WO2006092925A1 - Dispositif, procede et programme d'observation cellulaire et systeme microscopique - Google Patents

Dispositif, procede et programme d'observation cellulaire et systeme microscopique Download PDF

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Publication number
WO2006092925A1
WO2006092925A1 PCT/JP2006/301884 JP2006301884W WO2006092925A1 WO 2006092925 A1 WO2006092925 A1 WO 2006092925A1 JP 2006301884 W JP2006301884 W JP 2006301884W WO 2006092925 A1 WO2006092925 A1 WO 2006092925A1
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Prior art keywords
cell
time point
cells
imaging
division
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PCT/JP2006/301884
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English (en)
Japanese (ja)
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Satoshi Arai
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Olympus Corporation
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Publication of WO2006092925A1 publication Critical patent/WO2006092925A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms

Definitions

  • Cell observation device cell observation method, microscope system, and cell observation program
  • the present invention relates to a cell observation apparatus, a cell observation method, a microscope system, and a cell observation program for measuring parameters of living cells, in particular, measuring cell parameters indicating characteristics of individual cells over time. It is. Background art
  • Observation of cells using an optical microscope is a simple observation method that has been used for a long time, and is currently most widely used.
  • the culture device is used in combination, and the cells are kept in the culture device except during observation, so that the damage to the cells by the environment is minimized.
  • the image site meter “iCyte” manufactured by CompuCyte Inc., sold by Olympus Corporation has an imaging mechanism by laser scanning, and the amount of fluorescent light, area, perimeter, Parameters such as sphericity can be measured.
  • Patent Document 1 discloses a configuration for avoiding damage to cells due to environmental fluctuations by eliminating the trouble of taking in and out the culture apparatus. In this configuration, while the cells are cultured, the growth state is checked, and if necessary, the cells can be observed in the culture apparatus.
  • Patent Document 2 discloses a concept of capturing an image while appropriately maintaining the state of the cell, performing cell identification and parameter calculation by image processing, and tracking individual cells. It also presents specific applications that acquire cell parameters over time.
  • Patent Document 1 Special Table 2001-500744
  • the image cytometer “iCyte” is a device intended to measure statistical parameters of cells at high speed, and since it does not need to be traced originally, it is supposed to be used for repeated measurement of living cells. Not. Cells are usually discarded at the end of the measurement and have a culture mechanism to reduce environmental damage. The survival time of cells without a culture device is a few hours at most, and long-term observation is impossible. An external culture device can be used in combination, but in this case, as in the case of the observation system that combines the optical microscope and the culture device described above, it is difficult to keep the observation target the same. Arise
  • the present invention has been made in view of the above, and ensures a situation in which cell activity is not easily lost, and acquires cell parameters for the same cell over time while confirming the identity of the cell.
  • An object of the present invention is to provide a cell observation apparatus, a cell observation method, a microscope system, and a cell observation program.
  • a cell observation device is a cell observation device that measures a cell parameter indicating characteristics of a cell over time, using infrared light.
  • An imaging means for imaging a cell including at least an infrared imaging means for imaging a cell; a cell recognition means for recognizing a cell from cell image data of an image captured by the imaging means; and a cell recognized by the cell recognition means
  • Cell parameter measuring means for measuring cell parameters indicating the characteristics of the cell based on the cell image data, and images taken at different times
  • the cell image data force of the image comprises a cell tracking means for discriminating the identity of cells at different time points recognized based on the cell parameter.
  • a microscope system is a microscope system including the cell observation device, and includes an imaging optical system that magnifies and projects a cell, and the imaging unit forms an image of the imaging optical system. It is characterized in that a cell magnified and projected on a surface is imaged.
  • the cell observation device is a cell observation device that measures cell parameters indicating the characteristics of cells over time, and recognizes cells from the cell image data of an image obtained by imaging the cells.
  • Cell recognition means a cell parameter measurement means for measuring a cell meter indicating the characteristics of the cells recognized by the cell recognition means based on the cell image data, and the cells recognized by the cell recognition means are generated as a result of cell division.
  • Cell division detection means for detecting whether or not the target is a cell based on the cell parameter, the cell division detection means comprising a cell parameter indicating a cell area at a detection target time point and a threshold value relating to the area.
  • the cell observation device is a cell observation device that measures a cell parameter indicating the characteristics of the cell over time, and recognizes the cell from the cell image data of the image obtained by imaging the cell.
  • Cell recognition means a cell parameter measurement means for measuring a cell meter indicating the characteristics of the cells recognized by the cell recognition means based on the cell image data, and the cells recognized by the cell recognition means are generated as a result of cell division.
  • Cell division detection means for detecting whether or not the target is based on the cell parameter, and the cell division detection means is a circular shape of a cell at a time point chronologically prior to the detection target time point. It is characterized by detecting whether or not the cell at the detection target time point is a force resulting from cell division by referring to the value of the cell parameter indicating the degree.
  • the cell observation device is a cell observation device that measures cell parameters indicating the characteristics of cells over time, and recognizes cells from the cell image data of an image obtained by imaging the cells.
  • Cell recognizing means, and cells showing the characteristics of the cells recognized by the cell recognizing means A cell parameter measuring means for measuring a meter based on the cell image data, and a cell for detecting whether the cell recognized by the cell recognition means is a result of cell division based on the cell parameter.
  • Cell division detection means wherein the cell division detection means refers to the value of the cell parameter indicating the circularity of the cell at a time point chronologically prior to the detection target time point, and the cell parameter indicating the circularity
  • the threshold value for the circularity has a predetermined relationship with the threshold value! /
  • the determination reference time point at which the cell is imaged is extracted, and the cell parameter and area indicating the circularity of the cell at a time point near the determination reference time point are indicated. Based on the increasing / decreasing tendency of the value of the cell parameter, it is detected whether or not the cell at the detection target time point is a result of cell division.
  • the cell observation device is a cell observation device that measures cell parameters indicating the characteristics of cells over time, and recognizes cells from the cell image data of an image obtained by imaging the cells.
  • Cell recognition means a cell parameter measurement means for measuring a cell meter indicating the characteristics of the cells recognized by the cell recognition means based on the cell image data, and the cells recognized by the cell recognition means are generated as a result of cell division.
  • Cell division detection means for detecting whether or not the cell is a non-contained cell based on the cell parameters, the cell division detection means comprising the area of the cell nucleus of the cell at the time of detection and the presence of cytoplasm Based on the result of comparison with the area of the range, it is detected whether or not the cell at the detection target time point is a result of cell division.
  • the cell observation device is a cell observation device that measures the cell parameters indicating the characteristics of the cell over time, and recognizes the cell from the cell image data of the image obtained by imaging the cell.
  • Cell recognition means a cell parameter measurement means for measuring a cell meter indicating the characteristics of the cells recognized by the cell recognition means based on the cell image data, and the cells recognized by the cell recognition means are generated as a result of cell division.
  • Cell division detection means for detecting whether or not the target is based on the cell parameter, the cell division detection means obtaining a localized region of the microtubule of the cell at the detection target time point, and It is characterized by detecting whether or not the cell at the detection target time point is a result of cell splitting by detecting whether or not it is localized at a plurality of locations.
  • the cell observation device is capable of measuring cell parameters indicating characteristics of cells over time.
  • a cell recognition device for recognizing a cell, a cell recognition means for recognizing the cell, and a cell meter indicating the characteristics of the cell recognized by the cell recognition means.
  • Cell parameter measurement means for measuring based on image data
  • cell division detection means for detecting whether the cell recognized by the cell recognition means is a result of cell division based on the cell parameter
  • the cell division detection means includes a region parameter indicating a luminance sum total of the luminance values of each pixel for pixels having a luminance value higher than a threshold relating to luminance in a region corresponding to the cell of the captured image.
  • Data is obtained for a time point near the detection target time point, and the cell at the detection target time point is determined to be a cell based on the change in the luminance summation about the time point near the detection target time point. And detecting whether or not generated as a result of the crack.
  • the cell observation program according to the present invention is a cell observation program for observing cells with a cell observation device that measures the cell parameters indicating the characteristics of the cells over time.
