WO2015004917A1 - Procédé d'observation, dispositif d'observation, procédé de production de feuille de cellule et dispositif de production de feuille de cellule - Google Patents

Procédé d'observation, dispositif d'observation, procédé de production de feuille de cellule et dispositif de production de feuille de cellule Download PDF

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Publication number
WO2015004917A1
WO2015004917A1 PCT/JP2014/003656 JP2014003656W WO2015004917A1 WO 2015004917 A1 WO2015004917 A1 WO 2015004917A1 JP 2014003656 W JP2014003656 W JP 2014003656W WO 2015004917 A1 WO2015004917 A1 WO 2015004917A1
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Prior art keywords
observation
cell
cells
stage
detection
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PCT/JP2014/003656
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English (en)
Japanese (ja)
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福武　直樹
直志 相川
啓作 浜田
潤也 大川
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株式会社ニコン
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Priority to JP2015526172A priority Critical patent/JPWO2015004917A1/ja
Publication of WO2015004917A1 publication Critical patent/WO2015004917A1/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
    • G01N21/62—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
    • G01N21/65—Raman scattering
    • 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
    • G01N15/1429—Signal processing
    • G01N15/1433—Signal processing using image recognition
    • 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
    • G01N15/1434—Optical arrangements
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/16—Microscopes adapted for ultraviolet illumination ; Fluorescence microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements
    • G02B21/365—Control or image processing arrangements for digital or video microscopes
    • 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
    • G01N2015/1006—Investigating individual particles for cytology
    • 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
    • G01N2015/1488—Methods for deciding
    • 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
    • G01N2015/1497—Particle shape

Definitions

  • the present invention relates to an observation method, an observation apparatus, a cell sheet manufacturing method, and a cell sheet manufacturing apparatus.
  • Patent Document 1 Japanese Patent Application Laid-Open No. 2005-062155
  • CARS light is generated in a narrow region where excitation light is collected. For this reason, CARS observation has high resolution and composition (lipid or protein) selectivity. However, since it is an observation method using a non-linear process, the signal light is weak, and in exchange for high resolution, the area that can be observed per unit time is narrow, and observation takes time.
  • the first observation stage includes a first observation stage for observing the observation object and a second observation stage for observing the observation object based on the information obtained by the first observation stage. Includes a step of detecting a state of a cell included in the observation region, and the second observation step is a step of determining whether to observe the cell based on the state of the cell detected in the first observation step.
  • An observation method is provided.
  • the first observation stage includes a first observation stage for observing the observation object, and a second observation stage for observing the observation object based on information obtained by the first observation stage.
  • An observation method is provided that includes a step of determining whether or not.
  • a cell sheet comprising a preparation stage for isolating cells and preparing a cell line, a culture stage for culturing the cell line on a cell sheet, and an observation stage for observing the cells by the above observation method
  • a manufacturing method is provided.
  • a cell sheet comprising: a preparation unit for isolating cells and preparing a cell line; a culture unit for culturing the cell line on a cell sheet; and an observation unit for observing the cells by the observation method.
  • a manufacturing apparatus is provided.
  • the first observation unit includes a first observation unit that observes the observation target, and a second observation unit that observes the observation target based on the information obtained by the first observation unit.
  • the first observation unit includes a first observation unit that observes the observation target, and a second observation unit that observes the observation target based on information obtained by the first observation unit.
  • An observation apparatus having a determination unit for determining whether or not is provided.
  • FIG. 1 is a schematic diagram showing the structure of a laser microscope 101.
  • FIG. 2 is a block diagram of a control system of the laser microscope 101.
  • FIG. 3 is a flowchart showing an observation procedure using a laser microscope 101. It is a figure which shows the observation image by the detection of differential interference light.
  • FIG. 6 is a diagram illustrating a process of determining a detection area by a second detection system 150. It is a figure which shows the observation image of the living cell by the detection of CARS light. It is a figure which shows the observation image of the dead cell by the detection of CARS light.
  • 2 is a schematic diagram showing a structure of a laser microscope 102.
  • FIG. 1 is a schematic diagram showing the structure of the laser microscope 101.
  • the laser microscope 101 includes a stage 110, a laser device 120, an optical system 130, a first detection system 140, a second detection system 150, a control system 160, and a scanning system 180.
  • the laser microscope 101 can be used for both observation using differential interference light and observation using CARS light detection.
  • the stage 110 supports the sample 112 observed by the laser microscope 101.
  • the sample 112 supported by the stage 110 is irradiated with laser light as irradiation light from one surface (the lower surface in the drawing in the drawing).
  • an observation image is formed by detecting the exit light emitted by the linear optical phenomenon or the emergent light emitted by the nonlinear optical phenomenon from the side opposite to the side irradiated with the laser light with respect to the stage 110. Is done.
