WO2006092925A1 - Cell observation device, cell observation method, microscopic system and cell observation program - Google Patents

Cell observation device, cell observation method, microscopic system and cell observation program 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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French (fr)
Japanese (ja)
Inventor
Satoshi Arai
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Olympus Corporation
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Publication of WO2006092925A1 publication Critical patent/WO2006092925A1/en

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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.

Abstract

While ensuring conditions under which cells are hardly inactivated, the identity of cells is confirmed over a prolonged period of time and cell parameters of the same cells are acquired with the passage of time. Cells contained in a culture unit 101 for culturing cells are imaged and the cells are observed with the use of the cell image data thus obtained. Accordingly, observation can be easily conducted within a single visual scope over a prolonged period of time and, therefore, the identity of cells is confirmed over a prolonged period of time and cell parameters of the same cells can be easily acquired with the passage of time. In this case, the cells under culture are observed with an infrared imaging unit 211. Even though the observation or culture period is prolonged, therefore, the effects of the observation light on the cells can be lessened and thus the cells are hardly inactivated.

Description

明 細 書  Specification
細胞観察装置、細胞観察方法、顕微鏡システム、及び細胞観察プログラ ム  Cell observation device, cell observation method, microscope system, and cell observation program
技術分野  Technical field
[0001] 本発明は、生細胞のパラメータを計測、特に、個々の細胞の特徴を示す細胞パラメ 一タを経時的に計測する細胞観察装置、細胞観察方法、顕微鏡システム、及び細胞 観察プログラムに関するものである。 背景技術  TECHNICAL FIELD [0001] 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
[0002] これまで、生細胞を観察するために様々な試みがなされてきた。光学顕微鏡を用い た細胞の観察は、古くから用いられて来た簡便な観察手法であり、現在最も広く普及 している。通常は、培養装置を併せて使用し、観察時以外は培養装置内に細胞を保 存することで、環境が細胞に与えるダメージを最小限に抑えるようにする。  Until now, various attempts have been made to observe live cells. 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. Usually, 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.
[0003] また、ォリンパス株式会社から販売されている米国 CompuCyte社製の画像サイトメ 一ター「iCyte」は、レーザスキャニングによる画像化機構を備え、細胞の個体毎に蛍 光光量、面積、周囲長、真球度等のパラメータを測定することができる。  [0003] In addition, 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.
[0004] さらに、特許文献 1は、培養装置への出し入れの手間を省き、環境変動による細胞 へのダメージを回避するための構成を開示している。この構成では、細胞を培養しな がら育成状態をチェックし、必要に応じて培養装置内で観察できるようになつて 、る。  [0004] Further, 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.
[0005] また、特許文献 2は、細胞の状態を適切に維持したまま撮像し、画像処理によって 細胞の識別とパラメータ計算を行った上で、個々の細胞をトラッキングする概念を開 示し、さらに、細胞のパラメータを経時的に取得してなる具体的なアプリケーションも 提示している。  [0005] In addition, 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.
[0006] 特許文献 1：特表 2001— 500744号公報  [0006] Patent Document 1: Special Table 2001-500744
特許文献 2 :米国特許出願公開第 2004Z0128077号明細書  Patent Document 2: US Patent Application Publication No. 2004Z0128077
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problems to be solved by the invention
[0007] しかしながら、光学顕微鏡と培養装置とを組み合わせた観察系では、培養装置へ の出し入れの際、環境変動により細胞に大きなダメージを与えることが知られている。 また、対象を観察の度に顕微鏡に着脱するため、複数回の観察に渡って正確な観 察位置を再現することは困難である。すなわち、観察した細胞を次回の観察時には 見失ってしまい、観察対象の同一性を保つのが困難であった。観察毎に対象とする 細胞が異なる可能性が高ぐ得られるのは統計的な情報のみである。 [0007] However, in an observation system in which an optical microscope and a culture apparatus are combined, a culture apparatus is used. It is known that when cells are taken in and out, the cells are greatly damaged by environmental changes. In addition, since the object is attached to and detached from the microscope every time it is observed, it is difficult to reproduce the exact observation position over multiple observations. In other words, the observed cells were lost during the next observation, and it was difficult to maintain the identity of the observation target. It is only statistical information that the possibility of different target cells for each observation is high.
[0008] 画像サイトメーター「iCyte」は、細胞の統計的なパラメータを高速に測定することを 目的とした装置であり、元々追跡する必要がないため、生細胞を繰り返し測定すると いう用途は想定していない。細胞は、測定終了時に廃棄するのが普通であり、環境 ダメージを抑えるための培養機構も備えて 、な 、。培養装置無しでの細胞の生存期 間は高々 2, 3時間であり、長期間の観察は不可能である。外部の培養装置を併用 することも可能であるが、この場合、上述の光学顕微鏡と培養装置とを組み合わせた 観察系の場合と同様に、観察対象を同一に保つことが困難であるという問題が生じる [0008] 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
[0009] 特許文献 1や特許文献 2の技術では、共に細胞を長期的に観察するが、細胞の長 期培養を行う上で生じる問題である長期観察に伴う細胞活性の低下を抑制しながら 、効率よく所望の画像データを取得するための技術について、具体的な構成に欠け ている。 [0009] In the techniques of Patent Document 1 and Patent Document 2, cells are both observed for a long period of time, while suppressing a decrease in cell activity associated with long-term observation, which is a problem that occurs when performing long-term culture of cells. There is a lack of a specific configuration for a technique for efficiently obtaining desired image data.
[0010] 本発明は、上記に鑑みてなされたものであって、細胞の活性が失われにくい状況を 確保し、細胞の同一性を確認しながら同一の細胞についての細胞パラメータを経時 的に取得することができる細胞観察装置、細胞観察方法、顕微鏡システム、及び細 胞観察プログラムを提供することを目的とする。  [0010] 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.
課題を解決するための手段  Means for solving the problem
[0011] 上述した課題を解決し、目的を達成するために、本発明に係る細胞観察装置は、 細胞の特徴を示す細胞パラメータを経時的に計測する細胞観察装置であって、赤外 光線によって細胞を撮像する赤外光撮像手段を少なくとも含み細胞を撮像する撮像 手段と、該撮像手段で撮像した画像の細胞画像データから細胞を認識する細胞認 識手段と、該細胞認識手段で認識した細胞の特徴を示す細胞パラメータを前記細胞 画像データに基づき計測する細胞パラメータ計測手段と、異なる時点で撮像された 画像の前記細胞画像データ力 認識された異なる時点の細胞同士の同一性を前記 細胞パラメータに基づいて判別する細胞追跡手段と、を備えることを特徴とする。 [0011] In order to solve the above-described problems and achieve the object, a cell observation device according to the present invention 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.
[0012] また、本発明に係る顕微鏡システムは、前記細胞観察装置を備える顕微鏡システム であって、細胞を拡大投影する結像光学系を備え、前記撮像手段は、前記結像光学 系の結像面に拡大投影された細胞を撮像することを特徴とする。  [0012] Further, a microscope system according to the present invention 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.
[0013] また、本発明に係る細胞観察装置は、細胞の特徴を示す細胞パラメータを経時的 に計測する細胞観察装置であって、細胞を撮像した画像の細胞画像データ力ゝら細 胞を認識する細胞認識手段と、該細胞認識手段で認識した細胞の特徴を示す細胞 ノ メータを前記細胞画像データに基づき計測する細胞パラメータ計測手段と、前記 細胞認識手段で認識した細胞が細胞分裂の結果生じたものであるカゝ否かを前記細 胞パラメータに基づいて検出する細胞分裂検出手段と、を備え、前記細胞分裂検出 手段は、検出対象時点における細胞の面積を示す細胞パラメータと面積に関する閾 値とを比較するとともに、該検出対象時点における細胞の輝度を示す細胞パラメータ と該検出対象時点に対して比較の対象となる時点における細胞の輝度を示す細胞 ノ メータとを比較し、それぞれの比較結果に基づいて該検出対象時点における細 胞が細胞分裂の結果生じたものである力否かを検出することを特徴とする。  [0013] In addition, the cell observation device according to the present invention 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 parameter indicating the luminance of the cell at the detection target time point and the luminance of the cell at the time point to be compared with the detection target time point. It is characterized by detecting whether or not the cell at the detection target time point is a force generated as a result of cell division based on the respective comparison results.
[0014] また、本発明に係る細胞観察装置は、細胞の特徴を示す細胞パラメータを経時的 に計測する細胞観察装置であって、細胞を撮像した画像の細胞画像データ力ゝら細 胞を認識する細胞認識手段と、該細胞認識手段で認識した細胞の特徴を示す細胞 ノ メータを前記細胞画像データに基づき計測する細胞パラメータ計測手段と、前記 細胞認識手段で認識した細胞が細胞分裂の結果生じたものであるカゝ否かを前記細 胞パラメータに基づいて検出する細胞分裂検出手段と、を備え、前記細胞分裂検出 手段は、検出対象時点よりも時系列的に前の時点における細胞の円形度を示す細 胞パラメータの値を参照し、該検出対象時点における細胞が細胞分裂の結果生じた ものである力否かを検出することを特徴とする。  [0014] Further, the cell observation device according to the present invention 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.
[0015] また、本発明に係る細胞観察装置は、細胞の特徴を示す細胞パラメータを経時的 に計測する細胞観察装置であって、細胞を撮像した画像の細胞画像データ力ゝら細 胞を認識する細胞認識手段と、該細胞認識手段で認識した細胞の特徴を示す細胞 ノ メータを前記細胞画像データに基づき計測する細胞パラメータ計測手段と、前記 細胞認識手段で認識した細胞が細胞分裂の結果生じたものであるカゝ否かを前記細 胞パラメータに基づいて検出する細胞分裂検出手段と、を備え、前記細胞分裂検出 手段は、検出対象時点よりも時系列的に前の時点における細胞の円形度を示す細 胞パラメータの値を参照し、該円形度を示す細胞パラメータの値が円形度に関する 閾値と所定の関係を有して!/、る細胞を撮像した判定基準時点を抽出し、該判定基準 時点近傍の時点における細胞の円形度を示す細胞パラメータ及び面積を示す細胞 パラメータの値の増減傾向に基づいて、該検出対象時点における細胞が細胞分裂 の結果生じたものであるか否かを検出することを特徴とする。 [0015] In addition, the cell observation device according to the present invention 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! /, And 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.
[0016] また、本発明に係る細胞観察装置は、細胞の特徴を示す細胞パラメータを経時的 に計測する細胞観察装置であって、細胞を撮像した画像の細胞画像データ力ゝら細 胞を認識する細胞認識手段と、該細胞認識手段で認識した細胞の特徴を示す細胞 ノ メータを前記細胞画像データに基づき計測する細胞パラメータ計測手段と、前記 細胞認識手段で認識した細胞が細胞分裂の結果生じたものであるカゝ否かを前記細 胞パラメータに基づいて検出する細胞分裂検出手段と、を備え、前記細胞分裂検出 手段は、検出対象時点における細胞の細胞核の存在範囲の面積と細胞質の存在範 囲の面積とを比較した結果に基づいて、該検出対象時点における細胞が細胞分裂 の結果生じたものであるか否かを検出することを特徴とする。  [0016] The cell observation device according to the present invention 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.
[0017] また、本発明に係る細胞観察装置は、細胞の特徴を示す細胞パラメータを経時的 に計測する細胞観察装置であって、細胞を撮像した画像の細胞画像データ力ゝら細 胞を認識する細胞認識手段と、該細胞認識手段で認識した細胞の特徴を示す細胞 ノ メータを前記細胞画像データに基づき計測する細胞パラメータ計測手段と、前記 細胞認識手段で認識した細胞が細胞分裂の結果生じたものであるカゝ否かを前記細 胞パラメータに基づいて検出する細胞分裂検出手段と、を備え、前記細胞分裂検出 手段は、検出対象時点における細胞の微小管の局在領域を求め、細胞中の複数個 所に局在しているか否かを検出することで、該検出対象時点における細胞が細胞分 裂の結果生じたものであるか否かを検出することを特徴とする。  [0017] The cell observation device according to the present invention 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.
[0018] また、本発明に係る細胞観察装置は、細胞の特徴を示す細胞パラメータを経時的 に計測する細胞観察装置であって、細胞を撮像した画像の細胞画像データ力ゝら細 胞を認識する細胞認識手段と、該細胞認識手段で認識した細胞の特徴を示す細胞 ノ メータを前記細胞画像データに基づき計測する細胞パラメータ計測手段と、前記 細胞認識手段で認識した細胞が細胞分裂の結果生じたものであるカゝ否かを前記細 胞パラメータに基づいて検出する細胞分裂検出手段と、を備え、前記細胞分裂検出 手段は、撮像された画像の細胞に対応する領域内で輝度に関する閾値よりも高い輝 度値を示す画素について各画素の輝度値を合計した輝度総和を示す領域パラメ一 タを、検出対象時点近傍の時点について求め、該検出対象時点近傍の時点につい ての前記輝度総和の変化の様子に基づ 、て、該検出対象時点における細胞が細胞 分裂の結果生じたものであるか否かを検出することを特徴とする。 [0018] In addition, the cell observation device according to the present invention 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.