  • a cell recognition step for recognizing a cell from the cell image data of the image obtained by imaging the cell, and a cell parameter for measuring the cell parameter indicating the characteristic of the cell recognized in the cell recognition step based on the cell image data
  • the cell division detection step includes comparing a cell parameter indicating the area of the cell at the detection target time point with a threshold relating to the area, and The cell parameter indicating the brightness of the cell at the time of the image is compared with the cell parameter indicating the brightness of the cell at the time point to be compared with the detection time point, and the detection is performed based on the respective comparison results. It is a step for detecting whether or not the cell at the target time point is a force resulting from cell division.
  • the cell observation program according to the present invention is a cell observation program for observing cells with a cell observation device that measures cell parameters indicating the characteristics of cells over time.
  • a cell recognition step for recognizing a cell from the cell image data of the image obtained by imaging the cell, and a cell parameter for measuring the cell parameter indicating the characteristic of the cell recognized in the cell recognition step based on the cell image data Measuring step and said details
  • a cell division detection step for detecting whether or not the cell recognized in the cell recognition step is a result of cell division based on the cell parameter.
  • the step refers to the value of the cell parameter indicating the circularity of the cell at a time point chronologically prior to the detection target time point, and the force at which the cell at the detection target time point is a result of cell division. It is a step for detecting whether or not.
  • the cell observation program according to the present invention is a cell observation program for observing cells with a cell observation device that measures cell parameters indicating the characteristics of the cells over time.
  • a cell recognition step for recognizing a cell from the cell image data of the image obtained by imaging the cell, and a cell parameter for measuring the cell parameter indicating the characteristic of the cell recognized in the cell recognition step based on the cell image data
  • the cell division detection step refers to a value of a cell parameter indicating the circularity of the cell at a time point chronologically prior to the detection target time point.
  • Cell parameters indicating the circularity of cells at a time point in the vicinity of the determination reference time point are extracted by extracting a determination reference time point in which cells having a predetermined relationship with the threshold value regarding the circularity value. And a step of detecting whether or not the cell at the time of detection is a force resulting from cell division based on a tendency to increase or decrease the value of the cell parameter indicating the area.
  • the cell observation program is a cell observation program for observing cells with a cell observation device that measures the cell parameters indicating the characteristics of the cells over time.
  • a cell recognition step for recognizing a cell from the cell image data of the image obtained by imaging the cell, and a cell parameter for measuring the cell parameter indicating the characteristic of the cell recognized in the cell recognition step based on the cell image data
  • the step of detecting cell division includes the presence of cell nuclei at the time of detection. Based on the result of comparing the area of the range and the area of the cytoplasm existing range, it is a step of detecting whether or not the cell at the detection target time point is a result of cell division.
  • the cell observation program is a cell observation program for observing cells with a cell observation device that measures cell parameters indicating the characteristics of cells over time.
  • a cell recognition step for recognizing a cell from the cell image data of the image obtained by imaging the cell, and a cell parameter for measuring the cell parameter indicating the characteristic of the cell recognized in the cell recognition step based on the cell image data
  • the cell division detection step includes determining a localized region of the microtubule of the cell at the detection target time point and detecting whether the cell is localized at a plurality of locations in the cell. It is characterized by cells in the detected time is Sutetsu flop for detecting whether the power not arose result of cell division.
  • the cell observation program according to the present invention is a cell observation program for observing cells with a cell observation device that measures the cell parameters indicating the characteristics of the cells over time.
  • a cell recognition step for recognizing a cell from the cell image data of the image obtained by imaging the cell, and a cell parameter for measuring the cell parameter indicating the characteristic of the cell recognized in the cell recognition step based on the cell image data
  • the cell division detection step includes a step of detecting each pixel with respect to a pixel having a luminance value higher than a threshold relating to luminance in a region corresponding to the cell of the captured image.
  • a region parameter indicating the luminance sum total of the frequency values is obtained for a time point in the vicinity of the detection target time point, and based on the change in the luminance summation at a time point near the detection target time point, It is a step for detecting whether or not the cell at the detection target time point is a force resulting from cell division.
  • the cell observation method comprises a culture means for culturing cells and infrared light.
  • a cell observation device comprising at least an infrared imaging means for imaging cells and imaging the cells contained in the culture means, and measuring cell parameters indicating the characteristics of the cells over time.
  • the cultured cell imaging step, the cell recognition step, and the cell parameter measurement step are performed at a plurality of time points during the culture period, and are recognized from the captured images at the plurality of time points.
  • the cell observation method images a cell accommodated in the culture means, including at least a culture means for culturing the cells and an infrared light imaging means for imaging the cells with infrared rays.
  • a cell observation method for measuring cell parameters indicating the characteristics of a cell over time with a cell observation device comprising: an imaging means for culturing cells in culture while culturing the cells with the culture means A cultured cell imaging step of acquiring images of cells by imaging with the imaging means at a plurality of points in time during the period, and cell images of the images at each time point acquired in the cultured cell imaging step A cell recognition step for recognizing, a cell parameter measurement step for measuring cell parameters indicating characteristics of cells at each time point recognized in the cell recognition step based on the cell image data, and images taken at a plurality of time points Or A cell tracking step of discriminating the identity of the recognized cells based on the cell parameters, wherein the cells accommodated in the culture means are cultivated by the culture means during the culture of the culture of the
  • the cell observation device, the cell observation method, the microscope system, and the cell observation program according to the present invention can obtain cell parameters for the same cell while confirming the identity of the cell. It is possible to observe cells in culture with infrared imaging Therefore, even during the observation period or culture period, by observing the cells using the infrared light imaging means, the effect of the observation light on the cells can be suppressed to a low level, and the cell activity can be lost. And! /, Has the effect.
  • FIG. 1 is a schematic block diagram showing a configuration example of a cell observation device according to Embodiment 1 of the present invention.
  • FIG. 2 is a horizontal cross-sectional view showing a configuration example of a culture unit.
  • FIG. 3 is a longitudinal front view showing a configuration example of a culture unit.
  • FIG. 4 is a perspective view showing a configuration example of a current plate.
  • FIG. 5 is a cross-sectional view showing a heat insulation configuration example of a boundary portion between the culture unit side and the imaging unit side.
  • FIG. 6 is an explanatory diagram showing an example of a cell image in culture that has been subjected to fluorescence imaging.
  • FIG. 7 is a schematic flowchart showing an example of image data processing.
  • FIG. 8 is a diagram illustrating an example of weighting by a sharp edge filter.
  • FIG. 9 is a schematic flowchart showing a first method example of region integration.
  • FIG. 10 is a schematic flowchart showing a second method example of region integration.
  • FIG. 11 is an explanatory diagram showing an example of measurement results of cell parameters recorded in a recording unit.
  • FIG. 12 is an explanatory diagram showing the results of calculating evaluation values for possible combinations of m and n.
  • FIG. 13 is an explanatory diagram showing an example of processing result display.
  • FIG. 14 is an explanatory diagram showing an example of highlighting.
  • FIG. 15 is a schematic block diagram showing a configuration example of a cell observation device according to Embodiment 2 of the present invention.
  • FIG. 16 is a schematic flowchart showing an example of detection processing for occurrence of apoptosis.
  • FIG. 17 is a schematic block diagram showing a configuration example of a cell observation device according to Embodiment 3 of the present invention.
  • FIG. 18 is a schematic flowchart showing a first processing procedure of cell division determination processing.
  • FIG. 19 is a schematic flowchart showing a second processing procedure of cell division determination processing.
  • FIG. 20 is a schematic flowchart showing a third processing procedure of the cell division determination processing.
  • FIG. 21 is a schematic flowchart showing a fourth processing procedure of cell division determination processing.
  • FIG. 22 is a schematic flowchart showing a fifth processing procedure of cell division determination processing.
  • FIG. 24-1 is a plan view showing a state of cells other than the division phase.
  • FIG. 24-2 is a longitudinal front view showing a state of cells other than the division phase.
  • Fig. 24-3 shows the luminance distribution characteristics of the portion of Fig. 24-2.
  • FIG. 24-4 is a plan view showing the state of cells in the division phase.
  • FIG. 24-5 is a longitudinal front view showing the state of cells in the division phase.