  • differential interference light is used as the emitted light emitted by the linear optical phenomenon
  • CARS light is used as the emitted light emitted by the nonlinear optical phenomenon.
  • the laser device 120 includes a plurality of laser light sources 122, 124, 126 and a combiner 128.
  • the laser light source 122, 124, 126 generates separately a laser beam having different wavelengths lambda d, lambda P, the lambda S together.
  • a laser beam having a wavelength lambda d of one laser light source 122 is generated, the observation by detection of differential interference light is used as illumination light.
  • the other laser light sources 124 and 126 generate pulse lasers used for observation by CARS light detection.
  • the pulse lasers generated by the laser light sources 124 and 126 have wavelengths ⁇ P and ⁇ S.
  • the laser light sources 124 and 126 for example, a mode-locked picosecond Nd: YVO 4 laser, a mode-locked picosecond yttrium laser, or the like can be used.
  • One of the laser light sources 124 and 126 may be replaced with an optical parametric oscillator that converts the wavelength of the pulse laser generated by the other laser light sources 124 and 126.
  • the pulse laser having the shorter wavelength ⁇ P is used as pump light in CARS light observation.
  • the pulse laser having the longer wavelength ⁇ S is used as Stokes light in CARS light observation.
  • Laser beams emitted from the laser light sources 122, 124, and 126 are incident on the combiner 128 through the polarizing plates 121 and 123, respectively.
  • the polarizing plate 121 for pump light and Stokes light and the polarizing plate 123 for illumination light have polarization planes shifted by 45 degrees from each other.
  • the pump light, Stokes light, and illumination light become linearly polarized light having polarization planes C and D that are shifted from each other by 45 °.
  • the polarizing plate 123 of the laser light source 126 is also used as a polarizer in the case of performing observation with differential interference light.
  • the direction of the polarization plane C and the polarization plane D may be either P-polarized light or S-polarized light with respect to the reflecting mirrors such as the reflecting mirror 192 and the galvano scanner 182. If priority is given to the detection sensitivity of CARS light, the polarization plane C is preferably incident on these as P-polarized light or S-polarized light.
  • the combiner 128 integrates the laser beams generated by the plurality of laser light sources 122, 124, and 126 and emits them as irradiation light on a single optical path. Thereby, the irradiation light which combined pump light and Stokes light can be irradiated to the sample 112, and CARS light can be generated.
  • a dichroic mirror that can maintain the degree of polarization of the pump light and Stokes light used for CARS light observation and illumination light used for differential interference light observation.
  • the laser device 120 may be provided with a delay optical path for the purpose of synchronizing the pump light generated by the laser light sources 122 and 124 and the Stokes light.
  • the delay optical path can be formed by a plurality of reflecting mirrors whose intervals can be changed.
  • the Stokes light may be broadened using a photonic crystal fiber.
  • the scanning system 180 includes a galvano scanner 182 and a scan lens 184.
  • the galvano scanner 182 includes a reflecting mirror that swings around two axes whose directions are different from each other, and two-dimensionally displaces the optical path of incident irradiation light in a direction intersecting the optical axis.
  • the scan lens 184 focuses the irradiation light emitted from the galvano scanner 182 on a predetermined primary image plane 186. Thereby, the observation region of the sample 112 is scanned with the irradiation light emitted from the laser device 120, and the irradiation region can be irradiated with the irradiation region having a predetermined area.
  • the optical system 130 includes a pair of first objective lenses 132 and 134 disposed on the laser device 120 side with respect to the stage 110, and a condenser lens 136 disposed on the opposite side of the objective lens 132 and 134 with respect to the stage 110.
  • the objective lenses 132 and 134 focus the irradiation light irradiated toward the sample 112 within the observation region in the sample 112.
  • a reflecting mirror 192 is disposed on the optical path of the irradiation light between the laser device 120 and the objective lens 132.
  • the reflecting mirror 192 bends the optical path of the irradiation light to prevent the structure of the laser microscope 101 from becoming excessively high.
  • the reflecting mirror 192 a metal mirror that reflects incident light on the surface may be used instead of the total reflection prism. Thereby, the reflecting mirror 192 can alleviate the occurrence of a phase difference depending on the polarization direction.
  • the optical system 130 has a pair of birefringent prisms 172 and 174 arranged on the optical path of the irradiation light with the sample 112 supported by the stage 110 interposed therebetween.
  • One birefringent prism 172 disposed upstream of the sample 112 in the propagation direction of the irradiation light is disposed between the pair of objective lenses 132 and 134.
  • the other birefringent prism 174 disposed on the downstream side with respect to the sample 112 is disposed so as to sandwich the condenser lens 136 together with the sample 112.
  • the upstream birefringent prism 172 separates the irradiation light irradiated toward the sample 112 into a pair of light beams whose polarizations are orthogonal to each other.