[0019] また、本発明に係る細胞観察プログラムは、細胞の特徴を示す細胞パラメータを経 時的に計測する細胞観察装置で細胞の観察を行う細胞観察プログラムであって、前 記細胞観察装置に、細胞を撮像した画像の細胞画像データカゝら細胞を認識する細 胞認識ステップと、該細胞認識ステップで認識した細胞の特徴を示す細胞パラメータ を前記細胞画像データに基づ 、て計測する細胞パラメータ計測ステップと、前記細 胞認識ステップで認識した細胞が細胞分裂の結果生じたものであるカゝ否かを前記細 胞パラメータに基づ 、て検出する細胞分裂検出ステップと、を実行させるステップを 含み、前記細胞分裂検出ステップは、検出対象時点における細胞の面積を示す細 胞パラメータと面積に関する閾値とを比較するとともに、該検出対象時点における細 胞の輝度を示す細胞パラメータと該検出対象時点に対して比較の対象となる時点に おける細胞の輝度を示す細胞パラメータとを比較し、それぞれの比較結果に基づ ヽ て当該検出対象時点における細胞が細胞分裂の結果生じたものである力否かを検 出するステップであることを特徴とする。  [0019] Further, 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 A step of performing a measurement step and a cell division detection step of detecting, based on the cell parameters, whether or not the cell recognized in the cell recognition step is a result of cell division. 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.
[0020] また、本発明に係る細胞観察プログラムは、細胞の特徴を示す細胞パラメータを経 時的に計測する細胞観察装置で細胞の観察を行う細胞観察プログラムであって、前 記細胞観察装置に、細胞を撮像した画像の細胞画像データカゝら細胞を認識する細 胞認識ステップと、該細胞認識ステップで認識した細胞の特徴を示す細胞パラメータ を前記細胞画像データに基づ 、て計測する細胞パラメータ計測ステップと、前記細 胞認識ステップで認識した細胞が細胞分裂の結果生じたものであるカゝ否かを前記細 胞パラメータに基づ 、て検出する細胞分裂検出ステップと、を実行させるステップを 含み、前記細胞分裂検出ステップは、検出対象時点よりも時系列的に前の時点にお ける細胞の円形度を示す細胞パラメータの値を参照し、該検出対象時点における細 胞が細胞分裂の結果生じたものである力否かを検出するステップであることを特徴と する。 [0020] Further, 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.
[0021] また、本発明に係る細胞観察プログラムは、細胞の特徴を示す細胞パラメータを経 時的に計測する細胞観察装置で細胞の観察を行う細胞観察プログラムであって、前 記細胞観察装置に、細胞を撮像した画像の細胞画像データカゝら細胞を認識する細 胞認識ステップと、該細胞認識ステップで認識した細胞の特徴を示す細胞パラメータ を前記細胞画像データに基づ 、て計測する細胞パラメータ計測ステップと、前記細 胞認識ステップで認識した細胞が細胞分裂の結果生じたものであるカゝ否かを前記細 胞パラメータに基づ 、て検出する細胞分裂検出ステップと、を実行させるステップを 含み、前記細胞分裂検出ステップは、検出対象時点よりも時系列的に前の時点にお ける細胞の円形度を示す細胞パラメータの値を参照し、該円形度を示す細胞パラメ ータの値が円形度に関する閾値と所定の関係を有している細胞を撮像した判定基準 時点を抽出し、該判定基準時点近傍の時点における細胞の円形度を示す細胞パラ メータ及び面積を示す細胞パラメータの値の増減傾向に基づ 、て、該検出対象時点 における細胞が細胞分裂の結果生じたものである力否かを検出するステップであるこ とを特徴とする。  [0021] Further, 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 A step of performing a measurement step and a cell division detection step of detecting, based on the cell parameters, whether or not the cell recognized in the cell recognition step is a result of cell division. 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.
[0022] また、本発明に係る細胞観察プログラムは、細胞の特徴を示す細胞パラメータを経 時的に計測する細胞観察装置で細胞の観察を行う細胞観察プログラムであって、前 記細胞観察装置に、細胞を撮像した画像の細胞画像データカゝら細胞を認識する細 胞認識ステップと、該細胞認識ステップで認識した細胞の特徴を示す細胞パラメータ を前記細胞画像データに基づ 、て計測する細胞パラメータ計測ステップと、前記細 胞認識ステップで認識した細胞が細胞分裂の結果生じたものであるカゝ否かを前記細 胞パラメータに基づ 、て検出する細胞分裂検出ステップと、を実行させるステップを 含み、前記細胞分裂検出ステップは、検出対象時点における細胞の細胞核の存在 範囲の面積と細胞質の存在範囲の面積とを比較した結果に基づいて、該検出対象 時点における細胞が細胞分裂の結果生じたものである力否かを検出するステップで あることを特徴とする。 [0022] Further, 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 A step of performing a measurement step and a cell division detection step of detecting, based on the cell parameters, whether or not the cell recognized in the cell recognition step is a result of cell division. 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.
[0023] また、本発明に係る細胞観察プログラムは、細胞の特徴を示す細胞パラメータを経 時的に計測する細胞観察装置で細胞の観察を行う細胞観察プログラムであって、前 記細胞観察装置に、細胞を撮像した画像の細胞画像データカゝら細胞を認識する細 胞認識ステップと、該細胞認識ステップで認識した細胞の特徴を示す細胞パラメータ を前記細胞画像データに基づ 、て計測する細胞パラメータ計測ステップと、前記細 胞認識ステップで認識した細胞が細胞分裂の結果生じたものであるカゝ否かを前記細 胞パラメータに基づ 、て検出する細胞分裂検出ステップと、を実行させるステップを 含み、前記細胞分裂検出ステップは、検出対象時点における細胞の微小管の局在 領域を求めて、細胞中の複数個所に局在しているか否かを検出することで、該検出 対象時点における細胞が細胞分裂の結果生じたものである力否かを検出するステツ プであることを特徴とする。  [0023] Further, 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 A step of performing a measurement step and a cell division detection step of detecting, based on the cell parameters, whether or not the cell recognized in the cell recognition step is a result of cell division. 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.
[0024] また、本発明に係る細胞観察プログラムは、細胞の特徴を示す細胞パラメータを経 時的に計測する細胞観察装置で細胞の観察を行う細胞観察プログラムであって、前 記細胞観察装置に、細胞を撮像した画像の細胞画像データカゝら細胞を認識する細 胞認識ステップと、該細胞認識ステップで認識した細胞の特徴を示す細胞パラメータ を前記細胞画像データに基づ 、て計測する細胞パラメータ計測ステップと、前記細 胞認識ステップで認識した細胞が細胞分裂の結果生じたものであるカゝ否かを前記細 胞パラメータに基づ 、て検出する細胞分裂検出ステップと、を実行させるステップを 含み、前記細胞分裂検出ステップは、撮像された画像の細胞に対応する領域内で 輝度に関する閾値よりも高い輝度値を示す画素について各画素の輝度値を合計し た輝度総和を示す領域パラメータを、検出対象時点近傍の時点について求め、該検 出対象時点近傍の時点につ!、ての前記輝度総和の変化の様子に基づ 、て、該検 出対象時点における細胞が細胞分裂の結果生じたものである力否かを検出するステ ップであることを特徴とする。  [0024] Further, 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 A step of performing a measurement step and a cell division detection step of detecting, based on the cell parameters, whether or not the cell recognized in the cell recognition step is a result of cell division. And 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.
[0025] また、本発明に係る細胞観察方法は、細胞を培養する培養手段と、赤外光線により 細胞を撮像する赤外光撮像手段を少なくとも含み前記培養手段に収容されている細 胞を撮像する撮像手段と、を備えた細胞観察装置で細胞の特徴を示す細胞パラメ一 タを経時的に計測する細胞観察方法であって、前記培養手段で細胞を培養しながら 、培養中の細胞を前記撮像手段によって撮像して細胞の画像を取得する培養細胞 撮像工程と、該培養細胞撮像工程で取得した画像の細胞画像データ力ゝら撮像され た細胞を認識する細胞認識工程と、該細胞認識工程で認識した細胞の特徴を示す 細胞パラメータを前記細胞画像データに基づいて計測する細胞パラメータ計測工程 と、を含み、前記培養細胞撮像工程、前記細胞認識工程及び前記細胞パラメータ計 測工程を、培養期間中の複数時点で行い、撮像した複数時点の画像から認識した 細胞同士の同一性を前記細胞パラメータに基づいて判別する細胞追跡工程と、をさ らに含み、前記培養手段で細胞を培養している最中に、該培養手段に収容されてい る細胞を前記赤外光撮像手段によって撮像して観察することを特徴とする。 [0025] Further, the cell observation method according to the present invention 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. A cell observation method for obtaining a cell image by culturing cells with the culturing means and capturing an image of the cells by cultivating the cells with the imaging means, and the cultured cell imaging step. A cell recognition step for recognizing a captured cell, and a cell parameter measurement step for measuring a cell parameter indicating characteristics of the cell recognized in the cell recognition step based on the cell image data; 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. A cell tracking step of discriminating the identity between cells based on the cell parameter, and the cells accommodated in the culture means during the cultivation of the cells by the culture means It is characterized by imaging and observing with an infrared light imaging means.
[0026] また、本発明に係る細胞観察方法は、細胞を培養する培養手段と、赤外光線により 細胞を撮像する赤外光撮像手段を少なくとも含み前記培養手段に収容されている細 胞を撮像する撮像手段と、を備えた細胞観察装置で細胞の特徴を示す細胞パラメ一 タを経時的に計測する細胞観察方法であって、前記培養手段で細胞を培養しながら 、培養中の細胞を培養期間中の複数時点で前記撮像手段によって撮像して細胞の 画像を取得する培養細胞撮像工程と、該培養細胞撮像工程で取得した各時点の画 像の細胞画像データ力ゝら撮像された細胞を認識する細胞認識工程と、該細胞認識 工程で認識した各時点の細胞の特徴を示す細胞パラメータを前記細胞画像データ に基づ!/ヽて計測する細胞パラメータ計測工程と、撮像した複数時点の画像から認識 した細胞同士の同一性を前記細胞パラメータに基づいて判別する細胞追跡工程と、 を含み、前記培養手段で細胞を培養している最中に、該培養手段に収容されている 細胞を前記赤外光撮像手段によって撮像して観察することを特徴とする。  [0026] Further, the cell observation method according to the present invention 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 cells. It is characterized in that it is imaged and observed by an external light imaging means.
発明の効果  The invention's effect
[0027] 本発明に係る細胞観察装置、細胞観察方法、顕微鏡システム、及び細胞観察プロ グラムは、細胞の同一性を確認しながら同一の細胞についての細胞パラメータを取 得することができ、この場合、赤外光撮像手段で培養中の細胞を観察することが可能 であるため、観察期間ないし培養期間でも、赤外光撮像手段を用いて細胞を観察す ることで、細胞に対する観察光の影響を低く抑えて、細胞の活性が失われに《する ことができると!/、う効果を奏する。 [0027] 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.
図面の簡単な説明 Brief Description of Drawings
[図 1]図 1は、本発明の実施の形態 1に係る細胞観察装置の構成例を示す概略ブロッ ク図である。 FIG. 1 is a schematic block diagram showing a configuration example of a cell observation device according to Embodiment 1 of the present invention.
[図 2]図 2は、培養部の構成例を示す水平断面図である。  FIG. 2 is a horizontal cross-sectional view showing a configuration example of a culture unit.
[図 3]図 3は、培養部の構成例を示す縦断正面図である。 FIG. 3 is a longitudinal front view showing a configuration example of a culture unit.
[図 4]図 4は、整流板の構成例を示す斜視図である。 FIG. 4 is a perspective view showing a configuration example of a current plate.
[図 5]図 5は、培養部側と撮像部側との境界部分の断熱構成例を示す断面図である。  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.
[図 6]図 6は、蛍光撮像された培養中の細胞画像の一例を示す説明図である。  FIG. 6 is an explanatory diagram showing an example of a cell image in culture that has been subjected to fluorescence imaging.
[図 7]図 7は、画像データ処理例を示す概略フローチャートである。  FIG. 7 is a schematic flowchart showing an example of image data processing.
[図 8]図 8は、先鋭ィ匕フィルタによる重み付け例を示す図である。  FIG. 8 is a diagram illustrating an example of weighting by a sharp edge filter.
[図 9]図 9は、領域統合の第 1の手法例を示す概略フローチャートである。  FIG. 9 is a schematic flowchart showing a first method example of region integration.
[図 10]図 10は、領域統合の第 2の手法例を示す概略フローチャートである。  FIG. 10 is a schematic flowchart showing a second method example of region integration.
[図 11]図 11は、記録部に記録された細胞パラメータの計測結果例を示す説明図であ る。  FIG. 11 is an explanatory diagram showing an example of measurement results of cell parameters recorded in a recording unit.
[図 12]図 12は、 mと nの可能な組み合わせについて評価値を計算した結果を示す説 明図である。  [FIG. 12] FIG. 12 is an explanatory diagram showing the results of calculating evaluation values for possible combinations of m and n.
[図 13]図 13は、処理結果の表示の一例を示す説明図である。  FIG. 13 is an explanatory diagram showing an example of processing result display.
[図 14]図 14は、強調表示の一例を示す説明図である。 FIG. 14 is an explanatory diagram showing an example of highlighting.
[図 15]図 15は、本発明の実施の形態 2に係る細胞観察装置の構成例を示す概略ブ ロック図である。  FIG. 15 is a schematic block diagram showing a configuration example of a cell observation device according to Embodiment 2 of the present invention.
[図 16]図 16は、アポトーシスの発生の検出処理例を示す概略フローチャートである。  FIG. 16 is a schematic flowchart showing an example of detection processing for occurrence of apoptosis.
[図 17]図 17は、本発明の実施の形態 3に係る細胞観察装置の構成例を示す概略ブ ロック図である。 FIG. 17 is a schematic block diagram showing a configuration example of a cell observation device according to Embodiment 3 of the present invention.
[図 18]図 18は、細胞分裂判定処理の第 1の処理手順を示す概略フローチャートであ る。 [図 19]図 19は、細胞分裂判定処理の第 2の処理手順を示す概略フローチャートであ る。 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.
[図 20]図 20は、細胞分裂判定処理の第 3の処理手順を示す概略フローチャートであ る。  FIG. 20 is a schematic flowchart showing a third processing procedure of the cell division determination processing.