  • FIG. 24-6 is a diagram showing the luminance distribution characteristics of FIG. 24-5.
  • FIG. 25 is an explanatory diagram showing an example of display of observation results for times t to t.
  • FIG. 26 is an explanatory diagram showing an example of the highlighted display.
  • FIG. 27 is a schematic block diagram showing a configuration example of a microscope system according to Embodiment 4 of the present invention. Explanation of symbols
  • the cell observation device captures images of a plurality of living cells into which fluorescent proteins have been introduced while culturing them, recognizes individual cell regions, and tracks position changes over time, while Independently measure cell parameters indicating the characteristics of the cells.
  • FIG. 1 is a schematic block diagram showing a configuration example of the cell observation device according to the first embodiment.
  • the cell observation device according to the first embodiment generally includes a culture unit 101 for culturing cells, an imaging unit 201 for imaging cells contained in the culture unit 101, and the entire cell observation device.
  • a preprocessing unit 305 In addition to an input unit 303 that receives information input and a display unit 304 that displays various information such as image information and presents it to the operator, a preprocessing unit 305, a cell recognition unit 310, a parameter measurement unit 307, a cell A tracking unit 308, an exposure detection unit 309, an imaging number counting unit 310, an occupied area calculation unit 311, and a focus detection unit 312 are provided.
  • Each of these units 302 to 312 is connected to the control unit 301 and controlled by the control unit 301.
  • control unit 301 The control connection to the culture unit 101 and the imaging unit 201 is not particularly shown.
  • the control unit 301, the preprocessing unit 305, the cell recognition unit 306, the parameter measurement unit 307, the cell tracking unit 308, the exposure detection unit 309, the imaging number counting unit 310, the occupied area calculation unit 311, and the focus detection unit 312 Therefore, each processing performed is performed based on a processing program stored in a memory such as CPl ⁇ 3 ⁇ 4OM installed in the cell observation device while writing necessary data in a storage device such as RAM as appropriate. Is called.
  • the slide glass 102 holds a plurality of living cells C into which fluorescent proteins that are expressed without being localized are introduced in advance, and is installed in the culture unit 101.
  • the culture unit 101 has the same configuration as the culture vessel disclosed in, for example, Japanese Patent Application Laid-Open No. 2004-113175. In this case, it is possible to use general jellyfish-derived fluorescent proteins, etc., as long as the fluorescent protein does not localize. For example, BD Bioscience's Clontech's pEGFP-Nl can be used. .
  • FIG. 2 is a horizontal sectional view showing a configuration example of the culture unit 101
  • FIG. 3 is a longitudinal front view showing a configuration example of the culture unit 101.
  • the culture unit 101 as a culture means has a front and back through-hole 103 that can accommodate the slide glass 102 therein, and is made of a material having excellent heat conduction, such as a stainless or aluminum housing.
  • a body 104 an observation window 105 formed of two optically smooth glass plates that block the front and back through-holes 103 of the case 104, and a culture solution supply pipe that supplies the culture solution A into the case 104 106, a culture medium discharge pipe 107 for discharging the culture medium A that is no longer needed from the inside of the casing 104, and two rectifying plates 108 provided at the entrance of the culture medium A to the casing 104,
  • the culture solution supply pipe that supplies the culture solution A into the case 104 106
  • a culture medium discharge pipe 107 for discharging the culture medium A that is no longer needed from the inside of the casing 104
  • two rectifying plates 108 provided at the entrance of the culture medium A to the casing 104
  • a current plate 108 is installed in the vicinity of each Neuve 106, 107 so that the flow of the culture medium A can be uniformly dispersed and recovered.
  • FIG. 4 is a perspective view showing a configuration example of the rectifying plate 108.
  • the current plate 108 is a porous member in which a plurality of through holes 108a are formed in the thickness direction.
  • the rectifying plate 108 on the inlet side distributes the culture solution A flowing from the culture solution supply pipe 106 into a plurality of through holes 108a.
  • the rectifying plate 108 on the outlet side distributes and distributes the culture solution A to be discharged at once through the culture solution discharge pipe 107 to the plurality of through holes 108a.
  • the concentrated flow can be converted into a dispersed flow, and the culture solution A can flow at a constant flow rate and flow rate in the vicinity of the slide glass 102 in which the living cells C are arranged.
  • a temperature control unit 109 is attached to the culture unit 101, and a hot water channel 110 through which the hot water W is circulated is formed around the culture unit 101.
  • a hot water channel 110 through which the hot water W is circulated is formed around the culture unit 101.
  • the heat of the hot water is transferred to the culture solution A through the housing 104.
  • temperature information of a temperature sensor (not shown) is transmitted to the control unit 301 at predetermined time intervals so that the control unit 301 maintains the temperature in the culture unit 101 within a range of 37 ⁇ 0.5 ° C.
  • the temperature and flow rate of hot water W are controlled.
  • the pH information of the culture solution A is transmitted to the control unit 301 at predetermined time intervals by a pH sensor (not shown), and the control unit 301 maintains the culture solution so that the pH of the culture solution is maintained within a predetermined range. Control CO concentration in A.
  • the unused culture solution is stored in a culture solution storage unit (not shown), and is kept at about 4 ° C by a cold insulation mechanism (not shown) in order to suppress deterioration over time.
  • the culture medium that has been kept cool is heated to about 37 ° C. by a culture liquid heating mechanism (not shown), and then supplied to the housing 104 through the culture liquid supply pipe 106.
  • the culture solution discharged through the culture solution discharge pipe 107 is stored in a waste solution storage unit (not shown).
  • a part of the discharged culture solution may be mixed with fresh culture solution and supplied to the housing 104. In this case, the impact on the cells due to the replacement of the culture solution is reduced, and the culture is continued for a longer period. It becomes the composition suitable for.
  • FIG. 5 is a cross-sectional view showing a heat insulation configuration example of the boundary portion between the culture unit 101 side and the imaging unit 201 side.
  • the heat generated by the culture unit 101 is not transmitted to the imaging unit 201 side by providing the heat insulating unit 111 as a heat insulating unit.
  • the installation location of the heat insulating unit 111 that insulates between the culture unit 101 and the imaging unit 201 has various possible powers.In the first embodiment, the housing 104 of the culture unit 101 and the imaging device constituting the imaging unit 201 Insulating part 111 is installed between them.
  • the heat insulating part 111 is a sheet shape using a highly heat insulating and elastic member such as rubber, silicon, polyurethane, etc., and is provided with a through-hole 112 having approximately the same diameter as the objective lens 202. .
  • the culture unit 101 and the objective lens 202 are optically connected through the through-hole 112, and can freely exchange light rays.
  • most of the heat generated by the culture unit 101 is blocked by the heat insulating unit 111.
  • the optical system is adjusted on the assumption that it is used at around 25 ° C, and the performance assumed to be heated by the heat from the culture unit 101 cannot be exhibited.
  • a solid-state image sensor such as a CCD provided in the imaging unit 201 increases in noise and degrades SZN as the temperature rises. Therefore, it is necessary to keep the temperature as low as possible (but do not cause condensation) to capture weak fluorescence. is there.
  • the culture means it is more preferable to use a culture medium such as the culture unit 101 that can exchange the culture solution, but it is also possible to observe cells using a general well plate.
  • a general well plate when a general well plate is used, the culture solution cannot be changed while maintaining the environmental condition. Therefore, compared to the case where the culture unit 101 is used, the culture solution associated with cell replacement is used.
  • the culture period is limited to a short period of time due to the deterioration of.
  • Healer cells are used as an example of living cells serving as measurement samples.
  • HeLa cells are derived from cervical cancer and are widely used in drug discovery toxicity tests and the like.
  • the type of fluorescent protein to be introduced may be changed according to the contents of the assembly.
  • the imaging unit 201 includes an excitation light illumination unit 203, a dichroic aperture mirror 204, an objective optical system 205, an imaging optical system 206, a fluorescence imaging unit 207, an infrared light illumination unit 208, and a dichroic mirror 209. And an imaging optical system 210 and an infrared light imaging unit 211. That is, the imaging unit 201 of the first embodiment is configured to have a fluorescence imaging system and an infrared light imaging system.