  • the separated pair of light beams propagate in parallel in slightly spatially different positions and pass through the sample 112 very close, but different positions.
  • the distance between the pair of polarized light in the sample 112 is called a shear amount, and is about 0.2 ⁇ m, for example.
  • a Woraton prism that can take out incident linearly polarized light with an opening angle in a non-parallel manner can be used. Furthermore, a nomarski rhythm having the focal point of two divided polarized lights outside the prism can be preferably used.
  • the pair of polarized lights that have passed through different positions in the sample 112 are combined into one light beam in the birefringent prism 174 downstream of the sample 112.
  • the sample 112 since the sample 112 is transmitted through different positions, the combined polarized light propagates through different optical path lengths. For this reason, a phase difference is generated between the pair of polarized lights, and the intensity of the combined light beam is changed. In this way, an observation image that well reflects the shape of the sample 112 is obtained by detecting and imaging differential interference light that reflects the distribution of the optical characteristics of the sample 112.
  • the birefringent prisms 172 and 174 are exclusively used for observation by detecting differential interference light. Therefore, when the laser microscope 101 is observed by detecting CARS light, the birefringent prisms 172 and 174 may be removed from the optical path of the irradiation light. On the other hand, when the birefringent prisms 172 and 174 are inserted into and removed from the optical path, the characteristics of the optical system 130 may change and the observation position on the sample 112 may be shifted. Therefore, when the observation positions of the observation by differential interference light and the observation by CARS light detection are made to coincide with each other with high accuracy, the observation by CARS light detection may be performed while the birefringent prisms 172 and 174 are attached. .
  • a first detection system 140 and a second detection system 150 are arranged on the downstream side of the birefringent prism 174 along the propagation optical path of the irradiation light.
  • the first detection system 140 is used to detect differential interference light among the detection light emitted from the sample 112.
  • the second detection system 150 is used to detect CARS light out of the detection light emitted from the sample 112.
  • the first detection system 140 includes a polarizing plate 176, a condenser lens 142, a relay lens 144, and a photomultiplier tube 146.
  • the polarizing plate 176 is used as an analyzer in observation using differential interference light. Thereby, a pair of polarized light incident through different optical paths causes interference, and becomes differential interference light having light and dark according to the shape of the sample 112 that is transparent to the irradiation light.
  • the differential interference light is condensed by the condensing lens 142 and the relay lens 144 and is efficiently detected by the photomultiplier tube 146 to form an observation image.
  • observation with differential interference light has no restriction on the numerical aperture of the illumination, so that the resolution in the direction perpendicular to the optical axis can be exhibited to the limit of the microscope objective lens.
  • the observation image obtained by the differential interference light has no halo in the outline, high resolution can be obtained.
  • a reflecting mirror 194 is disposed between the condensing lens 142 and the relay lens 144 of the first detection system 140.
  • the reflecting mirror 194 bends the optical path of the detection light to prevent the structure of the laser microscope 101 from becoming excessively high.
  • the second detection system 150 includes a condenser lens 152, a relay lens 154, a photomultiplier tube 156, and a dichroic mirror 158.
  • the dichroic mirror 158 branches the CARS light emitted from the sample 112 on the upstream side of the polarizing plate 176 of the first detection system 140 and introduces it into the photomultiplier tube 156 through the condenser lens 152 and the relay lens 154. Therefore, the CARS light is received by the photomultiplier tube 156 without reducing the light amount.
  • the dichroic mirror 158 is used only when CARS light is detected. Therefore, when the differential interference light is observed, the dichroic mirror 158 may be extracted from the optical path of the detection light.
  • the incident angle of the detection light emitted from the sample 112 with respect to the dichroic mirror 158 is preferably smaller than 45 °. Thereby, CARS light can be extracted efficiently.
  • the control system 160 includes a control unit 162, a keyboard 164, a mouse 166, and a display unit 168.
  • the control unit 162 can be formed by mounting a program that causes a general-purpose personal computer to execute a control procedure described later.
  • the keyboard 164 and the mouse 166 are connected to the control unit 162 and operated when a user instruction is input to the control unit 162.
  • the display unit 168 returns feedback to the user's operation using the keyboard 164 and the mouse 166, and displays the image or character string generated by the control unit 162 toward the user.
  • the control unit 162 is connected to each of the laser device 120, the first detection system 140, the second detection system 150, and the scanning system 180, and controls each operation. Moreover, based on the detection results of the first detection system 140 and the second detection system 150, an image to be displayed on the display unit 168 is generated. In addition, the control unit 162 determines the state of the sample 112 based on the detection results of the first detection system 140 and the second detection system 150.
