
  • the extraction ranges R1-2 to R1-10 are reset by shifting the position by each correction amount.
  • the search process is performed on the extraction range R1-1 of the track number T1, and the same search process is performed on the most downstream extraction range R1-10 of the track number T1.
  • the other extraction ranges R1-2 to R1-9 of the track number T1 are reproduced with correction amounts obtained by proportionally distributing the correction amount of the most upstream extraction range R1-1 and the correction amount of the most downstream extraction range R1-10. It may be set.
  • the first extraction range R1-1 located on the most upstream side is reset to a position shifted by one pixel on the downstream side from the position set by the equation (1) by the correlation coefficient.
  • the second to ninth extraction ranges R1-2 to R1-9 is reset to positions shifted by 2 pixels, 3 pixels, 4 pixels, 5 pixels, 6 pixels, 7 pixels, 8 pixels, and 9 pixels downstream from the position set according to the expression (1).
  • the correction amount that is proportionally distributed to each extraction range is rounded off to the nearest decimal point and applied to each extraction range.
  • the extraction range set in the signal group of track numbers T2 to Tm is also corrected by the same processing. That is, for the p-th extraction range of the track number Ti, the above processing is performed using the p-th extraction range of the track number Ti-1 adjacent to the track number Ti in the radial direction Dr as a reference extraction range. Correction is performed from the extraction range set in (1).
  • the processing based on the correlation coefficient further suppresses the synchronization shift between the signal groups, so that a higher quality fluorescent image can be generated.
  • the process using the correlation coefficient is performed in a state where the extraction range is set in advance by the process according to Equation (1), the position where the correlation coefficient is maximized can be obtained without greatly expanding the search range of the correlation coefficient. You can search smoothly. Therefore, the extraction range resetting process based on the correlation coefficient can be smoothly performed while suppressing the processing load.
  • each extraction range is adjusted by the shift value Dp obtained by the equation (1) to correct the signal group synchronization deviation.
  • the signal group synchronization deviation is corrected by thinning out or interpolating the signal group of the track portion Ta having the track numbers T0, T1,... Shown in FIG.
  • the image processing unit 20 obtains an index Gi indicating the relationship between the extraction period C0 of the reference track number T0 and the extraction period Ci of the track number Ti to be corrected by the following equation (3).
  • the index Gi is +0.001
  • a process of thinning out signals every 1000 pixels from the pixel position on the most upstream side is performed on the signal group of the track number Ti, and the downstream side of the position of the thinned signal Is shifted upstream by one pixel.
  • the index Gi is ⁇ 0.001
  • the process of interpolating the signal for every 1000 pixels from the pixel position on the most upstream side is performed on the signal group of the track number Ti, and downstream from the position of the interpolated signal. Signal is shifted downstream by one pixel.
  • the signal to be interpolated is, for example, an average value of signals on both sides of the interpolated position.
  • the P1th pixel to the P11th pixel are performed on all the track number signal groups.
  • the signals in the range up to are extracted, stored in the fluorescent image memory area 220m, and mapped. That is, in the second modification, the memory area 220m for the fluorescence image is not divided into the plurality of divided areas Wp shown in FIG. 12, and the signals of the pixels in the P1st to P11th ranges are respectively represented by the corresponding lines Li. Is stored and mapped as it is.
  • a high-quality image can be obtained as in the above embodiment.
  • the synchronization shift is corrected for each extraction range corresponding to the divided region Wp, and thus more appropriate synchronization correction can be realized. Therefore, in order to obtain a higher-quality fluorescent image, it is preferable to use the synchronization correction according to the above-described embodiment, and further, by applying the processing based on the correlation coefficient according to the modified example 1, a higher-quality fluorescent image Obtainable.
  • the track area 102a of the sample storage disk 100 is divided into nine areas A0 to A8 in the disk circumferential direction Dc, and the number of areas in which the track area 102a of the sample storage disk 100 is divided in the disk circumferential direction Dc. Is not limited to this.
  • the shape and internal structure of the sample container 101b can be changed as appropriate other than the forms shown in FIGS. 1A and 1B.
  • a predetermined field can be appropriately deleted or changed from the format shown in FIG. 5A, or a new field can be added.
  • the content of the signal recorded in each field can be appropriately changed from that shown in FIG. 6A.
  • the shape of the sample storage portion 101b in plan view is not necessarily a trapezoidal shape, and may be another shape.
  • the laser beam scans the groove.
  • the laser beam may scan the land, or the laser beam may scan both the groove and the land.
  • FIG. 15 schematically shows another semi-permeable membrane 102d of the sample storage disk 100.
  • the same reference numerals are assigned to the same portions as those of the semipermeable membrane 102d shown in FIG.
  • a row of pits 113 arranged in a spiral shape is formed on the substrate 102, that is, the semipermeable membrane 102d, without forming grooves and lands.
  • a track 102 c is formed by a row of pits 113.
  • a signal is recorded by the pit 113 and the space between the pits 113 adjacent in the circumferential direction Dc, as in the above embodiment.
  • terms indicating directions such as “upper surface” and “upper side” indicate relative directions determined only by the relative positional relationship of the constituent members of the sample storage disk 100 such as the substrates 101 and 102, such as the vertical direction. It does not indicate the absolute direction.