  • the light emitted from the excitation light illuminating unit 203 is reflected by the dichroic mirror 204 and is applied to the slide glass 102 through the objective optical system 205 including the objective lens 202 and the observation window 105.
  • the irradiated light as excitation light
  • fluorescent protein force fluorescence introduced into the living cells C on the slide glass 102 is emitted, and both reflected light and fluorescence of the excitation light are emitted from the observation window 105.
  • the emitted light passes through the objective optical system 205 again and reaches the dichroic mirror 204, but only the fluorescence is transmitted, and the reflected light of the excitation light is blocked.
  • the fluorescence transmitted through the dichroic mirror 204 is reflected by the imaging optical system 206 as cell light imaging means.
  • the image is magnified and projected on a solid-state imaging device such as a CCD or CMOS provided in the fluorescence imaging unit 207.
  • FIG. 6 is an explanatory diagram showing an example of a cell image in culture that has been subjected to fluorescence imaging.
  • the light emitted from the infrared light illumination unit 208 is irradiated to the slide glass 102 through one observation window 105, and the transmitted light is emitted from the other observation window 105.
  • the emitted light passes through the objective optical system 205, and all the force infrared light that reaches the dichroic mirror 209 is reflected.
  • the reflected infrared light is enlarged and projected by the imaging optical system 210 onto a solid-state imaging device such as a CCD or CMOS provided in the infrared light imaging unit 211 as an infrared light imaging means.
  • An infrared light image of the formed measurement sample is converted into image data by a solid-state imaging device such as a CCD or CMOS provided in the infrared light imaging unit 211, and temporarily or temporarily in the recording unit 302 under the control of the control unit 301. Record permanently.
  • a solid-state imaging device such as a CCD or CMOS provided in the infrared light imaging unit 211, and temporarily or temporarily in the recording unit 302 under the control of the control unit 301. Record permanently.
  • fluorescent proteins cannot be uniformly introduced into all cells, and even if they can be introduced, they are not necessarily expressed immediately. Therefore, there is a need for a means for stably observing the entire cell over time.
  • infrared light has lower phototoxicity to living cells than visible light, it can maintain cell activity for a longer period of time compared to imaging using visible light.
  • the entire visible light range can be used as excitation light for fluorescence imaging, so that restrictions on available fluorescence proteins are relaxed.
  • the live cell C on the slide glass 102 is imaged by using the fluorescence imaging unit 207 and the infrared light imaging unit 211, so that the live cell C is captured. It is possible to acquire cell image data that is image data of an image obtained by imaging cell C.
  • imaging is automatically performed by the fluorescence imaging unit 207 at preset time intervals. And it is necessary when you want to observe the state of cells Accordingly, the user can observe the living cell C using the infrared light imaging unit 211 at a desired time.
  • the imaging by the infrared imaging unit 211 is performed at a timing synchronized with the imaging by the fluorescence imaging unit 207 by the control of the control unit 301 that is not performed by the user at a desired time, an infrared image is obtained.
  • a function of displaying the time when the fluorescent image or the infrared light image is captured on the display unit 304 may be added.
  • the configuration of the first embodiment includes the fluorescence imaging unit 207 and the infrared light imaging unit 211, the fluorescence image capturing and the infrared light image capturing can be performed in parallel. Compared with the case of imaging while switching, the time required for imaging is greatly reduced, and the switching drive unit is not required.
  • phase difference observation is performed instead of transmission observation. I can do it.
  • Phase contrast observation provides an image with higher contrast than transmission observation.
  • a polarizer and a DIC (Differential Interference Contrast) element are inserted into the infrared light illumination unit 208, and a DIC slider and an analyzer are inserted in the optical path from the dichroic mirror 209 to the imaging optical system 210, transmission is achieved.
  • Differential interference observation can be performed instead of observation. Differential interference observation provides a higher contrast image than transmission observation.
  • the stage conveyance mechanism 113 performs imaging of living cells C in a plurality of fields of view while changing the relative position of the slide glass 102 and the solid-state imaging device included in the fluorescence imaging unit 207 and the infrared light imaging unit 211. .
  • imaging range When capturing images while switching between multiple fields of view (imaging range), record the stage position in each field of view, and reproduce the stage position using the stage transport mechanism 113 prior to the second and subsequent imaging of each field of view. .
  • the exposure detection unit 309 detects whether or not the power at the time of image data capturing is appropriate. If the exposure at the time of imaging is inappropriate, the imaging of the improperly exposed part is performed again immediately or when imaging of any other observation part is completed. At this time, the exposure condition may be changed. Similarly, the focus detection unit 312 performs focusing when capturing image data. It detects whether it was appropriate. If the focusing at the time of imaging is inappropriate, the imaging of the inadequate in-focus area is performed again immediately or when imaging of any other observation area is completed. At this time, the focusing condition may be changed.
  • the circulation may be temporarily stopped in accordance with the timing of imaging. Thereby, it is possible to avoid fluctuations in the background at the time of imaging due to the circulation of the culture solution.
  • the number of times of imaging in a predetermined field of view is counted by an imaging number counting unit 310 as an imaging time point recognition unit, and after imaging a predetermined number of times (frames) of a predetermined field of view, an image is displayed on the display unit 304 as a notification unit. It may be displayed and the operator may be asked to confirm the contents. If the operator determines that there is no problem in the content, the process is continued. If it is determined that there is a problem, an instruction from the operator is accepted for resetting the imaging conditions. Alternatively, the process may simply be stopped. If there is no response from the operator even after a certain period of time, follow the predetermined instructions and choose to continue or stop the process.
  • the number of times of imaging by the fluorescence imaging unit 207 is counted, and the operator is asked to confirm the image after a predetermined number of times of imaging.
  • the time point of imaging not only based on the number of times of imaging, but also means of measuring the passage of a predetermined time from the start of observation (for example, acquiring information on the time when cell image data was captured and The image may be confirmed by providing (when the predetermined time is exceeded, recognizing the acquisition of cell image data at a predetermined time).
  • the culture period is long, the cells in culture may proliferate or die outside the assumed range, and the image brightness value may increase. At this time, an image suitable for cell observation may be obtained. It is also expected that it will be impossible to obtain. Therefore, as described above, by letting the operator recognize that the predetermined time has passed or by canceling the processing, the operator can check the image acquired so far and the image at that time. Thus, subsequent cell observation can be performed appropriately.
  • FIG. 7 is a schematic flowchart showing an example of image data processing executed by the preprocessing unit 305 and the like under the control of the control unit 301.
  • Imaging unit as described above
  • preprocessing is performed by the preprocessing unit 305 (step S2)
  • the cell is recognized by the cell recognition unit 306 as cell recognition means (step S3).
  • the cell parameter indicating the feature of the recognized cell is measured based on the cell image data by the parameter measuring unit 307 as a cell parameter measuring means (step S4).
  • the cell tracking unit 308 as a cell tracking means determines the identity of cells captured at different time points based on the cell parameters. .
  • the tracking result is further corrected (step S6).
  • the obtained tracking result is displayed on the display unit 304 (step S7), and the above processing steps are repeated in the same manner until the observation is completed (step S8; Yes).
  • step S1 (or step S1 and step S2) is performed in advance at a plurality of times, and step S2 and subsequent steps for image data acquired at each time ( Alternatively, the processing from step S3 onward may be performed collectively later.
  • image data captured at a plurality of times are preliminarily acquired and image data processing is performed later, imaging and image data processing are performed in parallel.
  • the system configuration is simplified and the responsiveness and stability can be improved by using an inexpensive computer.
  • step S2 the pre-processing unit 305 processes the image data picked up and recorded in the recording unit 302 as follows.
  • an edge-preserving low-pass filter is applied to the image data.
  • the edge-preserving low-pass filter suppresses the deterioration of the spatial frequency and high-frequency components at the edge, while providing a smoothing effect other than at the edge. Noise can be removed while preserving cell contour information. It is suitable for this method.
  • the image data after applying the edge preserving low-pass filter is further subjected to edge enhancement.
  • Apply a sharp edge filter is a filter that obtains the sum by weighting the target pixel and its neighboring 8 pixels as shown in Fig. 8, for example, and it is possible to realize sharpening processing by repeatedly executing this for each pixel. .