  • the control system 160 controls the overall operation of the laser microscope 101 in accordance with an instruction received from the user. Further, the control system 160 generates an observation image based on the detection results of the first detection system 140 and the second detection system 150. Further, the control system 160 may also output a character string or a code indicating a determination result reflecting the state of the sample 112.
  • the dichroic mirror 158 disposed for the purpose of separating the CARS light emitted from the sample 112 is arranged so that the differential interference light is P-polarized light or S-polarized light. Therefore, the polarization characteristic of the interference film formed on the dichroic mirror 158 is reduced with respect to the wavelength of the differential interference light, and the observation accuracy of observation using the differential interference light is prevented from being lowered.
  • the direction in which the dichroic mirror 158 and the reflecting mirror 192 are arranged may be different from that in FIG.
  • the dichroic mirror 158 is used only when CARS light is detected. Therefore, when importance is attached to the image quality of observation using differential interference light, the dichroic mirror 158 may be provided so as to be extracted from the optical path of the detection light.
  • FIG. 2 is a block diagram of a control system in the laser microscope 101.
  • the control unit 162 includes a first determination unit 310, a storage unit 320, a scanning control unit 330, a detection control unit 340, and a second determination unit 350.
  • the control unit 162 is coupled to the keyboard 164, the mouse 166, the display unit 168, the laser device 120, the first detection system 140, the second detection system, and the scanning system 180 through the input / output unit 163.
  • the first determination unit 310 determines the state of the cell based on the detection image of the sample 112 generated by the detection control unit 340 based on the detection result of the first detection system 140 and the reference information referenced from the storage unit 320. It is determined whether or not In this embodiment, the predetermined state is a state in which the cell is dead.
  • living cells are referred to as living cells, and dead cells are referred to as dead cells.
  • the first determination unit 310 determines whether the cell detected by the first detection system 140 is a live cell or a dead cell. For example, the first determination unit 310 acquires a detection image obtained from a known cell from the storage unit 320 as reference information, and evaluates the similarity between the detection image obtained from the sample 112 and the reference information. Cell viability may be determined.
  • the first determination unit 310 acquires feature items extracted from a detection image obtained from a known cell from the storage unit 320 as reference information, and features of the detection image obtained from the sample 112 apply to the reference information. Whether the cell is alive or not may be determined depending on whether or not it is.
  • the determination result by the first determination unit 310 is output to the outside through the display unit 168.
  • the first determination unit 310 may leave part or all of the determination process to the user by displaying the reference information and the detected image toward the user through the display unit 168.
  • the second determination unit 350 determines whether or not the cells detected by the first detection system 140 are detected by the second detection system 150 based on the state of the cells determined by the first determination unit 310. The second determination unit 350 determines whether or not to observe the cells determined by the first determination unit 310 as being in a predetermined state, that is, dead cells. In the present embodiment, the second determination unit 350 determines the viability state of the cells that are not determined to be dead cells by the first determination unit 310.
  • the second determination unit 350 observes a cell for which the determination of whether the cell is a live cell or a dead cell by the first determination unit 310 has not been clarified. Accordingly, since the first determination unit 310 can detect a cell whose life or death is unknown by the second detection system 150, it is possible to determine the life or death of the cell without omission.
  • the second determination unit 350 is a living cell. Determine if it is a dead cell.
  • the second determination unit 350 determines the determination based on the observation image generated by detecting the CARS light generated from the sample 112. Therefore, the determination by the second determination unit 350 has high accuracy based on the composition selectivity of CARS light observation.
  • a detection image obtained from a known cell is acquired from the storage unit 320 as reference information, and the life and death of the cell is determined by evaluating the similarity between the detection image obtained from the sample 112 and the reference information. You may judge.
  • the second determination unit 350 acquires the feature items extracted from the detection image obtained from the known cells from the storage unit 320 as reference information, and the feature of the detection image obtained from the sample 112 applies to the reference information. Whether the cell is alive or not may be determined depending on whether or not it is.
  • the second determination unit 350 compares the evaluation value calculated by image processing such as Fourier transform processing and differentiation processing on the detection image detected from the sample 112 with the evaluation value acquired from the storage unit 320, and is determined in advance. Whether the cells are alive or dead may be determined based on the threshold value.
  • the determination result by the second determination unit 350 is output to the outside through the display unit 168.
  • the second determination unit 350 may leave part or all of the determination process to the user by displaying the reference information and the detected image toward the user through the display unit 168.
  • the storage unit 320 stores a control program describing the control procedure of the control unit 162 itself.
  • the control unit 162 reads the control program stored in the storage unit 320 and controls the operation of the laser microscope 101.
  • the storage unit 320 stores the detection result detected by the first detection system 140 or the second detection system 150, and refers to it when the detection control unit 340 generates a detection image.
  • the storage unit 320 stores reference information that is referred to when determining whether a cell serving as the sample 112 is alive or dead.