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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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Abstract

L'invention concerne un disque de réception d'échantillon comprenant un substrat, des pistes formées sur la surface supérieure du substrat de manière à encercler le centre du disque et des parties de réception d'échantillon recevant un échantillon, lesdites parties de réception d'échantillon étant disposées sur les côtés supérieurs des pistes. Les pistes sont conçues de façon à être balayées dans la direction de balayage. Les pistes comprennent une pluralité de parties de piste séparées du centre du disque et s'étendant à travers les parties de réception d'échantillon alignées dans la direction radiale. Des signaux de réglage de synchronisation sont enregistrés sur le côté amont et le côté aval des parties de réception d'échantillon dans chacune des directions de balayage de partie de piste de la pluralité de parties de piste. Grâce audit disque de réception d'échantillon, une image de fluorescence favorable peut être créée même en cas d'irrégularité de rotation.
PCT/JP2018/000620 2017-01-19 2018-01-12 Disque de réception d'échantillon et détecteur de fluorescence WO2018135404A1 (fr)

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Cited By (1)

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WO2020195871A1 (fr) * 2019-03-25 2020-10-01 株式会社Ｊｖｃケンウッド Dispositif d'analyse et procédé d'analyse

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JP2005516336A (ja) * 2002-01-28 2005-06-02 バースタイン テクノロジーズ，インコーポレイティド 論理的なトリガのための方法および装置
US7014815B1 (en) * 1998-10-30 2006-03-21 Burstein Technologies, Inc. Trackable optical discs with concurrently readable nonoperational features
WO2013161543A1 (fr) * 2012-04-25 2013-10-31 三洋電機株式会社 Porte-échantillons et détecteur fluorimétrique l'utilisant
WO2014068951A1 (fr) * 2012-10-30 2014-05-08 パナソニック株式会社 Support d'échantillon et détecteur de fluorescence l'utilisant

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Publication number Priority date Publication date Assignee Title
US7014815B1 (en) * 1998-10-30 2006-03-21 Burstein Technologies, Inc. Trackable optical discs with concurrently readable nonoperational features
JP2005516336A (ja) * 2002-01-28 2005-06-02 バースタイン テクノロジーズ，インコーポレイティド 論理的なトリガのための方法および装置
WO2013161543A1 (fr) * 2012-04-25 2013-10-31 三洋電機株式会社 Porte-échantillons et détecteur fluorimétrique l'utilisant
WO2014068951A1 (fr) * 2012-10-30 2014-05-08 パナソニック株式会社 Support d'échantillon et détecteur de fluorescence l'utilisant

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020195871A1 (fr) * 2019-03-25 2020-10-01 株式会社Ｊｖｃケンウッド Dispositif d'analyse et procédé d'analyse
JP2020159733A (ja) * 2019-03-25 2020-10-01 株式会社Ｊｖｃケンウッド 分析装置及び分析方法
JP7310199B2 (ja) 2019-03-25 2023-07-19 株式会社Ｊｖｃケンウッド 分析装置
US12007325B2 (en) 2019-03-25 2024-06-11 Jvckenwood Corporation Analysis device and analysis method

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