  • step S3 the pre-processed image data is analyzed by the cell recognition unit 306 in the following procedure, and the area occupied by each cell is recognized. If this procedure is followed, the area occupied by individual cells can be recognized even when the cells are adjacent to each other and dense, not only when the cells are scattered without being adjacent to each other. It can also be applied to the case where the edge of the cell region is clear.
  • Dividing watershed region division is known as a process that satisfies such requirements.
  • This watershed region segmentation method is used as the cell recognition processing procedure in Embodiment 1 (Vincent & 3 ⁇ 4oille, Watersheds in Digital Spaces: An Efficient Algontnm Based
  • integration processing may be performed in which a plurality of cell regions are integrated into a new cell region according to the characteristics of adjacent cell regions. Since the result of the watershed area division process generally tends to be divided into small areas, the quality of the recognition result can be improved by performing the integration process.
  • I (P) is the luminance value of pixel P in the image after applying the edge-preserving low-pass filter
  • Zl (P) is the brightness of two vertices in the image after applying the edge-preserving low-pass filter.
  • the average of the degree values, ⁇ represents that the sum is obtained for all the pixels of the line segment connecting the vertices.
  • step S313 after obtaining the distance D between vertices for all combinations of adjacent cell regions, in step S314, the distance D between the distance D and a predetermined threshold V is calculated.
  • Step S314 If the result of the comparison is below the predetermined threshold V (Step S314; Yes
  • step S315) Integrate the cell areas into one area (step S315). This process is repeated in the same manner until all the combinations are completed (step S316; Yes).
  • FIG. 10 is a schematic flowchart showing a second method example of region integration.
  • an edge extraction filter such as a Sobel filter
  • the edge preserving low-pass filter is applied to the output result of the edge preserving low-pass filter to obtain an edge image (step S321).
  • step S322 select any two adjacent cell regions (step S322), and obtain the edge strength D defined by equation (2) (steps).
  • E (P) represents the luminance value of the pixel P in the edge image
  • ⁇ represents that the sum is obtained for all the pixels included in the boundary between the cell regions.
  • step S323 edge strength D for all combinations of adjacent cell regions
  • step S324 the edge strength D is compared with a predetermined threshold value V.
  • each cell region may be verified using luminance information.
  • a pixel having the maximum luminance value is obtained for each divided cell region, and when the luminance value is smaller than a predetermined threshold value Vtmin, the region is determined not to be a cell region, and the pixel to which it belongs is also included. Exclude it from subsequent processing. As a result, cells with insufficient introduction or expression of fluorescent protein, and background regions other than cells can be excluded.
  • the luminance of each pixel in the cell region may be compared with a predetermined threshold value Vpmin, and pixels having a luminance lower than the threshold value Vpmin may be excluded from the cell region force.
  • the pixels excluded in this way are not used for the subsequent processing.
  • the obtained cell region and a set of pixels belonging to each cell region are recorded in the recording unit 302.
  • the cell region can be recognized.
  • the total area of all cells in the image that is, in the image
  • the area corresponding to the cell occupancy value indicating the degree of occupancy in the cell area is calculated by the occupancy area calculation unit 311 as the occupancy area calculation means, and the area force occupied by the cell area in the image is determined with respect to the area of the image.
  • the control unit 301 is notified of the event.
  • the control unit 301 may further notify the operator through the display unit 304 as notification means according to the setting specified by force, and may change the control state of the culture unit 101. Yes. Alternatively, you can simply ignore the notification. With this function, if the culture is prolonged, the free space of the medium may decrease due to cell growth, etc., so there is not enough free space in the medium during the cell culture process! It is effective when notifying.
  • the occupied area calculation unit 311 obtains the area occupied by the cell region in the cell image as the cell occupation value.
  • the cell image may be either a fluorescent image or an infrared light image.
  • the area of the cell region is measured by the parameter measurement unit 307. Therefore, if the areas of all the cell regions in the image are summed, the area occupied by the cell region in the image can be obtained. it can.
  • the luminance value of the area where the cell exists is observed as a luminance value different from the background. Therefore, for each pixel in the image, a typical background brightness value P
  • the area occupied by the cell region in the image can be obtained.
  • the cell parameters measured at different times are not associated with each other, and it cannot be said that the measurement is performed with time. Therefore, it is necessary to associate cell regions between cell images taken at different times and associate cell parameters using the results.
  • the cell region association is executed in the cell tracking unit 308 as the processing of steps S5 and S6 as follows.
  • R is the cell region recognized at time t
  • t is the cell region recognized at time t
  • R denote the cell region recognized at 1 tl, m 2. However, time t is later than time t It is a later time in series.
  • m and n are the identification numbers of cell regions that do not overlap in the same image, l ⁇ m ⁇ M, l ⁇ n ⁇ N, and M and N are the cell regions recognized at time t and t, respectively.
  • Equation (3) an evaluation function related to the relationship between two cell regions R and R is defined by equation (3).
  • FIG. 12 is an explanatory diagram showing the results of calculating the evaluation values for possible combinations of m and n.
  • J (R, R) 3 ⁇ 4J is abbreviated.
  • region R at time t corresponding to region R at time t is determined according to equation (4).
  • R is t2, n tl, m 1 2 in the region at time t that minimizes the evaluation town between region R.
  • the evaluation function is applied to determine a combination that is smaller than the evaluation city 21S. Second evaluation value
  • the combination that is displayed and judged by the operator to be correct is input from the input unit 303, and the association is performed based on the input result.
  • the region R at time t and the region R at time t are the result of recognizing the same cell at different times.
  • both measured cell parameters can be regarded as measured values at the same time for the same cell. Therefore, the parameter measurement over time is completed by associating the value of the cell parameter with the cell image, the cell region, the association information of the cell region, and the time information together with the recording unit 302 as a recording means. .
  • the cells in the observation screen move to the outside of the observation screen, multiple cells overlap, or the cells die.
  • the number of cell regions recognized by the cell recognition unit 306 is reduced, there is no cell region at time t corresponding to the cell region at time t, or there is a duplication of time t.
  • the number of cells corresponds to one cell at time t.
  • a flag indicating that there is no corresponding area is recorded. If multiple cell areas correspond to one cell area, record all correspondences. A message may be displayed to the operator through the display unit 304, and the correspondence relationship may be corrected based on input by the operator.
  • the data representation when recording the correspondence of multiple cell regions is A tree structure is used in which the time is height and each cell region corresponds to a node. The degree of freedom of expression is higher, and a graph structure may be used.
  • cell region association may be a modified example with the following improvements.
  • the first variation is that if the minimum evaluation »is greater than a predetermined threshold V, its mapping jmax
  • mapping is considered invalid. In this case, region dmax
  • the meter measurement is discontinued until time tl. This modification is effective to reduce errors in the cell region association processing.
  • FIG. 13 is an explanatory diagram showing an example of a display of processing results.
  • the display screen 314 included in the display unit 304 has two display areas 314a and 314b, and individual cell areas recognized at the time of processing are displayed in the display area 314a.
  • a labeling process is applied to the cell region, and a color, brightness, line type, and pattern that can be identified for each region are given and displayed as, for example, label images a to e.
  • An infrared light image or a fluorescent image in the same display range as the label image may be displayed in conjunction with each other, or a plurality of label images, infrared light images, and fluorescent images may be displayed in an overlapping manner. Alternatively, superimpose display may be performed.
  • the measured cell parameters are displayed as a line chart with time on the horizontal axis and parameter values on the vertical axis in the display area 314b. Furthermore, if the display contents of both are synchronously highlighted according to the mouse operation or the like in the input unit 303 by the operator, the visibility of the display contents is improved.
  • FIG. 14 is an explanatory diagram showing an example in which a line chart of cell parameters corresponding to, for example, the case where the label image c is selected and specified as an instruction to be highlighted is also highlighted. is there. In this case, when the operator selects and emphasizes one of them, the corresponding other is also highlighted in synchronization.
  • Embodiment 1 fluorescent protein is introduced into living cells C and observed, but if a luminescent gene, for example, a luciferase gene is introduced instead of the fluorescent tank, a luminescent image is substituted for the fluorescent image. Can be imaged. In this case, the excitation light illumination unit 203 and the dichroic mirror 204 are unnecessary, and the configuration can be simplified.