[図 21]図 21は、細胞分裂判定処理の第 4の処理手順を示す概略フローチャートであ る。  FIG. 21 is a schematic flowchart showing a fourth processing procedure of cell division determination processing.
[図 22]図 22は、細胞分裂判定処理の第 5の処理手順を示す概略フローチャートであ る。  FIG. 22 is a schematic flowchart showing a fifth processing procedure of cell division determination processing.
[図 23]図 23は、輝度総和の時間的推移の一例を示す特性図である。  FIG. 23 is a characteristic diagram showing an example of temporal transition of the luminance sum.
[図 24-1]図 24— 1は、分裂期以外の細胞の様子を示す平面図である。  [FIG. 24-1] FIG. 24-1 is a plan view showing a state of cells other than the division phase.
[図 24-2]図 24— 2は、分裂期以外の細胞の様子を示す縦断正面図である。  [FIG. 24-2] FIG. 24-2 is a longitudinal front view showing a state of cells other than the division phase.
[図 24- 3]図 24— 3は、図 24— 2部分の輝度分布特性を示す図である。  [Fig. 24-3] Fig. 24-3 shows the luminance distribution characteristics of the portion of Fig. 24-2.
[図 24-4]図 24— 4は、分裂期の細胞の様子を示す平面図である。  [FIG. 24-4] FIG. 24-4 is a plan view showing the state of cells in the division phase.
[図 24-5]図 24— 5は、分裂期の細胞の様子を示す縦断正面図である。  [FIG. 24-5] FIG. 24-5 is a longitudinal front view showing the state of cells in the division phase.
[図 24- 6]図 24— 6は、図 24— 5部分の輝度分布特性を示す図である。  [FIG. 24-6] FIG. 24-6 is a diagram showing the luminance distribution characteristics of FIG. 24-5.
[図 25]図 25は、時刻 t〜tについての観察結果の表示の一例を示す説明図である。  FIG. 25 is an explanatory diagram showing an example of display of observation results for times t to t.
1 3  13
[図 26]図 26は、その強調表示の一例を示す説明図である。  FIG. 26 is an explanatory diagram showing an example of the highlighted display.
[図 27]図 27は、本発明の実施の形態 4に係る顕微鏡システムの構成例を示す概略 ブロック図である。 符号の説明  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
101 培養部  101 Incubator
104 筐体  104 housing
111 断熱部  111 Thermal insulation
201 撮像部  201 Imaging unit
206 結像光学系  206 Imaging optics
207 蛍光撮像部  207 Fluorescence imaging unit
210 結像光学系  210 Imaging optics
211 赤外光撮像部 302 記録部 211 Infrared light imaging unit 302 Recording section
303 入力部  303 Input section
304 表示部  304 Display
306 細胞認識部  306 Cell recognition unit
307 パラメータ計測部  307 Parameter measurement unit
308 細胞追跡部  308 Cell tracking unit
310 撮像回数計数部  310 Image count counter
311 占有面積算出部  311 Occupied area calculator
315 アポトーシス検出部  315 Apoptosis detector
316 細胞分裂検出部  316 Cell division detector
317 系譜作成部  317 Genealogy Department
401 顕微鏡  401 microscope
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0030] 以下に添付図面を参照して、本発明に係る好適な実施の形態について詳述する。  [0030] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the accompanying drawings.
[0031] (実施の形態 1)  [Embodiment 1]
本発明の実施の形態 1に係る細胞観察装置は、蛍光タンパクを導入した複数の生 細胞を培養したまま撮像し、個々の細胞の領域を認識し、経時的な位置変化を追尾 しつつ、個々の細胞の特徴を示す細胞パラメータを独立に計測する。  The cell observation device according to Embodiment 1 of the present invention 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.
[0032] 図 1は、本実施の形態 1に係る細胞観察装置の構成例を示す概略ブロック図である 。本実施の形態 1に係る細胞観察装置は、概略的には、細胞を培養する培養部 101 と、この培養部 101に収容されている細胞を撮像する撮像部 201と、細胞観察装置 の全体の処理及び動作を制御する制御部 301と、撮像部 201で撮像した画像のデ ータゃ処理後のデータなどの各種データを集中して一時的或いは永続的に記録す る記録部 302と、各種情報の入力を受ける入力部 303と、画像情報等の各種情報を 表示して操作者に提示する表示部 304とを備える他、前処理部 305、細胞認識部 3 06、パラメータ計測部 307、細胞追跡部 308、露出検出部 309、撮像回数計数部 31 0、占有面積算出部 311、合焦検出部 312を備える。これらの各部 302〜312は、制 御部 301に接続され、制御部 301により制御される。なお、図 1では、制御部 301か ら培養部 101や撮像部 201に対する制御接続は特に図示していない。なお、制御部 301、前処理部 305、細胞認識部 306、パラメータ計測部 307、細胞追跡部 308、露 出検出部 309、撮像回数計数部 310、占有面積算出部 311、合焦検出部 312によ つて行われる各処理は、細胞観察装置に搭載された CPl^¾OM等のメモリに記憶 された処理プログラムに基づ!/、て、適宜 RAM等の記憶装置に必要なデータを書き 込みながら行われる。 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 control unit 301 for controlling processing and operation, a recording unit 302 for centrally and permanently recording various data such as data after data processing of an image captured by the imaging unit 201, and various types 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. In FIG. 1, the 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 ^ ¾OM installed in the cell observation device while writing necessary data in a storage device such as RAM as appropriate. Is called.
[0033] まず、培養部 101について説明する。スライドガラス 102は、局在化せずに発現す る蛍光タンパクをあらかじめ導入した複数の生細胞 Cを保持し、培養部 101内に設置 されている。この培養部 101は、例えば特開 2004— 113175号公報によって開示さ れた培養容器と同様の構成である。この場合、蛍光タンパクは局在化しないものであ れば何でも良ぐ一般的なクラゲ由来の蛍光タンパク等が利用可能で、一例として B Dバイオサイエンス 'クロンテック社の pEGFP—Nlを使用することができる。  First, the culture unit 101 will be described. 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. .
[0034] 図 2は、培養部 101の構成例を示す水平断面図であり、図 3は、培養部 101の構成 例を示す縦断正面図である。培養手段としての培養部 101は、図 2及び図 3に示すよ うに、スライドガラス 102を内部に収容可能な表裏貫通孔 103を有する熱伝導に優れ た材質、例えば、ステンレス製又はアルミニウム製の筐体 104と、その筐体 104の表 裏貫通孔 103を塞ぐ光学的に平滑な 2枚のガラス板で形成された観察窓 105と、筐 体 104内部に培養液 Aを供給する培養液供給パイプ 106と、筐体 104内部から不要 となった培養液 Aを排出する培養液排出パイプ 107と、筐体 104への培養液 Aの出 入口に設けられた 2つの整流板 108とを備えて 、る。  FIG. 2 is a horizontal sectional view showing a configuration example of the culture unit 101, and FIG. 3 is a longitudinal front view showing a configuration example of the culture unit 101. As shown in FIGS. 2 and 3, 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
[0035] 生細胞 Cを健全に育成するためには、スライドガラス 102上の全域に渡って常に新 鮮な培養液 Aが供給されることが望ましい。しかし、培養液 Aの流れが急激な場合、 スライドガラス 102上に着床した生細胞 Cが剥離する可能性がある。このため、本実 施の形態 1は、各ノイブ 106, 107付近に整流板 108を設置し、培養液 Aの流れを 均一に分散、回収できるようにしている。  [0035] In order to grow the living cells C in a healthy manner, it is desirable to always supply a fresh culture solution A over the entire area of the slide glass 102. However, when the flow of the culture solution A is abrupt, there is a possibility that the living cells C that have been deposited on the slide glass 102 will be detached. For this reason, in the first embodiment, 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.
[0036] 図 4は、整流板 108の構成例を示す斜視図である。整流板 108は、図 4に示すよう に、厚さ方向に複数の貫通孔 108aを形成した多孔性の部材である。入口側の整流 板 108は、培養液供給パイプ 106から流入する培養液 Aを複数の貫通孔 108aに分 散させて流通させ、出口側の整流板 108は、培養液排出パイプ 107を経て一気に流 出しようとする培養液 Aを複数の貫通孔 108aに分散させて流通させる。これにより、 集中的な流れを分散流に変換し、生細胞 Cが配置されているスライドガラス 102近傍 にお 、て、一定の流速及び流量で培養液 Aを流動させることができる。 FIG. 4 is a perspective view showing a configuration example of the rectifying plate 108. As shown in FIG. 4, 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. As a result, 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.
[0037] 培養部 101には、温度制御ユニット 109が取り付けられ、培養部 101の周囲に温水 Wを流通させる温水流路 110を形成する。温水流路 110に温水 Wを循環させること により、筐体 104を介して温水の熱を培養液 Aに伝達させる。この時、図示しない温 度センサ力もの温度情報が制御部 301に所定時間間隔毎に伝達され、制御部 301 は培養部 101内の温度が 37±0. 5°Cの範囲に維持されるように温水 Wの温度と流 量を制御する。 [0037] 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. By circulating the hot water W through the hot water channel 110, the heat of the hot water is transferred to the culture solution A through the housing 104. At this time, 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.
[0038] また、図示しない pHセンサによって培養液 Aの pH情報が制御部 301に所定時間 間隔毎に伝達され、制御部 301は培養液の pHが所定の範囲に維持されるように培 養液 A内の CO濃度を制御する。  [0038] Further, 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.
2  2
[0039] 未使用の培養液は、図示しない培養液保存部に保存されており、経時的な劣化を 抑えるため、図示しない保冷機構で約 4°Cに保冷される。保冷された培養液は、図示 しな 、培養液加温機構によって約 37°Cに加温された後、培養液供給パイプ 106を 通じて筐体 104内に供給される。  [0039] 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.
[0040] 培養液排出パイプ 107を通じて排出された培養液は、図示しない廃液保存部に保 存される。排出された培養液の一部を新鮮な培養液と混合して筐体 104内に供給す る構成としても良ぐこの場合、培養液の交換に伴う細胞への衝撃を和らげ、より長期 の培養に適した構成となる。  [0040] 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.
[0041] 図 5は、培養部 101側と撮像部 201側との境界部分の断熱構成例を示す断面図で ある。培養部 101の発する熱は、断熱手段としての断熱部 111を設けることで、撮像 部 201側には伝達しな ヽようにする。培養部 101と撮像部 201との断熱を行う断熱部 111の設置個所は、種々考えられる力 本実施の形態 1では、培養部 101の筐体 10 4と撮像部 201を構成する撮像素子との間に断熱部 111を設置して 、る。断熱部 11 1は、断熱性が高く伸縮性のある部材、例えばゴム、シリコン、ポリウレタン等を使用し たシート状であって、対物レンズ 202と概略同じ直径の貫通孔 112が設けられている 。培養部 101と対物レンズ 202とは貫通孔 112を通して光学的に接続されており、自 由に光線をやり取りできる。一方、培養部 101の発する熱は断熱部 111によってその ほとんどが遮られる。一般に、光学系は 25°C前後での使用を前提として調整されて おり、培養部 101からの熱で加熱されると想定した性能を発揮できない。特に、撮像 部 201の備える CCD等の固体撮像素子は、高温になる程ノイズが増加して SZNが 劣化するため、微弱な蛍光を捉えるためにはできるだけ低温 (ただし、結露させない） に保つ必要がある。 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. On the other hand, most of the heat generated by the culture unit 101 is blocked by the heat insulating unit 111. In general, 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. In particular, 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.
[0042] 培養手段としては、培養部 101のように培養液の交換を行うことができるものを用い るとより好ましいが、一般的なゥエルプレートを用いて細胞を観察することも可能であ る。ただし、一般的なゥエルプレートを用いた場合には、環境状態を維持したまま培 養液を交換することができないため、培養部 101を用いる場合に比べて、細胞の代 謝に伴う培養液の劣化の影響で培養期間は短期に制限されてしまう。  [0042] As 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. The However, 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.
[0043] 以上の構成により、培養部 101内部の温度と培養液 Aの pHはほぼ一定に保たれ ている。測定試料となる生細胞の一例として、ヒーラ細胞を用いる。ヒーラ細胞は、子 宫頸癌由来であり、創薬毒性試験等で広く用いられている。導入する蛍光タンパクの 種類は、アツセィの内容に応じて変更しても良い。  [0043] With the above configuration, the temperature inside the culture unit 101 and the pH of the culture solution A are kept substantially constant. 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.
[0044] 次に、撮像部 201について説明する。撮像部 201は、励起光照明部 203と、ダイク 口イツクミラー 204と、対物光学系と 205と、結像光学系 206と、蛍光撮像部 207と、 赤外光照明部 208と、ダイクロイツクミラー 209と、結像光学系 210と、赤外光撮像部 211とを有して構成されている。すなわち、本実施の形態 1の撮像部 201は、蛍光撮 像系と赤外光撮像系とを有する構成とされている。  Next, the imaging unit 201 will be described. 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.
[0045] まず、励起光照明部 203から放射された光は、ダイクロイツクミラー 204によって反 射され、対物レンズ 202を含む対物光学系 205と観察窓 105を経てスライドガラス 10 2に照射される。照射された光を励起光として、スライドガラス 102上の生細胞 Cに導 入された蛍光タンパク力 蛍光が発せられ、励起光の反射光と蛍光は共に観察窓 10 5から射出される。射出された光は、再度対物光学系 205を通過し、ダイクロイツクミラ 一 204に到達するが、蛍光のみが透過し、励起光の反射光は遮断される。ダイクロイ ックミラー 204を透過した蛍光は、結像光学系 206によって細胞光撮像手段としての 蛍光撮像部 207が備える CCDや CMOS等の固体撮像素子上に拡大投影されて結 像する。 First, 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. Using 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.