  • the reference information may be a detection image detected from a cell whose life and death are known. Further, the reference information may be information representing a feature item of a detected image detected from a cell whose life and death are known. Furthermore, the reference information is information indicating an evaluation value calculated by performing image processing on a detected image detected from a cell whose life and death are known by Fourier transform processing, differentiation processing, and the like, a method for calculating the evaluation value, and the like. It may be.
  • the scanning control unit 330 When the scanning control unit 330 is instructed to use either the first detection system 140 or the second detection system 150, the scanning control unit 330 operates the scanning system 180 according to the detection system to be used. Thereby, the observation region of the sample 112 is scanned with the irradiation light, and the observation region predetermined in the sample 112 can be observed on one observation plane.
  • the detection control unit 340 instructs the detection timing of the first detection system 140 and the second detection system 150 with reference to the scanning timing of the irradiation light and the light emission timing of the laser device 120. Further, the detection control unit 340 acquires the detection result of the first detection system 140 or the second detection system 150 together with the irradiation position of the irradiation light on the sample 112. Accordingly, the detection control unit 340 generates the observation image of the sample 112 by mapping the light intensity of the detection light to the light emission position in the sample 112.
  • FIG. 3 is a flowchart showing a procedure for observing the sample 112 using the laser microscope 101.
  • the laser light source 126 and the first detection system 140 are enabled, and the sample 112 is observed with differential interference light (step S101).
  • the observation image can be generated by observing the sample 112 over a wide range and at high speed.
  • the observation image includes a plurality of cells.
  • the resolution of the observation image obtained from the sample 112 depends on the resolution of the differential interference light. Therefore, from the observation image, a relatively large organelle such as a cell nucleus can be observed in addition to the cell membrane showing the outline of the cell.
  • the first determination unit 310 performs the first dead cell determination based on the observation image obtained by the differential interference light (step S102).
  • apparent dead cells are determined such that the cell membrane, nucleus, etc. have already been destroyed.
  • the detection control unit 340 specifies an observation region by the second detection system 150 (step S103). At this stage, the cells determined to be dead cells in step S102 are excluded from the observation region by the second detection system 150.
  • the detection control unit 340 subsequently activates the laser light sources 122 and 124 and the second detection system 150 and performs observation by detecting CARS light (step S104).
  • the detection control unit 340 controls the scanning control unit 330 based on the observation region specified in step S103. Thereby, the cells that have already been determined to be dead cells in the first dead cell determination in step S102 are excluded from the observation target.
  • the CARS light when the CARS light is scanned on the observation target, it passes through without observing the place where the dead cell exists. For this reason, compared with the scanning speed when observing while acquiring image data, it is possible to increase the scanning speed of a place where dead cells exist. When passing through a place where dead cells exist, irradiation of CARS light to the place may be stopped. Therefore, the scanning time by the second detection system 150 is shorter than the case where all the observation target areas are observed by the second detection system 150 alone without using the first detection system 140. The time required for observation can be reduced.
  • the detection control unit 340 can quickly generate an observation image with high resolution using CARS light.
  • the second determination unit 350 performs the second dead cell determination based on the observation image obtained by the CARS light (step S105). Since the second dead cell determination is based on an observation image having information that can be detected up to individual molecular bonds, it is possible to accurately determine a cell to be observed. In this way, a wide region of the sample 112 can be determined with the observation image accuracy of CARS light having high composition selectivity. In addition, observation with CARS light has high resolution in the irradiation direction of irradiation light, that is, in the depth direction (depth direction) of the sample 112 that is the observation target. Therefore, the sample 112 can be observed with high resolution in the depth direction.
  • the first observation image detected by the first detection system 140 and the second observation image detected by the second detection system 150 are simultaneously It may be displayed.
  • the first observation image is stored in the storage unit 320, and the stored first observation image is acquired and acquired upon detection by the second detection system 150 or determination by the second determination unit 350.
  • a first observation image may be displayed.
  • the detection by the first detection system 140 is continued even after the detection by the second detection system 150 is started, and the first observation image is detected at the time of detection by the second detection system 150 or the determination by the second determination unit 350. You may display a 2nd observation image. Further, by displaying an image showing the observation region by the second detection system 150 on the first observation image, it is possible to know which position of the first observation image the second detection system 150 is detecting. Also good.
  • the sample 112 can be observed at high speed and with high accuracy using the laser microscope 101.
  • the sample 112 is a living cell, neither the observation with differential interference light nor the observation with CARS light damages the sample 112, so that the cell sheet before use can be observed.
  • the observation image generated by the differential interference light and the observation image generated by the detection of the CARS light are synthesized, and the outline of the cell detected by the differential interference light and the CARS light It is possible to generate an observation image that displays the distribution of lipids and the like detected by the above. This facilitates the determination of the viability of individual cells.