  • the luminescent image is captured by a fluorescence imaging unit 207 as a cell light imaging unit.
  • the light emission image may be processed in the same procedure as the fluorescence image. In this way, even when the cell emits light other than infrared light, for example, when the cell emits light or emits fluorescence, the cell image data can be acquired and the cell observation can be performed.
  • a fluorescent protein that is expressed without being localized in the cell is used.
  • the cell parameters measured by the parameter measuring unit 307 are not limited to those exemplified in the first embodiment, and are further defined as follows: area, perimeter, circumscribed rectangle position, X-direction ferret diameter, Y-direction Free diameter, minimum free diameter, maximum free diameter, average free diameter, convex circumference, roundness (roundness), number of holes, roughness (ratio of convex circumference to circumference), Euler number, Length, width, flatness, sum of brightness, minimum brightness, maximum brightness, average brightness, brightness standard deviation, brightness dispersion, entropy, center of gravity position, second moment, main axis direction, or more Also good.
  • the parameter measurement unit 307 performs the cell number, the minimum intercellular distance, the maximum intercellular distance, the average intercellular distance, the standard deviation of the intercellular distance, One or more of the dispersion of the intercellular distance and the minimum value, maximum value, average value, standard deviation, fractional difference, sum, intermediate value of each parameter measured for each cell may be obtained.
  • the living cells into which the fluorescent protein has been introduced are cultured for a long period of time, the influence of the observation light on the cells is suppressed to ensure a situation in which the cell activity is not easily lost. Track individual cells without losing sight and measure various cellular parameters over time A device can be realized.
  • the cell observation device like the first embodiment, images a plurality of living cells into which fluorescent proteins have been introduced while culturing them, recognizes the area of each cell, and determines the position over time. While tracking changes, cell parameters that characterize individual cells are independently measured, and cell apoptosis is further detected. Detection of cell apoptosis means detecting whether the cell has entered a mode of programmed death of the cell.
  • FIG. 15 is a schematic block diagram showing a configuration example of the cell observation device according to the second embodiment.
  • the connection from the control unit 301 for controlling the present cell observation apparatus to each part of the culture system and the imaging system is not particularly shown.
  • a culture including a slide glass 102 holding a plurality of living cells C preliminarily labeled with a fluorescent protein, an observation window 105, a stage transport mechanism 113, and the like.
  • Imaging unit 101 imaging unit 201 (objective optical system 205, imaging optical system 206, fluorescent imaging unit 207, infrared illumination unit 208, dichroic mirror 209, imaging optical system 210, infrared imaging unit 211, etc.), Control unit 301, recording unit 302, input unit 303, display unit 304, preprocessing unit 305, cell recognition unit 306, parameter measurement unit 307, cell tracking unit 3 08, exposure detection unit 309, imaging number counting unit 310, occupied area A calculation unit 311 and a focus detection unit 312 are provided, and these operate in the same manner as in the first embodiment.
  • the excitation light illumination unit 203 'and the dichroic mirror 204' have a wavelength selection function in addition to the functions of the illumination unit 203 and the dichroic mirror 204 in the first embodiment, and emit at least two different bands of fluorescence. It is possible. Further, as a unique configuration of the second embodiment, an apoptosis detection unit 315 is provided as an apoptosis detection means.
  • the living cells to be targeted in Embodiment 2 are those in which the cell membrane and the intracellular organelle are labeled with fluorescent proteins having different wavelength characteristics, respectively, and as a result, at least doubly labeled.
  • fluorescent proteins having different wavelength characteristics, respectively, and as a result, at least doubly labeled.
  • the measurement sample that is, the labeled live cell C
  • the measurement sample is cultured and imaged. This is performed over time, and the acquired fluorescent image and infrared light image are recorded in the recording unit 302.
  • fluorescent images of fluorescent proteins labeled with cell membranes hereinafter referred to as “cell membrane fluorescent images” t ⁇ ⁇
  • fluorescent images of fluorescent proteins labeled with intracellular organelles hereinafter referred to as “ Take at least two fluorescent images of “intracellular organelle fluorescence image” and i).
  • the excitation light illumination unit 203 ′ having a wavelength selection function and the dichroic mirror 204 ′ select excitation light and transmission wavelength suitable for excitation of each fluorescent protein.
  • the cell membrane fluorescent image is processed by the preprocessing unit 305 and the cell recognition unit 306 to recognize the cell region.
  • the processing procedure is the same as in the first embodiment.
  • the recognized cell area is recorded in the recording unit 302.
  • the parameter measuring unit 307 measures at least the area and the circularity among the parameters of the cell region shown in the first embodiment. Next, for each recognized individual cell region, the entropy in the corresponding region of the intracellular organelle fluorescence image is measured. Equation (6) is used to calculate entropy H. Where A is the area of the region of interest R, P is the luminance value at the position (X, y) in the region of interest R, and S is the luminance summation in the region of interest R as shown in equation (7).
  • Cell membrane fluorescence image power The parameters measured for the extracted cell region and the entropy measured for the corresponding region of the intracellular organelle fluorescence image are compared with the cell membrane fluorescence image, the intracellular organelle fluorescence image, and the cell region. By associating and recording in the recording unit 302, parameter measurement is completed.
  • cell regions extracted at different times are associated with the extracted cell regions.
  • the processing procedure is the same as in the first embodiment.
  • Mapping cell regions Information is recorded in the recording unit 302 in association with the cell parameter and time information measured by the cell membrane fluorescence image, the intracellular organelle fluorescence image, the cell region, and the parameter measurement unit 307.
  • the apoptosis detection unit 315 investigates whether or not apoptosis has occurred. Any one of the cell regions at time t recognized by the cell recognition unit 306 is manually or
  • FIG. 16 is a schematic flowchart showing a detection processing example of the occurrence of t4 apoptosis for the cell region R of interest, which is executed by the apoptosis detection unit 315. This process is
  • Step S9 This process is executed as step S9 in the flowchart shown in FIG. First, if the entropy H (R) of the cell region R t is less than the predetermined threshold V (Step S21; No),
  • step S25 It is determined that it is not! / ⁇ (negative) (step S25), and the apoptosis detection process is terminated.
  • step S21 If entropy! ⁇ ) Is greater than or equal to threshold V (step S21; Yes), cell region t4 H 4 at time t
  • a cell region R at time t corresponding to R is acquired from the recording unit 302.
  • the time t is a time (tast) that is a predetermined time interval before the time t in the time t4 3 t3 3 series.
  • the cell region is a time (tast) that is a predetermined time interval before the time t in the time t4 3 t3 3 series.
  • step S24 it is determined that apoptosis is positive (positive) (step S24). If at least one of them is not established, it is determined that the apoptosis is negative (negative) (step S25).
  • the result of apoptosis determination is displayed on the display unit 304.
  • This can be done using any display format that can distinguish between cell areas that were positive in apoptosis determination and cell areas that were negative.
  • the marker is superimposed on only the positive or negative cell area
  • the different marker is superimposed on the positive and negative cell area
  • only the positive or negative cell area is displayed, and the positive and negative cell areas are displayed.
  • Techniques such as changing display attributes such as color 'pattern' blinking, changing response contents to operator operations in positive and negative cell regions, etc. can be applied, and these may be switched between each other.
  • the determination results for a plurality of images acquired at different times are sequentially switched and displayed, it is possible to visually grasp the occurrence of apoptosis over time.
  • the cell observation device captures images while culturing a plurality of living cells into which a fluorescent protein has been introduced, recognizes the area of each individual cell, and determines the position over time. While tracking changes, cell parameters indicating individual cell characteristics are independently measured, and cell division is further detected to generate a cell lineage.
  • FIG. 17 is a schematic block diagram showing a configuration example of the cell observation device according to the third embodiment.
  • the present Embodiment 3 is a culture in common with Embodiment 1, including a glass slide 102 holding a plurality of viable cells C preliminarily labeled with a fluorescent protein, an observation window 105, a stage transport mechanism 113, etc.