[0046] 結像した測定試料の蛍光像を蛍光撮像部 207が備える固体撮像素子によって画 像データに変換し、制御部 301による制御の下に記録部 302において一時的或い は永続的に記録する。図 6は、蛍光撮像された培養中の細胞画像の一例を示す説 明図である。  [0046] The fluorescence image of the imaged measurement sample is converted into image data by the solid-state imaging device included in the fluorescence imaging unit 207, and is temporarily or permanently recorded in the recording unit 302 under the control of the control unit 301. To do. FIG. 6 is an explanatory diagram showing an example of a cell image in culture that has been subjected to fluorescence imaging.
[0047] また、赤外光照明部 208から放射された光は、一方の観察窓 105を経てスライドガ ラス 102に照射され、その透過光が他方の観察窓 105から射出される。射出された 光は、対物光学系 205を通過し、ダイクロイツクミラー 209に到達する力 赤外光は全 て反射される。反射された赤外光は、結像光学系 210によって赤外光撮像手段とし ての赤外光撮像部 211が備える CCDや CMOS等の固体撮像素子上に拡大投影さ れて結像する。結像した測定試料の赤外光像を赤外光撮像部 211が備える CCDや CMOS等の固体撮像素子によって画像データに変換し、制御部 301による制御の 下に、記録部 302において一時的或いは永続的に記録する。  In addition, 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.
[0048] 一般に、蛍光タンパクは全ての細胞に均等に導入できる訳ではなぐまた導入でき た場合でも直ちに発現するとは限らないため、細胞の全体像を経時的に安定して観 察する手段が必要であり、本実施の形態では赤外光像がこれに当たる。赤外光像を 表示する場合、初期状態では蛍光タンパクが殆ど或いは全く発現して 、な 、測定試 料であっても、細胞像を視認しながら観察範囲の確認や調整を行える。  [0048] In general, 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. There is an infrared light image in this embodiment. In the case of displaying an infrared light image, the fluorescent protein is expressed almost or not in the initial state, and even if it is a measurement sample, the observation range can be confirmed and adjusted while visually recognizing the cell image.
[0049] 赤外光は、可視光と比較して生細胞に対する光毒性が低いため、可視光を用いて 撮像した場合と比較して、より長期間細胞の活性を維持することができる。また、蛍光 撮像用の励起光として可視光全域を使用できるようになるため、利用可能な蛍光タン パクの制約が緩和される。  [0049] Since 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. In addition, the entire visible light range can be used as excitation light for fluorescence imaging, so that restrictions on available fluorescence proteins are relaxed.
[0050] このように、本実施の形態 1の撮像部 201によれば、蛍光撮像部 207や赤外光撮 像部 211を用いてスライドガラス 102上の生細胞 Cを撮像することで、生細胞 Cを撮 像した画像の画像データである細胞画像データを取得することが可能である。本実 施の形態 1では、制御部 301の制御により、あら力じめ設定した時間間隔で自動的に 蛍光撮像部 207によって撮像を行う。そして、細胞の様子を観察したいときに、必要 に応じて、ユーザが所望の時期に赤外光撮像部 211を用いた生細胞 Cの観察を行う ことができる。なお、赤外光撮像部 211による撮像は、ユーザが所望の時期に行うば 力りではなぐ制御部 301の制御によって、蛍光撮像部 207による撮像と同期したタ イミングで行えば、赤外光像と蛍光像との対応付けが容易になるだけでなぐ両画像 に含まれる生細胞 C同士の対応付けも容易になる。その結果、生細胞 Cを長期に培 養しながら観察する場合に、細胞の活性低下を抑えながら、効率よく生細胞 Cの観察 を行うことが可能となる。蛍光像や赤外光像を撮像した時間を、表示部 304に表示す る機能を付加してもよい。 As described above, according to the imaging unit 201 of the first embodiment, 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. In the first embodiment, under the control of the control unit 301, 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. Note that if 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. In addition to facilitating the association between a fluorescent image and a fluorescent image, it is also easy to associate living cells C included in both images. As a result, when observing the living cells C while culturing them for a long period of time, it is possible to efficiently observe the living cells C while suppressing the decrease in cell activity. 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.
[0051] 本実施の形態 1の構成は、蛍光撮像部 207と赤外光撮像部 211とを備えるため、 蛍光像の撮像と赤外光像の撮像とを並行して行うことができ、両者を切り替えながら 撮像する場合と比較して、撮像に要する時間が大幅に短縮され、切り替え用の駆動 部も不要である。 [0051] Since 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.
[0052] 赤外光照明部 208にリング絞りをカ卩え、ダイクロイツクミラー 209から結像光学系 21 0に至る光路に位相板を挿入すれば、透過観察に代えて位相差観察を行うことがで きる。位相差観察は、透過観察と比較して、よりコントラストの高い画像が得られる。  [0052] If a phase stop is inserted in the optical path from the dichroic mirror 209 to the imaging optical system 210 when a ring stop is provided in the infrared light illumination unit 208, 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.
[0053] 赤外光照明部 208にポラライザと DIC (Differential Interference Contrast)素子を揷 入し、ダイクロイツクミラー 209から結像光学系 210に至る光路に DICスライダとアナラ ィザを挿入すれば、透過観察に代えて微分干渉観察を行うことができる。微分干渉 観察は、透過観察と比較して、よりコントラストの高い画像が得られる。  [0053] If 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.
[0054] ステージ搬送機構 113によって、スライドガラス 102と、蛍光撮像部 207や赤外光 撮像部 211が備える固体撮像素子との相対位置を変化させながら、複数の視野で生 細胞 Cの撮像を行う。複数の視野 (撮像範囲）を切り替えながら撮像する場合は、各 視野でのステージ位置を記録しておき、各視野の 2回目以降の撮像に先立って、ス テージ搬送機構 113によりステージ位置を再現する。  [0054] 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. . 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. .
[0055] ここで、露出検出部 309により画像データ撮像時の露出が適正であった力否かを 検出する。撮像時の露出が不適正だった場合、直ちに或いは他の任意の観察部位 の撮像が完了した時点で、露出不適正部位の撮像をやり直す。この際、露出条件を 変更しても良い。同様に、合焦検出部 312により画像データ撮像時の焦点合わせが 適正であつたか否かを検出する。撮像時の焦点合わせが不適正だった場合、直ちに 或いは他の任意の観察部位の撮像が完了した時点で、合焦不適正部位の撮像をや り直す。この際、焦点合わせの条件を変更しても良い。 Here, 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.
[0056] また、培養部 101内は培養液 Aが循環して ヽるが、撮像を行うタイミングに合わせて 一時的に循環を停止させても良い。これにより、培養液の流通に由来する撮像時の 背景の揺らぎを回避することができる。  [0056] Although the culture medium A circulates in the culture unit 101, 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.
[0057] さらに、所定の視野における撮像回数を撮像時点認識手段としての撮像回数計数 部 310により計数し、所定の視野の所定回数 (フレーム)の撮像後、報知手段として の表示部 304に画像を表示し、その内容について操作者に確認を求めるようにして もよい。内容に問題が無いと操作者が判断した場合は処理を継続し、問題ありと判断 した場合は、撮像条件の再設定について操作者力もの指示を受け付ける。或いは、 単に処理を中止してもよい。一定時間経っても操作者からの応答が無い場合は、既 定の指示に従い、処理の継続或いは中止を選択する。  [0057] Further, 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.
[0058] なお、本実施の形態 1では、蛍光撮像部 207 (又は、赤外光撮像部 211)による撮 像回数を計数し、所定回数の撮像後に操作者に画像の確認を求めるようにしたが、 撮像回数を基準にするだけではなぐ撮像時点認識手段として、観察開始から所定 時間の経過を計測する手段 (例えば、細胞画像データを撮像した時刻の情報も取得 して、その時刻があら力じめ定めた時刻を超えた場合に、所定時点の細胞画像デー タを取得したことを認識する）を設けることで、画像の確認を行うようにしてもよい。  In the first embodiment, the number of times of imaging by the fluorescence imaging unit 207 (or the infrared light imaging unit 211) is counted, and the operator is asked to confirm the image after a predetermined number of times of imaging. However, as a means of recognizing 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).
[0059] 培養期間が長期となると、培養中の細胞に関して、想定した範囲を逸脱した増殖、 死滅、画像輝度値の過大等が起こる可能性があり、或る時点で細胞観察にとって適 切な画像を取得できなくなる場合も予想される。そこで、上述のように、所定の時点が 経過したことを操作者に認識させたり、処理を中止したりすることで、操作者はそれま でに取得した画像や、その時点の画像を確認して、その後の細胞観察を適切に行う ことが可能となる。  [0059] If 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.
[0060] っ 、で、撮像した画像のうち、蛍光撮像部 207で取得した画像データの処理手順 について説明する。図 7は、制御部 301による制御の下に、前処理部 305等により実 行される画像データ処理例を示す概略フローチャートである。前述したような撮像部 201による細胞画像の撮像 (ステップ SI)に引き続き、前処理部 305で前処理を行い (ステップ S2)、細胞認識手段としての細胞認識部 306で細胞を認識する (ステップ S 3)。ついで、認識した細胞の特徴を示す細胞パラメータを細胞パラメータ計測手段と してのパラメータ計測部 307で細胞画像データに基づき計測する (ステップ S4)。さら に、細胞追跡手段としての細胞追跡部 308で、異なる時点で撮像された画像の細胞 画像データ力 認識された異なる時点の細胞同士の同一性を細胞パラメータに基づ いて判別する (ステップ S5)。或いは、さらに追跡結果を修正する (ステップ S6)。そし て、得られた追跡結果を表示部 304に表示させ (ステップ S7)、観察が終了するまで (ステップ S8 ; Yes)、上述の処理ステップを同様に繰り返す。 Thus, the processing procedure of the image data acquired by the fluorescence imaging unit 207 among the captured images will be described. 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 Subsequent to the imaging of the cell image by step 201 (step SI), preprocessing is performed by the preprocessing unit 305 (step S2), and the cell is recognized by the cell recognition unit 306 as cell recognition means (step S3). Next, 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). In addition, 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. . Alternatively, the tracking result is further corrected (step S6). Then, 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).
[0061] なお、ステップ S1 (又は、ステップ S 1及びステップ S2)の処理を、複数の時期にお いてあら力じめ行っておいて、各時期に取得した画像データに対するステップ S 2以 降 (又は、ステップ S3以降）の処理を後にまとめて行うようにしてもよい。このように、 複数の時期において撮像した画像データをあらカゝじめ取得しておいて、後にまとめて 画像データの処理を行うようにする場合、撮像と画像データの処理とを並行して行う 場合と比較し、装置構成が単純化され、安価な計算機を用いて応答性と安定性を向 上させることができる。 [0061] It should be noted that the processing of 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. As described above, when 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. Compared to the case, the system configuration is simplified and the responsiveness and stability can be improved by using an inexpensive computer.
[0062] 各処理ステップの内容を個別に説明する。撮像し記録部 302に記録された画像デ ータを、ステップ S2では、前処理部 305において以下のように処理する。まず、画像 データにエッジ保存型のローパスフィルタを適用する。エッジ保存型のローパスフィ ルタは、エッジ部における空間周波数高周波成分の劣化を抑えつつ、エッジ部以外 に平滑ィ匕の効果をもたらすものであり、細胞の輪郭情報を保存したままノイズ除去が できる点で、本手法に好適である。  [0062] The contents of each processing step will be described individually. In step S2, the pre-processing unit 305 processes the image data picked up and recorded in the recording unit 302 as follows. First, 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.
[0063] このような要件を満たすフィルタとして、バイラテラルフィルタ（Tomasi & Manduchi," Bilateral Filtering [0063] As a filter satisfying such requirements, a bilateral filter (Tomasi & Manduchi, "Bilateral Filtering
for ray and Color Images , Proceedings of the 1998 IEEE International  for ray and Color Images, Proceedings of the 1998 IEEE International
Conference on Computer Vision, Bombay, India参照)が知られており、本手法でもこ れを使用する。  Conference on Computer Vision, Bombay, India) is known and is used in this method.
[0064] 次に、エッジ保存型ローパスフィルタ適用後の画像データに、さらにエッジ強調のた めの先鋭ィ匕フィルタを適用する。先鋭ィ匕フィルタは、注目画素とその近傍の 8画素に 例えば図 8に示すような重み付けを行って総和を求めるフィルタであり、これを画素毎 に反復実行することで、先鋭化処理が実現できる。 [0064] Next, the image data after applying the edge preserving low-pass filter is further subjected to edge enhancement. Apply a sharp edge filter. The sharpness 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. .
[0065] ステップ S3では、前処理後の画像データを、細胞認識部 306において以下のよう な手順で分析し、個々の細胞の占める領域を認識する。この手順に従えば、細胞が 互いに隣接せずに散在する場合だけでなぐ細胞が互いに隣接し、密集している場 合にも個々の細胞の占める領域を認識できる。また、細胞領域のエッジが明瞭でな Vヽ場合にも適用することができる。  [0065] In 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.
[0066] まず、画像を高輝度画素の集中する領域毎に領域分割する。一般に、蛍光画像に おいて、細胞は高輝度画素の塊の様相を呈するため、高輝度画素の集中する領域（ 塊）毎に領域分割することは、画像を細胞毎の領域に分割することに相当する。  First, the image is divided into regions where high luminance pixels are concentrated. In general, in a fluorescent image, cells exhibit the appearance of a cluster of high-luminance pixels. Therefore, dividing an area into areas (lumps) where high-luminance pixels are concentrated means dividing the image into areas for each cell. Equivalent to.