  • a step of detecting the spectrum of the cell nucleus in the second dead cell determination may be provided for the cell nucleus detected in the first dead cell determination (step S102).
  • the viability of cells can be confirmed by different methods.
  • the spectrum of the cell nucleus can be measured by providing a polychromator in the second detection system 150 in place of the photomultiplier tube 156 or in addition to the photomultiplier tube 156, for example.
  • the observation process shown in FIG. 3 when the first one of a plurality of cell sheets manufactured by the same process is observed, and as a result, the number or ratio of dead cells is smaller than a predetermined value
  • the cells determined to be dead cells by the first detection system 140 may be detected at the time of detection by the second detection system 150 at the time of observing each of the remaining plurality of cell sheets. Observation of either the first detection system 140 or the second detection system 150 may be omitted when observing the cell sheet.
  • the second detection system 150 again inspects the cells determined to be dead cells by the first detection system 140.
  • the observation by the first detection system 140 may be omitted, and the observation process shown in FIG. 3 may be performed when observing another cell sheet. That is, the second determination unit 350 may determine that the second detection system 150 observes a cell that is determined to be a dead cell by the first determination unit 310.
  • FIG. 4 and 5 are diagrams illustrating the observation image generated based on the differential interference light in step S101.
  • the observed sample 112 is a mixture of live and dead cells.
  • some of the cells include a plurality of small particles having high brightness.
  • Such cells are presumed to be dead cells or dying cells. That is, the first determination unit 310 determines whether or not a cell is in a dying state, and determines that the cell is a dead cell when it is determined that the cell is in a dying state. Therefore, the cells determined to be dead cells in step S102 are excluded from the CARS light observation target in step S103. Moreover, the space
  • the scanning time by the second detection system 150 is shortened as compared with the case where all regions to be observed are observed only by the second detection system 150 without using the first detection system 140.
  • the area of the region observed with the CARS light is smaller than the case where all the regions to be observed are observed only with the second detection system 150 without using the first detection system 140. Therefore, the time required to complete the observation with CARS light can be shortened.
  • FIG. 6 is a diagram showing an observation image generated by CARS light detection using the second detection system 150 of the laser microscope 101.
  • the illustrated detection image was generated for a single observation plane perpendicular to the optical axis in a cell sheet formed by living cells.
  • the sample 112 was irradiated with pump light having a wavelength of 1064 nm and Stokes light having a wavelength of 1550 nm from the laser device 120 of the laser microscope 101. Thereby, in each of the cells included in the sample 112, CARS light having a wavelength of 820 nm is generated by the CH bond included in the lipid.
  • the sample 112 was repeatedly irradiated with the irradiation light while displacing the optical path by the scanning system 180, and the CARS light emitted from the square region having a side of 50 ⁇ m on the observation plane was detected by the photomultiplier tube 156.
  • the detection control unit 340 generates an observation image by plotting the light intensity of the detected CARS light according to the scanning by the scanning system 180.
  • the detection image generated by the detection control unit 340 has bright spots reflecting the distribution of lipids contained in the cell sheet. Further, a plurality of black spots appearing in the detection image correspond to the arrangement of cell nuclei not containing lipid. Therefore, for example, by counting these spots, the characteristics of lipid distribution in living cells can be detected. In this way, it is possible to accurately determine that the observation target is a living cell for each individual cell that cannot be determined by detection of differential interference light.
  • a threshold value may be set in advance, and if the spatial frequency of a detected image detected from an unknown sample is lower than the threshold value, it may be determined that the sample cell is a living cell.
  • FIG. 7 is a diagram showing another observation image generated by CARS light detection using the second detection system 150 of the laser microscope 101.
  • the illustrated detection image was generated for a single observation plane perpendicular to the optical axis in a cell sheet formed by necrotic dead cells.
  • the observation conditions in the laser microscope 101 were the same as those in the case where the cell sheet of live cells shown in FIG. 6 was observed, including the wavelength of irradiation light. Therefore, the detection image shown in FIG. 8 reflects the lipid distribution in dead cells.
  • a detected image detected from dead cells as described above is subjected to Fourier transform, it is characterized by high spatial frequency components. Therefore, when a threshold value is set in advance and the spatial frequency of a detected image detected from an unknown sample exceeds the threshold value, it can be determined that the sample cell is a dead cell. In this way, it is possible to accurately determine that the observation target is a living cell for each individual cell that cannot be determined by detection of differential interference light.
  • the sample was irradiated with irradiation light that generates Raman scattered light in the molecules contained in the lipid.
  • irradiation light that generates Raman scattered light in the molecules contained in the lipid.
  • other types of molecules that can generate Raman scattered light may be selected as detection targets depending on the type of cells contained in the sample.
  • an observation image in which the protein is detected may be generated, and noise may be removed by utilizing the exclusive distribution of the lipid and the protein.