  • imaging unit 201 excitation light illumination unit 203, dichroic mirror 204, objective optical system 205, imaging optical system 206, fluorescence imaging unit 207, infrared illumination unit 208, dichroic mirror 209, imaging optics System 210, infrared imaging unit 211, etc.
  • control unit 301 recording unit 302, input unit 303, display unit 304, preprocessing unit 305, cell recognition unit 306, parameter measurement unit 307, cell tracking unit 308, exposure detection Part 309, Counting the Number of Imaging A unit 310, an occupied area calculation unit 311, and a focus detection unit 312 are provided and operate in the same manner as in the first embodiment.
  • a cell division detection unit 316 as a cell division detection unit and a genealogy generation unit 317 as a cell lineage generation unit are provided.
  • the measurement sample that is, the live cell C with the label
  • the measurement sample is cultured and imaged over time, and the obtained fluorescence image and infrared light image are obtained.
  • the cell fluorescence image is processed by the preprocessing unit 305 and the cell recognition unit 306 to recognize the cell region.
  • the processing procedure is the same as in the first embodiment.
  • the recognized cell area is recorded in the recording unit 302.
  • the parameter measurement unit 307 measures the cell parameters in the cell region shown in the first embodiment. The necessary cell parameters differ depending on the operation of the cell division detection unit 316 described later.
  • the measured cell parameters are recorded in the recording unit 302.
  • the cell tracking unit 308 associates the cell regions from which the cell fluorescence image forces at different times are also extracted.
  • the processing procedure is the same as in the first embodiment.
  • the cell region association information is recorded in the recording unit 302 in association with the cell image, the cell region, the cell parameter measured by the parameter measuring unit 307, and the time information.
  • the cell division detection unit 316 determines the presence or absence of cell division according to the procedure described later.
  • parent-child correspondence When cell division is determined, in addition to the normal relationship between cells over time (hereinafter referred to as “time relationship”), one parent cell before cell division and two after division
  • time relationship the normal relationship between cells over time
  • parent-child correspondence relationship may be recorded as additional information in the temporal correspondence relationship, or may be recorded separately from the temporal correspondence relationship.
  • the former can save the storage area, and the latter makes the operation for referring to information simple.
  • a tree structure is used in which each generation corresponds to a height and each cell region corresponds to a node.
  • the cell division determination in the cell division detection unit 316 includes a plurality of processing procedures as described below, and the determination processing is performed using one or more of these procedures. This process is executed as the process of step S10 in the flowchart shown in FIG.
  • FIG. 18 is a schematic flowchart showing a first processing procedure of the cell division determination processing.
  • the schematic flow chart shown in Fig. 31 shows that if the daughter cell area after cell division is smaller than the area of a normal cell and the sum of the brightness of the corresponding cells before and after cell division is approximately equal, it is based on ⁇ ⁇ characteristics.
  • nl t2, n2 Al is small or at least one area is larger than the threshold value V (step S31;
  • step S35 it is determined that it is not cell division (step S35).
  • the threshold V is 0.5 times the average area of the cell region, and the threshold V is 0.9 times.
  • Step S35 If threshold V or less (step S33; Yes), cells
  • step S34 It is determined that it is a split.
  • FIG. 19 is a schematic flowchart showing a second processing procedure of the cell division determination processing.
  • the schematic flowchart shown in FIG. 19 exemplifies a cell division determination processing procedure based on the characteristic that a cell immediately before cell division contracts with the passage of time and becomes a substantially spherical shape and then undergoes cell division.
  • S is cell area
  • L is cell contour length
  • step S41 Is obtained (step S41), and it is determined whether or not there is a frame whose circularity C exceeds a predetermined threshold V (step S42). If such a frame does not exist (step S42; No), it is determined that the cell division is not! / (Step S46).
  • step S42 If it exists (step S42; Yes), the roundness of the region R exceeds the predetermined threshold.
  • step S43 and S44 it is determined whether or not the condition that the former is approximately monotonically increasing and the latter is approximately monotonically decreasing is satisfied.
  • the circularity and area transitions by regression analysis are approximated by a straight line, and the slope of the approximate straight line of the circularity transition is greater than a predetermined positive value V and the area transition. Near You can see if the slope of the straight line is less than a predetermined negative value V.
  • step S43 When the two conditions are both satisfied (step S43; Yes, step S44; Yes), it is determined that the cell is split (step S45). If not, it is determined that the cell is not divided (step S46).
  • FIG. 20 is a schematic flowchart showing a third processing procedure of the cell division determination processing.
  • the schematic flowchart shown in FIG. 20 illustrates the procedure for determining cell division based on the characteristics when the nuclear membrane disappears in the cell immediately before cell division and the components in the cell nucleus diffuse throughout the cytoplasm! .
  • cell nuclei and cytoplasm are preliminarily labeled so that regions can be extracted independently of each other.
  • a subcellular localization vector “BDLivingColorsSubcellularLocalizationVectorJ” licensed by BD Biosciences, Clontech can be used.
  • FIG. 21 is a schematic flowchart showing a fourth processing procedure of the cell division determination processing.
  • the schematic flow chart shown in Fig. 21 is based on the characteristic that microtubules form two spindles in the cell immediately before cell division, and there is almost no area other than this spindle region! The determination processing procedure is illustrated.
  • intracellular microtubules are labeled in advance so as to be optically distinguishable.
  • An example of such a label is the BD Biosciences licensed by Clontech! /
  • Step S61 the generated density distribution map is subjected to spatial low-pass filter processing (step S62), and the density distribution map power after filtering is Is detected (step S63), and it is determined whether or not there are two detected maximum points (step S64). If there are two maximum points (step S64; Yes), that is, if the concentration is localized at two locations in the region, it is determined that the cell is dividing (step S65). Otherwise (step S64; No), it is determined that cell division is not! / (Step S66).
  • FIG. 22 is a schematic flowchart showing the fifth processing procedure of the cell division determination processing.
  • the schematic flowchart shown in FIG. 22 exemplifies a cell division determination processing procedure based on the characteristics when the three-dimensional shape of the cell changes before and after cell division.
  • FIG. 23 is a characteristic diagram showing an example of temporal transition of luminance sum S, tl, m0 L L.
  • the luminance sum S increases rapidly as shown in Fig. 23.
  • step S 74 When adding, it is determined that the cell is dividing (step S 74), and the time when the luminance sum S has changed most rapidly is taken as the division time.
  • a predetermined threshold V is prepared, and the luminance sum S is the threshold V.
  • step S73 If it is determined that the range exceeding the period is the mitotic period (step S73), the processing can be realized easily. This j
  • step S75 if there is no range exceeding the threshold V, it means no cell division (step S75).
  • the cells are shown in the plan view shown in Fig. 24-1, the longitudinal front view shown in Fig. 24-2, and the portion of Fig. 24-2 shown in Fig. 24-3. It is observed as a luminance distribution characteristic.
  • the mitotic phase M phase
  • the microtubules that maintain the shape of the cells are diverted to the formation of the spindle, so that the cells cannot maintain their previous shape and are affected by the surface tension of the cell membrane. It becomes a shape close to a sphere.
  • the cells are observed as shown in the plan view shown in Fig. 24-4, the longitudinal front view shown in Fig. 24-5, and the luminance distribution characteristics of Fig. 24-5 shown in Fig. 24-6.
  • the part involved in the luminance sum S varies depending on the three-dimensional shape.
  • the luminance sum S is calculated by using only the part exceeding the threshold V as in this method, the part A involved in the sum in the mitotic period M becomes larger than the part B involved in the sum other than the mitotic period M. . Therefore, the luminance sum S is as shown in Fig. 23. Change. In other words, the period in which the luminance sum S shows a unimodal transition is the cell cycle.
  • this processing procedure 5 is also effective as a method for detecting the M phase in the cell cycle.
  • any of the first processing procedure power and the fifth processing procedure described above may be used. Furthermore, it is possible to combine two or more processing procedures and combine the determination results. In this case, determination with higher accuracy is possible.
  • the presence or absence of cell division can be determined, and the parent-child relationship of cells can be known.
  • the parent-child relationship of cells can be known.
  • This cell lineage is generated by the lineage creation unit 317 associating cells before and after division based on the detection result of the cell division detection unit 316.