[0067] このような要件を満たす処理として、分水嶺領域分割が知られて 、る。本実施の形 態 1の細胞認識の処理手順として、この分水嶺領域分割方式を使用する (Vincent & ¾oille , Watersheds in Digital Spaces: An Efficient Algontnm Based  [0067] 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 & ¾oille, Watersheds in Digital Spaces: An Efficient Algontnm Based
on Immersion Simulations", IEEE TRANSACTIONS ON PATTERN ANALYSIS AND MACHINE INTELLIGENCE, VOL.13, NO.6, JUNE 1991参照)。原論文における分 水嶺領域分割は、画像を低輝度画素の集中する領域に分割するものであるが、ここ では輝度を反転して考え、高輝度領域の分割に適用する。得られた領域分割結果 における個々の領域が細胞領域となる。  on Immersion Simulations ", IEEE TRANSACTIONS ON PATTERN ANALYSIS AND MACHINE INTELLIGENCE, VOL.13, NO.6, JUNE 1991). However, in this case, the luminance is reversed and applied to the division of the high luminance region, and each region in the obtained region division result becomes the cell region.
[0068] ここで、隣接する細胞領域の特性に応じて複数の細胞領域を統合し新たな 1つの 細胞領域とするような、統合処理を行っても良い。分水嶺領域分割処理の結果は、 一般に、小領域に分割され易い傾向があるため、統合処理を行うことで認識結果の 品質を高めることができる。  [0068] Here, 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.
[0069] 領域統合の第 1の手法を、図 9を参照して説明する。図 9は、領域統合の第 1の手 法例を示す概略フローチャートである。まず、各細胞領域において輝度が最大となる 点、すなわち輝度の頂点を求める (ステップ S311)。次に、隣接する任意の 2つの細 胞領域を選択し (ステップ S312)、それらの頂点間を結ぶ線分に沿った道のり距離 D を求める (ステップ S313)。道のり距離 D の計算には式 (1)を用いる。 [0070] [数 1] [0069] A first method of region integration will be described with reference to FIG. FIG. 9 is a schematic flowchart showing a first example of region integration. First, the point where the luminance is maximum in each cell region, that is, the vertex of the luminance is obtained (step S311). Next, any two adjacent cell regions are selected (step S312), and the distance D along the line connecting the vertices is obtained (step S313). Equation (1) is used to calculate the distance D. [0070] [Equation 1]
DUW =∑(I(P)— …（1 ) D UW = ∑ (I (P) —… (1)
p  p
[0071] ここで、 I(P)は、エッジ保存型ローノ スフィルタ適用後の画像における画素 Pの輝度 値、 Zl(P )は、エッジ保存型ローパスフィルタ適用後の画像における 2つの頂点の輝 Here, I (P) is the luminance value of pixel P in the image after applying the edge-preserving low-pass filter, and Zl (P) is the brightness of two vertices in the image after applying the edge-preserving low-pass filter.
S  S
度値の平均、∑は、頂点間を結ぶ線分の全画素について総和を求めることを表す。  The average of the degree values, ∑, represents that the sum is obtained for all the pixels of the line segment connecting the vertices.
[0072] ステップ S313で、隣接する細胞領域の全ての組み合わせについて頂点間の道の り距離 D を求めた後、ステップ S314で、この道のり距離 D と所定の閾値 V との [0072] In 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.
UW UW UW UW UW UW
比較を行う。比較の結果、所定の閾値 V 以下である場合には (ステップ S314 ; Yes  Make a comparison. If the result of the comparison is below the predetermined threshold V (Step S314; Yes
UW  UW
)、細胞領域同士を 1つの領域に統合する (ステップ S315)。このような処理を全組み 合わせについて完了するまで (ステップ S316 ; Yes)、同様に繰り返す。  ), 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).
[0073] 領域統合の第 2の手法を、図 10を参照して説明する。図 10は、領域統合の第 2の 手法例を示す概略フローチャートである。まず、エッジ保存型ローパスフィルタの出 力結果にエッジ抽出フィルタ、例えば Sobelフィルタを適用してエッジ画像を得る (ステ ップ S321)。任意の隣接する細胞領域間の境界を考えて、隣接する任意の 2つの細 胞領域を選択し (ステップ S322)、式 (2)で定義されるエッジ強度 D を求める (ステツ [0073] A second method of region integration will be described with reference to FIG. FIG. 10 is a schematic flowchart showing a second method example of region integration. First, an edge extraction filter, such as a Sobel filter, is applied to the output result of the edge preserving low-pass filter to obtain an edge image (step S321). Considering the boundary between any adjacent cell regions, select any two adjacent cell regions (step S322), and obtain the edge strength D defined by equation (2) (steps
UE  UE
プ S323)。  S323).
[0074] [数 2] [0074] [Equation 2]
DUE =∑E(P) … ）DUE = ∑E (P)…)
P P
[0075] ここで、 E(P)は、エッジ画像における画素 Pの輝度値、∑は、細胞領域間の境界に 含まれる全画素につ 、て総和を求めることを表す。 Here, E (P) represents the luminance value of the pixel P in the edge image, and ∑ represents that the sum is obtained for all the pixels included in the boundary between the cell regions.
[0076] ステップ S323で、隣接する細胞領域の全ての組み合わせについてエッジ強度 D [0076] In step S323, edge strength D for all combinations of adjacent cell regions
UE  UE
を求めた後、ステップ S324で、このエッジ強度 D と所定の閾値 V との比較を行う。  In step S324, the edge strength D is compared with a predetermined threshold value V.
UE UE  UE UE
比較の結果、所定の閾値 V 以下である場合には (ステップ S324 ; Yes)、細胞領域  If the result of the comparison indicates that the threshold value is less than or equal to the predetermined threshold V (Step S324; Yes),
UE  UE
同士を 1つの領域に統合する (ステップ S325)。このような処理を全組み合わせにつ いて完了するまで (ステップ S326 ; Yes)、同様に繰り返す。 [0077] これらの第 1,第 2の領域統合の手法は、それぞれ別個に使用しても良いし、任意 の順番で連続して用いても良い。 Merge them into one area (step S325). This process is repeated until all the combinations are completed (step S326; Yes). [0077] These first and second region integration methods may be used separately or sequentially in any order.
さらに、輝度情報を用いて各細胞領域の妥当性を検証しても良い。そのためには、 分割された細胞領域毎に輝度値が最大となる画素を求め、その輝度値が所定の閾 値 Vtminより小さい場合、その領域は細胞領域ではないと判定し、所属する画素も含 めて以降の処理の対象から除外する。これにより、蛍光タンパクの導入又は発現が不 十分な細胞、及び細胞ではな 、背景領域を除外することができる。  Further, the validity of each cell region may be verified using luminance information. For this purpose, 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.
[0078] さらに、細胞領域内の各画素の輝度を所定の閾値 Vpminと比較し、閾値 Vpminより 輝度の小さい画素を細胞領域力も除外しても良い。こうして除外された画素は、以降 の処理には使用しない。これにより、細胞領域の中でも SZNの低い低輝度部位を除 外することができ、細胞領域の境界形状をより正確に認識することが可能となる。 得られた細胞領域、及び各細胞領域に属する画素の集合を記録部 302に記録す る。  [0078] Furthermore, 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. As a result, it is possible to exclude a low-luminance part having a low SZN in the cell region and to recognize the boundary shape of the cell region more accurately. The obtained cell region and a set of pixels belonging to each cell region are recorded in the recording unit 302.
[0079] なお、赤外光撮像部 211が撮像した赤外光画像を用いて細胞領域を認識すること も可能である。赤外光画像が位相差画像である場合、細胞の存在する領域の輝度値 は、背景とは異なる輝度値として観察される。したがって、画像内の各画素について 代表的な背景の輝度値 P  [0079] Note that it is also possible to recognize a cell region using an infrared light image captured by the infrared light imaging unit 211. When the infrared light image is a phase difference image, the luminance value of the area where the cells exist is observed as a luminance value different from the background. Therefore, for each pixel in the image, the representative background brightness value P
BGとの差を求め、差が所定の閾値 V  Find the difference from BG, and the difference is the predetermined threshold V
PGより大きな画素のみを 抽出し、一般的なラベリング処理を行って隣接する画素を統合すれば、細胞領域を 認識することができる。  If only pixels larger than PG are extracted and general labeling processing is performed to integrate adjacent pixels, the cell region can be recognized.
[0080] 図 7の処理に戻り、ステップ S4では、パラメータ計測部 307において、細胞認識部 3 06で認識した細胞領域毎に細胞パラメータを計測し、その計測結果を記録部 302に 記録する。図 11は、記録部 302に記録された細胞パラメータの計測結果例を示す説 明図である。ただし、 Mは認識された細胞領域の数である。本実施の形態 1では、細 胞パラメータは、例えば、重心位置、面積、円形度、輝度の総和、平均輝度、輝度の 標準偏差を測定項目対象としており、画像データ及び細胞領域と関連付けて記録部 302に記録する。アツセィの内容に応じて、周囲長、フェレ径、長さ、幅、最大輝度等 の一般的な測定項目を追加しても良 、。  Returning to the processing of FIG. 7, in step S 4, the parameter measurement unit 307 measures the cell parameter for each cell region recognized by the cell recognition unit 320, and records the measurement result in the recording unit 302. FIG. 11 is an explanatory diagram showing an example of measurement results of cell parameters recorded in the recording unit 302. Where M is the number of recognized cell regions. In the first embodiment, the cell parameters are, for example, the center of gravity position, area, circularity, total luminance, average luminance, and standard deviation of luminance as measurement items, and are associated with the image data and the cell region and are recorded in the recording unit. Record in 302. Depending on the content of the assembly, general measurement items such as perimeter, ferret diameter, length, width, and maximum brightness may be added.
[0081] ここで、本実施の形態 1は、画像内の全ての細胞の面積の総和、すなわち画像内 で細胞領域の占める度合 、を示す細胞占有値に相当する面積を占有面積算出手 段としての占有面積算出部 311で算出し、画像内で細胞領域の占める面積力 画像 の面積に対して所定の割合を超えた場合、制御部 301にその事象を通知する。この 場合、制御部 301はあら力じめ指定された設定に従い、報知手段としての表示部 30 4を通じて操作者にさらに報知しても良 ヽし、培養部 101の制御状態を変更しても良 い。或いは単に通知を無視しても構わない。この機能は、培養が長期になると、細胞 の増殖等によって培地の空きスペースが減少することがあるので、細胞培養の過程 にお!/、て培地の空きスペースが不足してきて!/、ることを通知する場合に有効である。 [0081] Here, in the first embodiment, 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. When the ratio is exceeded, the control unit 301 is notified of the event. In this case, 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.
[0082] 占有面積計算部 311は、細胞画像内で細胞領域の占める面積を細胞占有値とし て求めるものである。細胞画像は、蛍光画像或いは赤外光画像のいずれであっても 構わない。蛍光画像を対象とする場合、パラメータ計測部 307により細胞領域の面積 が計測されているので、画像内の全ての細胞領域の面積を合計すれば、画像内で 細胞領域の占める面積を求めることができる。赤外光画像を対象とする場合、細胞の 存在する領域の輝度値は、背景とは異なる輝度値として観察される。したがって、ま ず画像内の各画素について代表的な背景の輝度値 P 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. When targeting a fluorescent 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. When targeting an infrared light image, 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
BGとの差を求め、差が所定の 閾値 V より大きな画素のみを抽出する。さらに、画像内で抽出された画素の数を計 Find the difference from BG, and extract only the pixels whose difference is larger than the predetermined threshold value V. In addition, count the number of pixels extracted in the image.
PG PG
数すれば、画像内で細胞領域の占める面積が得られる。  If counted, the area occupied by the cell region in the image can be obtained.
[0083] 以上の手順により、複数の細胞画像を含む単一の細胞画像に関して、個々の細胞 の領域を求めた上で細胞パラメータを計測することができる。所定の時間間隔 A t毎 に細胞画像の撮像とパラメータ計測とを反復して行うことで、細胞パラメータを時間経 過に伴って蓄積することができる。 [0083] With the above procedure, it is possible to measure cell parameters after obtaining individual cell regions for a single cell image including a plurality of cell images. By repeatedly capturing a cell image and measuring parameters at predetermined time intervals At, cell parameters can be accumulated with time.
[0084] ここで、このままの処理では、異なる時刻に計測された細胞パラメータ同士が関連 付けられておらず、経時的に計測した状態とは言えない。そこで、異なる時刻に撮影 された細胞画像間において、細胞領域の対応付けを行い、その結果を用いて細胞 パラメータ同士の関連付けを行う必要がある。 Here, in the process as it is, 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.
[0085] 細胞領域の対応付けは、細胞追跡部 308において、ステップ S5, S6の処理として 以下のように実行される。ここで、時刻 tにお ヽて認識された細胞領域を R 、時刻 t The cell region association is executed in the cell tracking unit 308 as the processing of steps S5 and S6 as follows. Here, R is the cell region recognized at time t, and t
1 tl,m 2 において認識された細胞領域を R と表すものとする。ただし、時刻 tは時刻 tより時 系列的に後の時刻である。 m, nは同一画像内で重複の無い細胞領域の識別番号 で、 l≤m≤M, l≤n≤Nであり、 Mと Nはそれぞれ時刻 t , tで認識された細胞領 Let 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.
1 2  1 2
域の数を表す。  Represents the number of areas.
[0086] まず、 2つの細胞領域 R と R の関連性に関する評価関数を式 (3)で定義する。  [0086] First, an evaluation function related to the relationship between two cell regions R and R is defined by equation (3).
tl,m t2,n  tl, m t2, n
式 (3)で計算される評価街力 S小さい程、 2つの領域は関連性が高ぐ同一の細胞を  The smaller the evaluation city force S calculated by Eq. (3), the smaller the two cells
1  1
示して 、る可能性が高 、と言える。  It can be said that the possibility is high.