  • the index for determining the state of the cell based on the detected image is not limited to the above example.
  • life / death is determined based on the feature that a living cell moves but a dead cell does not move.
  • the same observation region of the sample cell can be observed twice or more at time intervals, and the image obtained by observation can be compared to determine that a cell having no change is a dead cell.
  • a determination index based on cell movement pay particular attention to whether or not the position of organelles such as mitochondria changes, and based on images that detect the distribution of lipids that are abundant in organelles. Thus, the movement of cells can be easily detected.
  • matters that can be determined using the laser microscope 101 are not limited to cell life and death. For example, even for the same dead cell, it is possible to determine whether the dead cell is a necrotic death or an apoptotic death according to the characteristics of the detected image.
  • apoptotic dead cells approaches the shape of a round circle when compared to living cells that were flat and elongated when viewed two-dimensionally.
  • nuclear concentration occurs inside the cell.
  • the cell membrane shrinks smaller than the living state.
  • several organelles in the cytoplasm are trapped in the lipid membrane, peeled off from other surrounding cells, and eventually become apoptotic endoplasmic reticulums divided into multiple.
  • FIG. 8 is a diagram schematically showing the structure of another laser microscope 102.
  • the laser microscope 102 has the same structure as the laser microscope 101 shown in FIG. Therefore, common elements are denoted by the same reference numerals, and redundant description is omitted.
  • the laser microscope 102 is different from the laser microscope 101 in that a third detection system 210 is provided. Similar to the second detection system 150, the third detection system 210 includes a dichroic mirror 218, a condenser lens 212, a relay lens 214, and a photomultiplier tube 216. However, the wavelength of light reflected by the dichroic mirror 218 is different from that of the dichroic mirror 158 of the second detection system 150.
  • the dichroic mirror 218 in the third detection system 210 of the laser microscope 102 reflects a band including the third harmonic generated in the sample 112 by pump light or Stokes light irradiated from either of the laser light sources 122 and 124. Therefore, in the third detection system 210, the photomultiplier tube 216 receives and detects the third harmonic collected by the condenser lens 212 and the relay lens 214.
  • the third harmonic is generated according to the third-order susceptibility ⁇ (3) in the sample 112. Therefore, for example, when a cell is observed as the sample 112, a third harmonic is generated at the interface of the cell membrane, cell nucleus, or the like. Therefore, by selectively detecting the third harmonic in the third detection system 210, the contours of cells, nuclei, and the like can be detected with a resolution comparable to the observation by detection of CARS light.
  • the position of the cell membrane is known in advance by observation of differential interference light, only the relevant part is irradiated with a pulsed laser generated by the laser light sources 122 and 124 so that the outline of the cell is high-resolution. Can be detected. Therefore, even if the step of detecting the cell membrane by the third detection system 210 is added, the time required for the observation is greatly reduced as compared with the case where the entire observation region is scanned with a pulse laser.
  • the dichroic mirror 218 is used only when the third harmonic is detected. Therefore, when observing differential interference light, the dichroic mirror 218 may be extracted from the optical path of the detection light.
  • the incident angle of the detection light emitted from the sample 112 with respect to the dichroic mirror 218 is preferably smaller than 45 °. Thereby, the third harmonic can be extracted efficiently.
  • the laser microscope 102 has a structure in which a third detection system 210 is provided in addition to the first detection system 140 and the second detection system 150.
  • the third detection system 210 may be provided in place of either the first detection system 140 or the second detection system 150.
  • the first detection system 140, the second detection system 150, and the third detection system 210 may be formed as individual microscopes.
  • the third detection system 210 may be formed using a detection system that detects stimulated Raman scattering light, which is one of the third-order nonlinear optical effects.
  • dead cells are excluded from detection targets by the second detection system 150 by excluding cells determined to be dead cells by the first determination unit 310 from the observation region of the second detection system 150.
  • dead cells may be excluded from detection targets of the second detection system 150 as follows.
  • the first determination unit 310 determines whether the cells included in the observation region of the first detection system 140 are in a predetermined state, that is, dead cells, and the second determination unit 350 Based on the number of cells determined to be dead cells in the observation area or the ratio of dead cells to the total number of cells in the observation area of the first detection system 140, the first detection system 140 observes the number of dead cells. It is determined whether or not the observed area is observed by the second detection system 150.
  • the second determination unit 350 sets the observation region of the first detection system 140 to the second It is determined that the observation is not performed by the detection system 150. That is, the observation region including the cells determined to be dead cells is excluded from the observation target of the second detection system 150.
  • the ratio of dead cells to the total number of cells when the observation region of the first detection system 140 is excluded from the observation target of the second detection system 150 is, for example, 30% or more, and more preferably 10% or more.