  • a cell line that connects two or more generations of parent-child relationship information is called a “genealogy”, and the parent-child relationship information between two generations is the smallest genealogy.
  • the genealogy information generated by the genealogy creation unit 317 is held in the recording unit 302.
  • the screen 314 of the display device included in the display unit 304 is divided into four display areas 314c to 314f, and each of the display areas 314c to 314e includes a time point t that is a detection target time point. 3 time points
  • Image information indicating each cell region of t, t, and t is displayed.
  • each time point indicating each cell region of t, t, and t is displayed.
  • each cell area is displayed in a tree structure (tree structure), and the genealogy information of the cells generated by cell division is displayed at the same time.
  • the contents to be displayed are selected according to the operator's instruction and the capability of the display unit 304.
  • a labeling process is applied to the cell area, and a color, brightness, line type, and pattern that can be identified for each area are given and displayed as a label image.
  • a cellular infrared light image or a fluorescence image in the same display range may be displayed, or the three may be switched.
  • the tripartite A plurality of them may be displayed in conjunction with each other, or may be displayed in a superimposed manner. As shown in FIG. 38, the visibility is further improved if the shape of the label image corresponds to the shape of the actual cell.
  • the display contents of both are synchronously highlighted in accordance with an operator instruction using the input unit 303 such as a mouse, the display contents can be confirmed more easily.
  • the selected object is highlighted and the other corresponding display object is also highlighted.
  • the ancestors (including parents) and descendants (including children) of the selected cell are also emphasized, it becomes easier to visually grasp the cell lineage.
  • FIG. 26 shows an example in which the operator selects cell region 321 at time t.
  • the region 321 is highlighted by increasing the width of the outline, and the cell region 322 at time t, which corresponds to an ancestor on the genealogy, the cell region 323 at time t, which corresponds to a descendant on the genealogy, and the genealogy
  • the corresponding nodes and the connection lines between the nodes are highlighted in synchronization.
  • a plurality of living cells labeled with a fluorescent substance are imaged over time, the individual cells are recognized, and the position change with time is tracked.
  • Cell lineage information can be acquired.
  • cell division occurs during long-term culture of cells, this can be detected, and if cell division occurs during long-term culture of cells, this can be detected.
  • the accuracy of cell observation is improved.
  • cell division can be detected with high accuracy.
  • Embodiment 4 of the present invention shows an application example to a microscope system in which the functions of Embodiments 1 to 3 are combined.
  • the present microscope system uses fluorescent proteins. After culturing multiple introduced living cells, recognizing the area of each cell, tracking changes in position over time, and independently measuring cell parameters indicating the characteristics of each cell, Apoptosis test similar to that in Embodiment 2 or cell splitting detection similar to that in Embodiment 3 is performed.
  • FIG. 27 is a schematic block diagram showing a configuration example of the microscope system according to the fourth embodiment.
  • the same configuration as in the first embodiment includes a slide glass 102 holding a plurality of living cells C labeled with a fluorescent protein, an observation window 105, a stage transport mechanism 113, and the like.
  • Control unit 301 Control unit 301, recording unit 302, input unit 303, display unit 304, preprocessing unit 305, cell recognition unit 306, parameter measurement unit 307, cell tracking unit 308, exposure detection unit 309, imaging number counting unit 310, An occupied area calculation unit 311 and a focus detection unit 312 are provided, which operate in the same manner as in the first embodiment.
  • an excitation light illumination unit 203 ′, a dichroic mirror 204 ′, and an apoptosis detection unit 315 are provided, which operate in the same manner as in the second embodiment. Since the excitation light illumination unit 203 ′ and the dichroic mirror 204 ′ include the functions of the illumination unit 203 and the dichroic mirror 204 in the first embodiment, they can be used even when apoptosis detection is not performed. Instead of using the apoptosis detection function, the same illumination unit 203 and dichroic mirror 204 as in the first embodiment may be mounted. Further, as a configuration common to the third embodiment, a cell division detection unit 316 and a western genealogy plan 317 are provided, which operate in the same manner as the third embodiment.
  • the measurement sample that is, the live cell that has been labeled is used.
  • C is cultured and imaged over time, and the acquired fluorescence image and infrared light image are recorded in the recording unit 302.
  • the captured fluorescent image is processed by the preprocessing unit 305 and the cell recognition unit 306 to recognize the cell region.
  • the processing procedure is the same as in the first embodiment.
  • the recognized cell area is recorded in the recording unit 302.
  • apoptosis is detected, a cell membrane fluorescence image is taken as in the second embodiment, and cell recognition is performed based on this.
  • the parameter measurement unit 307 measures the cell parameters of the cell region described in the first embodiment. Necessary cell parameters can be changed by an instruction from the input unit 303. When performing apoptosis detection or cell division detection, measure the cell parameters required for each treatment. These are as described in Embodiment 2 or 3. The measured cell parameters are recorded in the recording unit 302.
  • the cell tracking unit 308 associates the cell regions from which the cell image forces at different times are extracted.
  • the processing procedure is the same as in the first embodiment.
  • the cell region association information is recorded in the recording unit 302 in association with the cell image, the cell region, the cell parameter measured by the parameter measuring unit 307, and the time information.
  • apoptosis detection unit 315 detects cell apoptosis. Whether or not apoptosis detection is performed can be selected at least by an instruction from the input unit 303, and the detection result is recorded in the recording unit 302. Further, the detection result may be displayed on the display unit 304. Further, the cell division detection unit 316 detects cell division. Whether or not to perform cell division detection can be selected at least by an instruction from the input unit 303, and the detection result is recorded in the recording unit 302. Further, the detection result may be displayed on the display unit 304. Therefore, the input unit 303 of the present embodiment also functions as a detection function selection unit.
  • FIG. 27 illustrates a configuration in which both the apoptosis detection unit 315 and the cell division detection unit 316 are mounted, a configuration in which at least one of them is omitted may be used. The effect of improvement and cost reduction can be obtained.
  • the input unit 303 is not open to the public! !
  • a plurality of living cells labeled with a fluorescent substance are imaged over time, the individual cells are recognized, and the position change with time is tracked.
  • the function of detecting tosis or cell division and acquiring cell lineage information can be realized as a relatively inexpensive and reliable apparatus based on the microscope 401.
  • the processing procedures by each of the above-described cell recognition unit 306, parameter measurement unit 307, cell tracking unit 308, apoptosis detection unit 315, cell division detection unit 316, etc. are all prepared by a cell observation program prepared in advance. May be realized by executing the above with a microphone computer such as the control unit 301.
  • This cell observation program can also be distributed through a network such as the Internet.
  • this cell observation program can be executed by being recorded on a recording medium that can be read by a microcomputer such as a hard disk, FD, CD-ROM, MO, or DVD, and read from the recording medium by a microcomputer. .
  • the cell observation device, the cell observation method, the microscope system, and the cell observation program according to the present invention are useful for time-dependent measurement of cell parameters indicating the characteristics of living cells. Suitable for observation of cells inside.

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Abstract

Tout en garantissant des conditions dans lesquelles les cellules sont difficilement inactivées, la présente invention permet de confirmer l'identité de cellules pendant une période prolongée et les paramètres cellulaires de ces mêmes cellules sont acquis au fil du temps. Les cellules contenues dans une unité de culture 101 sont visualisées et les cellules sont observées grâce à l'utilisation des données d'imagerie cellulaire ainsi obtenues. Par conséquent, l'observation peut être réalisée facilement avec un seul oscilloscope pendant une période prolongée et, de ce fait, l'identité des cellules est confirmée pendant une période prolongée et les paramètres cellulaires de ces mêmes cellules peuvent être acquis facilement à mesure que le temps passe. En l'occurrence les cellules cultivées sont observées à l'aide d'une unité d'imagerie infrarouge 211. Bien que l'observation ou la culture se prolonge, les effets de la lumière d'observation sur les cellules peuvent être, de ce fait, atténués et les cellules sont ainsi difficilement inactivées.
PCT/JP2006/301884 2005-03-03 2006-02-03 Dispositif, procede et programme d'observation cellulaire et systeme microscopique WO2006092925A1 (fr)

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