[0087] 〔数 3〕  [0087] [Equation 3]
J =J (R , R )=k δ +k δ +k δ …… (3)  J = J (R, R) = k δ + k δ + k δ …… (3)
1 1 tl，m t2,n d d a a c c  1 1 tl, m t2, n d d a a c c
δ ：重心間の距離  δ: Distance between centroids
d  d
δ ：面積の差  δ: Area difference
a  a
δ ：円形度の差  δ: Difference in circularity
k , k , k：所定の重み付け係数  k, k, k: predetermined weighting factors
d a c  d a c
[0088] 図 12は、 mと nの可能な組み合わせについて評価値を計算した結果を示す説明図 である。ただし、図 12では記述の簡便のため、 J (R , R )¾J と簡略表記している  FIG. 12 is an explanatory diagram showing the results of calculating the evaluation values for possible combinations of m and n. However, in FIG. 12, for the sake of simplicity, J (R, R) ¾J is abbreviated.
1 tl,m t2,n m,n  1 tl, m t2, n m, n
[0089] ここで、時刻 tの領域 R に対応する時刻 tの領域 R を式 (4)に従って決定する。 Here, region R at time t corresponding to region R at time t is determined according to equation (4).
1 tl,m 2 t2,n  1 tl, m 2 t2, n
すなわち、 R とは、領域 R との間の評価街を最小化するような時刻 tの領域で t2,n tl,m 1 2 ある。  That is, R is t2, n tl, m 1 2 in the region at time t that minimizes the evaluation town between region R.
[0090] [数 4]  [0090] [Equation 4]
Rt2,fi： where ( い m,Rt2，fi) = η ^ ，！^ ） -(4) R t2 , fi : where (i m , R t2 , fi ) = η ^ ,! ^)-(4)
1 い 1 1<η<Ν 1 ζ 1 1 1 <η <Ν 1 ζ
[0091] 評価 »が最小となる rTが複数存在する場合、それらに対して式 (5)に示す第 2の [0091] If there are multiple rTs that have the smallest evaluation », the second
1  1
評価関数を適用し、評価街 21S より小さくなる組み合わせを決定する。第 2の評価値 The evaluation function is applied to determine a combination that is smaller than the evaluation city 21S. Second evaluation value
Jが最小となる組み合わせも複数あった場合、操作者へのメッセージを表示部 304にIf there are multiple combinations that minimize J, a message to the operator is displayed on display 304.
2 2
表示し、操作者が正しいと判断する組み合わせを入力部 303より入力させ、入力結 果に基づ ヽて対応付けを行う。  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.
[0092] 〔数 5〕 [0092] [Equation 5]
J =J (R , R )=k δ +k δ +k δ …… (5)  J = J (R, R) = k δ + k δ + k δ …… (5)
2 2 tl，m t2,n s s m m v v δ ：輝度の総和の差 2 2 tl, m t2, nssmmvv δ: Difference in total luminance
δ ：平均輝度の差  δ: Difference in average luminance
δ ：輝度の標準偏差の差  δ: Difference in luminance standard deviation
k , k , k：所定の重み付け係数  k, k, k: predetermined weighting factors
[0093] ただし、評価街 , Jの両方を求めず、どちらか一方のみを用いることで処理を高速  [0093] However, both the evaluation city and J are not obtained, and only one of them is used to speed up the processing.
1 2  1 2
化しても良い。また、操作者へのメッセージ表示、及び操作者力もの入力ステップを 省略し、評価街又〖おを最小化する複数の対応関係を全て記録するようにしても良  May be used. Also, it is also possible to omit the message display to the operator and the operator's input step, and to record all the correspondences that minimize the evaluation town or the street.
1 2  1 2
い。  Yes.
[0094] 時刻 tの領域 R と時刻 tの領域 R は、同一の細胞を異なる時刻に認識した結  [0094] The region R at time t and the region R at time t are the result of recognizing the same cell at different times.
1 tl 2 t2,n  1 tl 2 t2, n
果と考えられるから、両者の計測済みの細胞パラメータも同一の細胞に対する異なる 時刻での計測値とみなせる。そこで、細胞パラメータの値を、細胞画像、細胞領域、 細胞領域の対応付け情報、時刻情報と関連付け、併せて記録手段としての記録部 3 02に記録することで、経時的なパラメータ計測が完了する。  Therefore, 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. .
[0095] 経時的に、蛍光タンパクが新たに発現し蛍光を発するようになった場合、観察画面 外にあった細胞が観察画面内へと移動した場合、重なり合つていた複数の細胞が分 かれた場合、又は細胞が分裂した場合は、細胞認識部 306において認識される細胞 領域の数が増加するため、時刻 tの細胞領域に対応する時刻 tの細胞領域が存在 [0095] When a fluorescent protein is newly expressed and emits fluorescence over time, when cells outside the observation screen move into the observation screen, a plurality of overlapping cells are separated. If the cell is divided or the cell divides, the number of cell regions recognized by the cell recognition unit 306 increases, so that there is a cell region at time t corresponding to the cell region at time t.
2 1  twenty one
しない場合や、時刻 tの複数の細胞が時刻 tの 1つの細胞に対応する場合が発生す  Or when multiple cells at time t correspond to one cell at time t.
2 1  twenty one
る。  The
[0096] また、経時的に、蛍光タンパクの蛍光強度が低下した場合、観察画面内にあった細 胞が観察画面外へと移動した場合、複数の細胞が重なり合った場合、又は細胞が死 滅した場合は、細胞認識部 306にお 、て認識される細胞領域の数が減少するため、 時刻 tの細胞領域に対応する時刻 tの細胞領域が存在しない場合や、時刻 tの複 [0096] In addition, when the fluorescence intensity of the fluorescent protein decreases with time, the cells in the observation screen move to the outside of the observation screen, multiple cells overlap, or the cells die. In this case, since 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.
1 2 1 数の細胞が時刻 tの 1つの細胞に対応する場合が発生する。 1 2 1 The number of cells corresponds to one cell at time t.
2  2
[0097] 対応する細胞領域が無!、場合は、対応領域が無 、ことを意味するフラグを記録す る。 1つの細胞領域に複数の細胞領域が対応する場合は、全ての対応関係を記録 する。表示部 304を通じて操作者にメッセージを表示し、操作者力もの入力を元に対 応関係を修正しても良い。複数の細胞領域の対応を記録する際のデータ表現は、各 時刻を高さに、各細胞領域を節点に対応させた木構造を用いる。表現の自由度がよ り高 、グラフ構造を用いても良 、。 [0097] If there is no corresponding cell area, 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.
[0098] なお、細胞領域の対応付けに関しては、以下のような改良を加えた変形例であっても よい。  [0098] It should be noted that the cell region association may be a modified example with the following improvements.
第 1の変形例は、最小の評価 »が所定の閾値 V より大きい場合、その対応付け jmax  The first variation is that if the minimum evaluation »is greater than a predetermined threshold V, its mapping jmax
は無効とみなす。この場合、領域 R に対応する時刻 tの領域は発見できなかったと tl,m 2  Is considered invalid. In this case, the region at time t corresponding to region R was not found and tl, m 2
し、領域 R に対する経時的パラメータ計測は時刻 tまでで打ち切る。この変形例は tl,m 1  However, the time-lapse parameter measurement for region R is terminated by time t. This variant is tl, m 1
、ノイズの影響を低減させるために有効である。  This is effective for reducing the influence of noise.
[0099] 第 2の変形例は、領域 R と対応する領域 R の重心間距離を求め、重心間距離 tl,m t2,n [0099] In the second modification, the distance between the centers of gravity of the region R and the corresponding region R is obtained, and the distance between the centers of gravity tl, m t2, n
が所定の閾値 V より大きカゝつた場合、その対応付けは無効とみなす。この場合、領 dmax  If is greater than the predetermined threshold V, the mapping is considered invalid. In this case, region dmax
域 R に対応する時刻 tの領域は発見できなカゝつたとし、領域 R に対する経時的 tl,m 2 tl,m  Suppose that the region at time t corresponding to region R cannot be found, and that tl, m 2 tl, m
ノ メータ計測は時刻 tlまでで打ち切る。この変形例は、細胞領域対応付け処理の 誤りを低減させるために有効である。  The meter measurement is discontinued until time tl. This modification is effective to reduce errors in the cell region association processing.
以上の手順により、細胞のパラメータを経時的に計測することができる。  With the above procedure, cell parameters can be measured over time.
[0100] 最後に、図 7の処理に戻り、ステップ S7の処理として、細胞パラメータ表示手段とし ての表示部 304にて、認識した細胞領域と計測した細胞パラメータを表示する。図 1 3は、処理結果の表示の一例を示す説明図である。表示部 304が備える表示画面 3 14は、 2つの表示領域 314a, 314bを有し、表示領域 314aには、処理対象時点に おいて認識された個々の細胞領域が表示される。ここで、細胞領域にはラベリング処 理を適用し、領域毎に識別可能な色、輝度、線種、パターンを与え、例えばラベル画 像 a〜eとして表示する。ラベル画像と同じ表示範囲の赤外光画像或いは蛍光画像を 連動して表示させても良いし、ラベル画像、赤外光画像、蛍光画像のうち複数を重ね 合わせて表示しても良い。或いは、スーパーインポーズ表示を行っても良い。計測し た細胞パラメータは、表示領域 314bにおいて、時間を横軸、パラメータ値を縦軸とし た折れ線チャートとして表示する。さらに、操作者による入力部 303中のマウス操作 等に応じて両者の表示内容を同期して強調表示すれば、表示内容の視認性が向上 する。図 14は、例えばラベル画像 cを強調表示の指示対象として選択指定した場合 に対応する細胞パラメータの折れ線チャートも強調表示される一例を示す説明図で ある。この場合、操作者が片方を選択強調した場合、対応する他方も同期して強調 表示する。 [0100] Finally, returning to the processing of FIG. 7, as the processing of step S7, the recognized cell region and the measured cell parameters are displayed on the display unit 304 as cell parameter display means. 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. Here, 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.
[0101] 本実施の形態 1では、生細胞 Cに蛍光タンパクを導入し観察しているが、蛍光タン ノ クに代えて発光遺伝子、例えばルシフェラーゼ遺伝子を導入すれば、蛍光画像に 代えて発光画像が撮像できる。この場合、励起光照明部 203及びダイクロイツクミラ 一 204は不要であり、構成を簡略化できる。発光画像は、細胞光撮像手段としての 蛍光撮像部 207によって撮像される。発光画像に対しては、蛍光画像と同じ手順で 処理を行えば良い。このように、例えば細胞が自発光する場合や蛍光を発する場合 など、細胞が赤外光以外の光を発する場合でも細胞画像データを取得して細胞観 察を行うことができる。  [0101] In 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.
[0102] また、本実施の形態 1では、細胞内に局在せずに発現する蛍光タンパクを使用して いるが、細胞核、細胞質、核膜、細胞膜、或いはオルガネラに局在して発現する蛍光 タンパクであっても構わな 、。  [0102] In the first embodiment, a fluorescent protein that is expressed without being localized in the cell is used. However, fluorescence that is expressed in a localized manner in the cell nucleus, cytoplasm, nuclear membrane, cell membrane, or organelle. It can be protein.
[0103] なお、パラメータ計測部 307において計測する細胞パラメータは、本実施の形態 1 に例示したものに限定されず、さら〖こ、面積、周囲長、外接矩形位置、 X方向フェレ 径、 Y方向フ レ径、最小フ レ径、最大フ レ径、平均フ レ径、凸周囲長、円形度 (真円度)、孔の数、ラフネス (凸周囲長と周囲長の比)、オイラー数、長さ、幅、扁平 度、輝度の総和、最小輝度、最大輝度、平均輝度、輝度の標準偏差、輝度の分散、 エントロピー、重心位置、 2次モーメント、主軸方向、のいずれか或いは複数であって も良い。  [0103] Note that 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.
[0104] さらに、パラメータ計測部 307は、任意の複数の細胞力 なるグループに対して、細 胞数、最小細胞間距離、最大細胞間距離、平均細胞間距離、細胞間距離の標準偏 差、細胞間距離の分散、並びに個々の細胞に対して計測した各パラメータの最小値 、最大値、平均値、標準偏差、分差、総和、中間値、のいずれか或いは複数を求め ても良い。  [0104] Further, 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.
[0105] 以上、本実施の形態 1によれば、蛍光タンパクを導入した生細胞を長期間培養しな がら、細胞に対する観察光の影響を抑制して細胞の活性が失われにくい状況を確保 し、個々の細胞を見失うこと無く追跡し、種々の細胞パラメータを経時的に計測する 装置を実現できる。 [0105] As described above, according to the first embodiment, while 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.
[0106] (実施の形態 2)  [Embodiment 2]
本発明の実施の形態 2の細胞観察装置は、実施の形態 1と同様、蛍光タンパクを導 入した複数の生細胞を培養したまま撮像し、個々の細胞の領域を認識し、経時的な 位置変化を追尾しつつ、個々の細胞の特徴を示す細胞パラメータを独立に計測した 上で、さらに細胞のアポトーシスを検出する。細胞のアポトーシスの検出とは、細胞が 該細胞のプログラム死のモードに入つたかを検出することを意味する。  The cell observation device according to the second embodiment of the present invention, 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.
[0107] 図 15は、本実施の形態 2に係る細胞観察装置の構成例を示す概略ブロック図であ る。図中、本細胞観察装置を制御する制御部 301から培養系、撮像系各部への接続 は特に図示していない。本実施の形態 2は、実施の形態 1と共通の構成として、あら 力じめ蛍光タンパクで標識した複数の生細胞 Cを保持したスライドガラス 102、観察 窓 105、ステージ搬送機構 113等を含む培養部 101、撮像部 201 (対物光学系 205 、結像光学系 206、蛍光撮像部 207、赤外光照明部 208、ダイクロイツクミラー 209、 結像光学系 210、赤外光撮像部 211等）、制御部 301、記録部 302、入力部 303、 表示部 304、前処理部 305、細胞認識部 306、パラメータ計測部 307、細胞追跡部 3 08、露出検出部 309、撮像回数計数部 310、占有面積算出部 311、合焦検出部 31 2を備え、これらは実施の形態 1と同様に作用する。  FIG. 15 is a schematic block diagram showing a configuration example of the cell observation device according to the second embodiment. In the figure, 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. In the second embodiment, as a configuration common to the first embodiment, 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. 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.