  • the second determination unit 350 determines the observation region of the first detection system 140 from the observation target of the second detection system 150 when the number or ratio of cells determined to be dead cells is equal to or less than a predetermined value. Observe with the second detection system without exclusion. At this time, it may be determined that cells other than the cells determined to be dead cells among the cells in the observation region of the first detection system 140 are observed by the second detection system 150.
  • the second detection system 150 passes without observation.
  • the second detection system 150 may be observed at a location or area where dead cells exist.
  • data processing such as data processing for life / death determination and cell nucleus spectrum detection by the second determination unit 350 is not performed.
  • the entire data processing time by the second detection system 150 is shortened as compared with the case where the data detected by the second detection system 150 is processed for all regions to be observed. Can be shortened.
  • Image data obtained by observing a place or area where a dead cell exists is stored in the storage unit 320, and when performing a reexamination of a cell determined to be a dead cell, the image data is acquired from the storage unit 320. Data processing.
  • the second determination unit 350 When the second determination unit 350 observes an observation area where the number or ratio of dead cells is equal to or less than a predetermined value, and the second determination unit 350 determines whether the number or ratio of dead cells is equal to or less than a predetermined value On the other hand, the second determination unit 350 determines that the observation region is good, and if it is equal to or greater than a predetermined value, the second determination unit 350 determines that the observation region is defective.
  • the cell observation method can be used in a production apparatus for producing a cell sheet. That is, a manufacturing apparatus including a preparation unit that isolates cells and prepares a cell line, a culture unit that cultures the cell line on a cell sheet, and a determination unit that determines whether the cell sheet is alive or dead by the cell observation method. Moreover, by using the cell observation method, a high-quality cell sheet with few dead cells or many living cells can be efficiently produced. In this production apparatus, the cell observation method may be applied to observation of cells in culture or may be used in other production stages.

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Abstract

La présente invention porte sur un procédé d'observation qui comprend une première étape d'observation destinée à observer un sujet à observer et une seconde étape d'observation destinée à observer le sujet sur la base de données obtenues dans la première étape d'observation, la première étape d'observation mettant en jeu une étape de détection de l'état de cellules comprises dans une zone d'observation et la seconde étape d'observation mettant en jeu une étape de détermination du point de savoir si ou non les cellules sont à observer en fonction de l'état de cellules détecté dans la première étape d'observation. Dans le procédé d'observation mentionné ci-dessus, la première étape observation peut mettre en jeu une étape de jugement du point de savoir si ou non les cellules comprises dans la zone d'observation sont dans un état défini et la seconde étape observation peut mettre en jeu une étape de détermination du point de savoir si ou non la zone d'observation est à observer en fonction du nombre des cellules ayant été jugées comme étant dans l'état défini ou le rapport de ces cellules sur le nombre de ces cellules dans la zone d'observation.
PCT/JP2014/003656 2013-07-10 2014-07-10 Procédé d'observation, dispositif d'observation, procédé de production de feuille de cellule et dispositif de production de feuille de cellule WO2015004917A1 (fr)

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JP2016192923A (ja) * 2015-03-31 2016-11-17 株式会社ニコン 判定装置、判定システム、判定プログラム、細胞の製造方法、及び細胞
WO2016208356A1 (fr) * 2015-06-26 2016-12-29 株式会社ニコン Dispositif de détermination, programme de détermination, procédé de détermination, dispositif de fabrication de feuille de cellules, et procédé de fabrication de feuille de cellules
EP3315595A4 (fr) * 2015-06-26 2018-07-18 Nikon Corporation Dispositif de détermination, programme de détermination, procédé de détermination, dispositif de fabrication de feuille de cellules, et procédé de fabrication de feuille de cellules
JP2019058156A (ja) * 2017-09-28 2019-04-18 オリンパス株式会社 画像処理装置および細胞観察システム
CN112119339A (zh) * 2018-04-23 2020-12-22 生德奈股份有限公司 用于检查包含至少一种细胞和/或至少一种颗粒的液体的方法
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JP2021524593A (ja) * 2018-07-13 2021-09-13 ダンマークス テクニスク ユニバーシテットＤａｎｍａｒｋｓ Ｔｅｋｎｉｓｋｅ Ｕｎｉｖｅｒｓｉｔｅｔ 偏光分解ラマン分光法を実施するための装置
JP7330538B2 (ja) 2018-07-13 2023-08-22 ダンマークス テクニスク ユニバーシテット 偏光分解ラマン分光法を実施するための装置
WO2020162601A1 (fr) * 2019-02-08 2020-08-13 国立大学法人 筑波大学 Procédé d'estimation de type de cellule, dispositif d'estimation de type de cellule, procédé de production de cellule, dispositif de production de cellule, procédé de surveillance, dispositif de surveillance, procédé de production de modèle appris et dispositif de production de modèle appris

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