[0108] 励起光照明部 203'、ダイクロイツクミラー 204'は実施の形態 1における照明部 203 、ダイクロイツクミラー 204の機能に加えて波長選択機能を有し、少なくとも 2つの異な る帯域の蛍光に対応可能である。さらに、本実施の形態 2の特有の構成として、アポ トーシス検出手段としてのアポトーシス検出部 315を備える。  [0108] 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.
[0109] 本実施の形態 2で対象とする生細胞は、細胞膜と細胞内のオルガネラをそれぞれ 波長特性の異なる蛍光タンパクで標識し、結果として少なくとも二重に標識されたも のである。生細胞の細胞膜及び細胞内のオルガネラを蛍光タンパクで標識するには 、一例として BDバイオサイエンス 'クロンテック社がライセンスしている細胞内局在化 ベクター「BD  [0109] 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. To label live cell membranes and intracellular organelles with fluorescent proteins, as an example, BD Biosciences' subcellular localization vector “BD” licensed by Clontech
Livingし olors buocellular Localization VectorJ 使用すること力 21 (？さる。 Living olors buocellular Localization VectorJ Using power 21 (?
[0110] 実施の形態 1と同様に測定試料、すなわち標識を行った生細胞 Cの培養と撮像とを 経時的に行い、取得した蛍光画像と赤外光画像とを記録部 302に記録する。ただし 、同一の撮像領域に対して、細胞膜を標識した蛍光タンパクの蛍光画像 (以下、「細 胞膜蛍光画像」 t ヽぅ）及び細胞内のオルガネラを標識した蛍光タンパクの蛍光画像 (以下、「細胞内オルガネラ蛍光画像」と ヽぅ）の、少なくとも 2枚の蛍光画像を撮像す る。この際、波長選択機能を有する励起光照明部 203'、ダイクロイツクミラー 204'に よって、各蛍光タンパクの励起に適切な励起光と透過波長をそれぞれ選択する。 [0110] As in Embodiment 1, the measurement sample, that is, the labeled live cell C, 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. However, fluorescent images of fluorescent proteins labeled with cell membranes (hereinafter referred to as “cell membrane fluorescent images” t ヽ ぅ) and 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). At this time, 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.
[0111] 撮像した蛍光画像のうち、細胞膜蛍光画像を前処理部 305及び細胞認識部 306 で処理し、細胞領域を認識する。処理手順は実施の形態 1と同様である。認識した細 胞領域を記録部 302に記録する。  [0111] Among the captured fluorescent images, 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.
[0112] パラメータ計測部 307において、実施の形態 1で示した細胞領域のパラメータのう ち、少なくとも面積と円形度を計測する。次に、認識した個々の細胞領域毎に、細胞 内オルガネラ蛍光画像の対応する領域でのエントロピーを計測する。エントロピー H の計算には式 (6)を使用する。ここで、 Aは注目領域 Rの面積、 P は注目領域 R内 の位置 (X, y)における輝度値、 Sは式 (7)で示すような注目領域 R内の輝度総和をそ  [0112] 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).
R  R
れぞれ表す。ただし、輝度総和 S力^の場合は、エントロピー Hの値を 1とする。  Represent each. However, the value of entropy H is 1 in the case of the luminance sum S power ^.
R  R
[0113] [数 6]
Figure imgf000030_0001
[0113] [Equation 6]
Figure imgf000030_0001
[0114] [数 7] [0114] [Equation 7]
SR = ∑PX,Y …(力 SR = ∑P X , Y … (force
(x,y)eR  (x, y) eR
[0115] 細胞膜蛍光画像力 抽出した細胞領域について計測したパラメータと、細胞内ォ ルガネラ蛍光画像の対応する領域にっ 、て計測したエントロピーを、細胞膜蛍光画 像、細胞内オルガネラ蛍光画像、細胞領域と関連付けて記録部 302に記録すること で、パラメータ計測が完了する。 [0115] 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.
[0116] 細胞追跡部 308において、異なる時刻の細胞膜蛍光画像力 抽出された細胞領 域の対応付けを行う。処理手順は実施の形態 1と同様である。細胞領域の対応付け 情報を、細胞膜蛍光画像、細胞内オルガネラ蛍光画像、細胞領域、パラメータ計測 部 307にお 、て計測した細胞パラメータ、時刻情報と関連付けて記録部 302に記録 する。 [0116] In the cell tracking unit 308, 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.
[0117] 次に、アポトーシス検出部 315において、アポトーシス発生の有無を調査する。細 胞認識部 306において認識された、時刻 tの細胞領域のうち、任意の 1つを手動又  Next, 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
4  Four
は自動で選択する（以下、 R と記述する)。  Is automatically selected (hereinafter referred to as R).
t4  t4
[0118] 図 16は、アポトーシス検出部 315により実行される、注目する細胞領域 R に対する t4 アポトーシスの発生の検出処理例を示す概略フローチャートである。この処理は、図 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
7に示したフローチャート中のステップ S9の処理として実行される。まず、細胞領域 R t のエントロピー H(R )が所定の閾値 V未満の場合 (ステップ S21 ;No)、アポトーシ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),
4 t4 H 4 t4 H
スではな!/ヽ（陰性）と判定し (ステップ S25)、アポトーシス検出処理を終了する。  It is determined that it is not! / ヽ (negative) (step S25), and the apoptosis detection process is terminated.
[0119] エントロピー !^ )が閾値 V以上の場合 (ステップ S21； Yes)、時刻 tの細胞領域 t4 H 4 [0119] If entropy! ^) Is greater than or equal to threshold V (step S21; Yes), cell region t4 H 4 at time t
R に対応する時刻 tの細胞領域 Rを記録部 302から取得する。ただし、時刻 tは時 t4 3 t3 3 系列的に時刻 tより所定の時間間隔だけ前の（過去の）時刻である。次に、細胞領域  A cell region R at time t corresponding to R is acquired from the recording unit 302. However, 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. Next, the cell region
4  Four
R のエントロピー H(R )と円形度 C(R ),細胞領域 R のエントロピー H(R )と円形度 t3 t3 t3 t4 t4 R entropy H (R) and circularity C (R), cell region R entropy H (R) and circularity t3 t3 t3 t4 t4
C(R )に関して、次の条件式 (8)及び (9)が共に成立する場合 (ステップ S22 ; Yes, S t4 When both of the following conditional expressions (8) and (9) are satisfied for C (R) (Step S22; Yes, S t4
23； Yes)、アポトーシスである（陽性）と判定する (ステップ S24)。少なくとも一方が成 立しな 、場合、アポトーシスではな ヽ（陰性）と判定する (ステップ S25)。  23; Yes), 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).
[0120] 〔数 8〕 [0120] [Equation 8]
H(R )<H(R )…… (8)  H (R) <H (R) …… (8)
t3 t4  t3 t4
〔数 9〕  [Equation 9]
C(R )< C(R )…… (9)  C (R) <C (R) …… (9)
t3 t4  t3 t4
なお、円形度 Cは、 C =4 SZL2で定義されるものであって、 0≤C≤1である。た だし、 Sは細胞の面積、 Lは細胞の輪郭の長さ (周囲長）である。 The circularity C is defined by C = 4 SZL 2 and 0≤C≤1. Where S is the area of the cell and L is the length of the cell outline (perimeter).
[0121] 以上のアポトーシス判定手順を時刻 tにおける全ての細胞領域に対して反復して [0121] The above apoptosis determination procedure is repeated for all cell regions at time t.
4  Four
行うことで、単一画像内の全てのアポトーシスを検出することができる。さらに、撮像し た全ての時刻の画像に対してアポトーシスの検出を反復して行 ヽ、全ての画像内の アポトーシスを検出する。判定結果は、細胞領域と関連付けて記録部 302に記録す る。 By doing so, all apoptosis in a single image can be detected. Furthermore, the detection of apoptosis is repeated for all images taken at the time, and apoptosis is detected in all images. The determination result is recorded in the recording unit 302 in association with the cell region. The
[0122] 最後に、アポトーシス判定結果を表示部 304にて表示する。これには、アポトーシス 判定で陽性であった細胞領域と、陰性であった細胞領域を区別可能な任意の表示 形式を使用できる。例えば、陽性或いは陰性の細胞領域にのみマーカを重畳表示 する、陽性と陰性の細胞領域にそれぞれ異なるマーカを重畳表示する、陽性或いは 陰性の細胞領域のみを表示する、陽性と陰性の細胞領域で表示色'パターン '点滅 等の表示属性を変える、陽性と陰性の細胞領域で操作者操作への応答内容を変え る、等の手法が適用可能であり、これらを相互に切り替えても構わない。また、異なる 時刻に取得した複数の画像に対する判定結果を順次切り替えて表示すれば、経時 的なアポトーシスの発生状況を視覚的に把握可能となる。  Finally, 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. For example, 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. In addition, if 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.
[0123] 以上、本実施の形態 2によれば、蛍光タンパクを導入した生細胞を長期間培養しな がら、個々の細胞を見失うこと無く追跡し、種々の細胞パラメータを経時的に計測し た上で、さらに長期に亘つて細胞を培養していくうちにアポトーシスが発生すれば、こ のようなアポトーシスの発生を経時的に検出することができる。  [0123] As described above, according to the second embodiment, while culturing a living cell into which a fluorescent protein has been introduced for a long period, individual cells are traced without losing sight, and various cell parameters are measured over time. On the other hand, if apoptosis occurs while the cells are cultured for a longer period of time, the occurrence of such apoptosis can be detected over time.
[0124] (実施の形態 3)  [0124] (Embodiment 3)
本発明の実施の形態 3の細胞観察装置は、実施の形態 1と同様、蛍光タンパクを導 入した複数の生細胞を培養したまま撮像し、個々の細胞の領域を認識し、経時的な 位置変化を追尾しつつ、個々の細胞の特徴を示す細胞パラメータを独立に計測した 上で、さらに細胞の分裂を検出し、細胞の系譜を生成するものである。  The cell observation device according to the third embodiment of the present invention, as in the first embodiment, 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.
[0125] 図 17は、本実施の形態 3に係る細胞観察装置の構成例を示す概略ブロック図であ る。図中、本細胞観察装置を制御する制御部 301から培養系、撮像系各部への接続 は特に図示していない。本実施の形態 3は、実施の形態 1と共通の構成として、あら 力じめ蛍光タンパクで標識した複数の生細胞 Cを保持したスライドガラス 102、観察 窓 105、ステージ搬送機構 113等を含む培養部 101、撮像部 201 (励起光照明部 2 03、ダイクロイツクミラー 204、対物光学系 205、結像光学系 206、蛍光撮像部 207、 赤外光照明部 208、ダイクロイツクミラー 209、結像光学系 210、赤外光撮像部 211 等）、制御部 301、記録部 302、入力部 303、表示部 304、前処理部 305、細胞認識 部 306、パラメータ計測部 307、細胞追跡部 308、露出検出部 309、撮像回数計数 部 310、占有面積算出部 311、合焦検出部 312を備え、これらは実施の形態 1と同 様に作用する。さらに、本実施の形態 3の特有の構成として、細胞分裂検出手段とし ての細胞分裂検出部 316と細胞系譜作成手段としての系譜作成部 317とを備える。 FIG. 17 is a schematic block diagram showing a configuration example of the cell observation device according to the third embodiment. In the figure, 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. 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. 101, 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. Furthermore, as a unique configuration of the third 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.
[0126] 本実施の形態 3においても、実施の形態 1と同様に測定試料、すなわち標識を行つ た生細胞 Cの培養と撮像を経時的に行 ヽ、取得した蛍光画像と赤外光画像を記録 部 302に記録する。撮像した画像のうち、細胞蛍光画像を前処理部 305及び細胞認 識部 306で処理し、細胞領域を認識する。処理手順は実施の形態 1と同様である。 認識した細胞領域を記録部 302に記録する。パラメータ計測部 307において、実施 の形態 1で示した細胞領域の細胞パラメータを計測する。必要な細胞パラメータは、 後述する細胞分裂検出部 316の動作に応じて異なる。計測した細胞パラメータは記 録部 302に記録する。 [0126] Also in the third embodiment, as in the first embodiment, the measurement sample, that is, the live cell C with the label, is cultured and imaged over time, and the obtained fluorescence image and infrared light image are obtained. Is recorded in the recording unit 302. Among the captured images, 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.
[0127] 細胞追跡部 308にお 、て、異なる時刻の細胞蛍光画像力も抽出された細胞領域の 対応付けを行う。処理手順は実施の形態 1と同様である。細胞領域の対応付け情報 を、細胞画像、細胞領域、パラメータ計測部 307において計測した細胞パラメータ、 時刻情報と関連付けて記録部 302に記録する。  [0127] 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.
[0128] 時刻 tにおける異なる 2つの細胞領域 R , R 力 共に時刻 tの同一の領域 R  [0128] Two different cell regions R and R force at time t are the same region R at time t
2 t2,nl t2,n2 1 tl,m 2 t2, nl t2, n2 1 tl, m
0に対応付けられた場合、細胞分裂発生の可能性ありとみなし、細胞分裂検出部 316 により、後述の手順に従って細胞分裂の有無を判定する。 If it is associated with 0, it is considered that there is a possibility of cell division, and the cell division detection unit 316 determines the presence or absence of cell division according to the procedure described later.
[0129] 細胞分裂と判定された場合、通常の経時的な細胞の対応関係（以下、「経時的対 応関係」という）に加え、細胞の分裂前の 1つの親細胞と分裂後の 2つの娘細胞の親 子間の対応関係 (以下、「親子対応関係」という)も記録する。親子対応関係は、経時 的対応関係に追加情報として記録しても良いし、経時的対応関係とは別に記録して も良い。前者は記憶領域を節約することができ、後者は情報を参照する際の操作が 簡便になる。親子対応関係を記録する際のデータ表現は一例として、各世代を高さ に、各細胞領域を節点に対応させた木構造を用いる。  [0129] 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 The correspondence between the parent and child of the daughter cell (hereinafter referred to as “parent-child correspondence”) is also recorded. The 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. As an example of the data representation when recording the parent-child correspondence, a tree structure is used in which each generation corresponds to a height and each cell region corresponds to a node.
[0130] 細胞分裂検出部 316における細胞分裂判定には、以下に示すように複数の処理 手順が存在し、これらのうち 1つ以上の手順を用いて判定処理を行う。この処理は、 図 7に示したフローチャート中のステップ S 10の処理として実行される。 [0131] 図 18は、細胞分裂判定処理の第 1の処理手順を示す概略フローチャートである。 図 31に示す概略フローチャートは、細胞分裂後の娘細胞の面積が通常の細胞の面 積よりも小さぐかつ細胞分裂前後でそれぞれ対応する細胞の輝度の総和が概ね等 しくなると ヽぅ特性に基づ ヽた細胞分裂判定処理手順を例示する。 [0130] 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 ヽ ぅ characteristics. An example of the procedure for determining cell division.
[0132] まず、細胞領域 R , R の面積を所定の閾値 V , V とそれぞれ比較する (ステ  [0132] First, the areas of the cell regions R 1 and R 2 are respectively compared with predetermined threshold values V 1 and V 2 (step
t2,nl t2,n2 Al A2  t2, nl t2, n2 Al A2
ップ S31, S32)。細胞領域 R , R のうちの少なくとも一方の面積が閾値 V より  S31, S32). The area of at least one of the cell regions R and R is greater than the threshold V
t2,nl t2,n2 Al 小さい場合、或いは少なくとも一方の面積が閾値 V より大きい場合 (ステップ S31 ;  t2, nl t2, n2 Al is small or at least one area is larger than the threshold value V (step S31;
A2  A2
No,又はステップ S32 ;No)、細胞分裂ではないと判定する（ステップ S35)。一例と して、閾値 V は細胞領域の平均的な面積の 0.5倍、閾値 V は 0.9倍の値とする。  No, or step S32; No), it is determined that it is not cell division (step S35). As an example, the threshold V is 0.5 times the average area of the cell region, and the threshold V is 0.9 times.
Al A2  Al A2
[0133] さらに、細胞領域 R , R の輝度総和を合算したものと領域 R の輝度総和と  [0133] Furthermore, the sum of the luminance sums of the cell regions R 1 and R and the sum of the luminances of the region R
t2,nl t2,n2 tl，m0  t2, nl t2, n2 tl, m0
の差を求め、この差が所定の閾値 Vより大きい場合 (ステップ S33 ;No)、細胞分裂  If this difference is greater than the predetermined threshold V (Step S33; No), cell division
D  D
ではないと判定する (ステップ S35)。閾値 V以下の場合 (ステップ S33 ; Yes)、細胞  (Step S35). If threshold V or less (step S33; Yes), cells
D  D
分裂であると判定する (ステップ S34)。  It is determined that it is a split (step S34).
[0134] また、図 19は、細胞分裂判定処理の第 2の処理手順を示す概略フローチャートで ある。図 19に示す概略フローチャートは、細胞分裂直前の細胞が時間の経過と共に 収縮しながら略球体の形状となった後、細胞分裂を行うという特性に基づいた細胞分 裂判定処理手順を例示する。 [0134] 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.
[0135] まず、細胞領域 R の属する時刻 tのフレームから所定の N フレーム前まで遡り、 [0135] First, go back from the frame at time t to which the cell region R belongs to a predetermined N frames,
F1  F1
領 ただし: Sは細胞の面積、 Lは細胞の輪郭の長
Figure imgf000034_0001
Where: S is cell area, L is cell contour length
Figure imgf000034_0001
さ（周囲長）である。）を取得し (ステップ S41)、円形度 Cが所定の閾値 Vを超えるフ c レームが存在するか否かを求める（ステップ S42)。そのようなフレームが存在しない 場合 (ステップ S42 ;No)、細胞分裂ではな!/、と判定する (ステップ S46)。  (Perimeter length). ) 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).
[0136] 存在する場合 (ステップ S42 ; Yes)、領域 R の円形度が所定の閾値を超えた最 [0136] If it exists (step S42; Yes), the roundness of the region R exceeds the predetermined threshold.
tl,m0  tl, m0
初のフレーム力 さらに所定の N フレーム前まで遡り、領域 R の円形度及び面積  Initial frame force Go back N frames, and circularity and area of region R
F2 tl'mO  F2 tl'mO
の時間的推移を求め、前者が概略単調増加であると同時に後者が概略単調減少で あるという条件が成り立つか否かを求める（ステップ S43, S44)。この条件を判定す るためには、例えば回帰分析による円形度と面積の推移をそれぞれ直線近似し、円 形度の推移の近似直線の傾きが所定の正の値 V より大きぐかつ面積の推移の近 似直線の傾きが所定の負の値 V より小さいかどうかを見れば良い。 Then, it is determined whether or not the condition that the former is approximately monotonically increasing and the latter is approximately monotonically decreasing is satisfied (steps S43 and S44). In order to determine this condition, for example, 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.
T2  T2
[0137] 2つの条件が共に成り立つ場合 (ステップ S43 ;Yes、ステップ S44 ; Yes)、細胞分 裂であると判定する (ステップ S45)。成り立たない場合、細胞分裂ではないと判定す る（ステップ S46)。  [0137] 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).
[0138] また、図 20は、細胞分裂判定処理の第 3の処理手順を示す概略フローチャートで ある。図 20に示す概略フローチャートは、細胞分裂直前の細胞内では核膜が消失し 、細胞核内の構成要素が細胞質全体に拡散すると!ヽぅ特性に基づ ヽた細胞分裂判 定処理手順を例示する。  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! .
[0139] この処理手順では、あら力じめ細胞核と細胞質を互いに独立に領域抽出可能なよ うに標識しておく。このような標識には、一例として BDバイオサイエンス 'クロンテック 社がライセンスして 、る細胞内局在化ベクター「BDLivingColorsSubcellularLocalizati onVectorJを使用することができる。  [0139] In this processing procedure, cell nuclei and cytoplasm are preliminarily labeled so that regions can be extracted independently of each other. As an example of such a label, a subcellular localization vector “BDLivingColorsSubcellularLocalizationVectorJ” licensed by BD Biosciences, Clontech, can be used.
[0140] まず、細胞領域 R において、細胞核の存在範囲の面積と細胞質の存在範囲の tl,m0  [0140] First, in the cell region R, the area of the cell nucleus existing area and the cytoplasm existing area tl, m0
面積をそれぞれ求め、前者を後者で割った比を計算し、この比が所定の閾値 V 以  Each area is calculated and the ratio of the former divided by the latter is calculated.
R1 上で、所定の閾値 V 以下の場合 (ステップ S51； Yes)、細胞分裂であると判定する（ If it is below the predetermined threshold V on R1 (step S51; Yes), it is determined that the cell is dividing (step S51; Yes)
2  2
ステップ S52)。そうでな 、場合 (ステップ S51； No)、細胞分裂ではな!/、と判定する（ ステップ S53)。ただし、 V ≤V である。  Step S52). If not (step S51; No), it is determined that cell division is not! / (Step S53). However, V ≤ V.
Rl 2  Rl 2
[0141] また、図 21は、細胞分裂判定処理の第 4の処理手順を示す概略フローチャートで ある。図 21に示す概略フローチャートは、細胞分裂直前の細胞内では微小管が 2つ の紡錘体を形成するとともに、この紡錘体の領域以外には殆ど存在しないという特性 に基づ!/ヽた細胞分裂判定処理手順を例示する。  [0141] 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.
[0142] この処理手順では、あらかじめ細胞内の微小管を光学的に識別可能なように標識 しておく。このような標識には、一例として BDバイオサイエンス 'クロンテック社がライ センスして!/、る細胞内局在化ベクター「BD  [0142] In this processing procedure, 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! /
Livingし olors buocellular Localization VectorJ 使用すること力 21 (？さる。 Living olors buocellular Localization VectorJ Using power 21 (?
[0143] まず、細胞領域 R において、微小管の濃度分布を 2次元的又は 3次元的に可視 tl,m0 [0143] First, in the cell region R, the microtubule concentration distribution is visible two-dimensionally or three-dimensionally tl, m0
化した濃度分布マップを生成し (ステップ S61)、生成した濃度分布マップに空間ロー パスフィルタ処理を施し (ステップ S62)、フィルタ処理後の濃度分布マップ力も濃度 の極大点を検出し (ステップ S63)、検出された極大点が 2つである力否かを判断する (ステップ S64)。極大点が 2つである場合 (ステップ S64 ; Yes)、すなわち、領域内の 2個所に濃度が局在していれば、細胞分裂であると判定する (ステップ S65)。そうで な 、場合 (ステップ S64 ;No)、細胞分裂ではな!/、と判定する (ステップ S66)。 (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).
[0144] また、図 22は、細胞分裂判定処理の第 5の処理手順を示す概略フローチャートで ある。図 22に示す概略フローチャートは、細胞分裂前後において細胞の立体形状が 変化すると ヽぅ特性に基づ ヽた細胞分裂判定処理手順を例示する。  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.
[0145] まず、細胞領域 R において、輝度が所定の閾値 VLを超える画素のみを対象とし tl,m0  [0145] First, in the cell region R, only pixels whose luminance exceeds a predetermined threshold VL are targeted. Tl, m0
て輝度値を合計し、輝度総和 Sを求める (ステップ S71)。輝度総和 Sを、細胞領域 し し  The luminance values are summed to obtain the luminance sum S (step S71). Luminance sum S
R の属する時刻 tのフレーム力 所定の N フレーム前まで遡りながら求め、領域 tl,m0 1 F3  Frame force at time t to which R belongs. Calculated by going back to the previous N frames, area tl, m0 1 F3
R の輝度総和 Sの変化の様子を調べる（ステップ S72)。図 23は、輝度総和 Sの tl,m0 L L 時間的推移の一例を示す特性図である。輝度総和 Sが図 23に示すように急激に増 し  The state of change of the luminance sum S of R is examined (step S72). 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.
加する場合、細胞分裂であると判定し (ステップ S 74)、最も急激に輝度総和 Sが変 し 化した時刻を分裂時刻とする。ここで、所定の閾値 Vを用意し、輝度総和 Sが閾値 V  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. Here, a predetermined threshold V is prepared, and the luminance sum S is the threshold V.
J L  J L
を超えている範囲を分裂期と判定する (ステップ S73)と簡便に処理を実現できる。こ j  If it is determined that the range exceeding the period is the mitotic period (step S73), the processing can be realized easily. This j
の場合、閾値 Vを超える範囲が無ければ細胞分裂なし (ステップ S75)を意味する。  In the case of, if there is no range exceeding the threshold V, it means no cell division (step S75).
j  j
[0146] 分裂期（細胞周期における M期）以外では、細胞は、図 24— 1に示す平面図、図 2 4- 2に示す縦断正面図、図 24— 3に示す図 24— 2部分の輝度分布特性のように観 察される。しかし、分裂期（M期）においては、細胞の形状を維持するための微小管 が紡錘体の形成に転用されるため、細胞はそれまでの形状を維持できず、細胞膜の 表面張力等の影響で球形に近い形状となる。この状態では、細胞は、図 24— 4に示 す平面図、図 24— 5に示す縦断正面図、図 24— 6に示す図 24— 5部分の輝度分布 特性のように観察される。  [0146] Except for the mitotic phase (M phase in the cell cycle), 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. However, in 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. In this state, 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.
[0147] 閾値 Vを超える画素のみを用いて輝度総和 Sを求めた場合、細胞の体積や発現 し し  [0147] When the luminance sum S is obtained using only pixels that exceed the threshold V, the cell volume and expression
している蛍光タンパクの量が同じであっても、立体形状によって輝度総和 Sに関与す し る部位が変化する。本手法のように閾値 Vを超える部位のみを用いて輝度総和 Sを し し 求めた場合、分裂期 Mにおいて総和に関与する部位 Aは、分裂期 M以外において 総和に関与する部位 Bより大きくなる。したがって、輝度総和 Sは図 23に示したように 変化する。すなわち、輝度総和 Sが単峰性の推移を示している期間が、細胞周期に し Even if the amount of fluorescent protein is the same, the part involved in the luminance sum S varies depending on the three-dimensional shape. When 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.
おける M期に対応する。  Corresponds to M period.
以上の説明から明らかなように、本処理手順 5は細胞周期における M期の検出手 法としても有効である。  As is clear from the above explanation, this processing procedure 5 is also effective as a method for detecting the M phase in the cell cycle.
[0148] 細胞分裂を判定する際には、上述の第 1の処理手順力 第 5の処理手順までのい ずれを用いても良い。さらに、 2つ以上の処理手順を実施して判定結果を組み合わ せても良ぐその場合、さらに精度の高い判定が可能となる。  [0148] In determining cell division, 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.
[0149] 以上の処理手順により、細胞分裂の有無を判定すると共に、細胞の親子関係を知 ることができる。細胞の親子関係を複数の時刻において判定し記録部 302に蓄積す ることで、個々の細胞の世代間の繋がり、すなわち細胞系譜を得ることができる。この 細胞系譜は、系譜作成部 317が細胞分裂検出部 316の検出結果に基づいて、分裂 前後の細胞を関連付けることによって生成する。ここでは、細胞の親子関係情報が 2 世代以上に渡って繋がったものを「系譜」と呼称し、 2世代間の親子関係情報は最小 の系譜である。系譜作成部 317によって生成された系譜情報は、記録部 302で保持 される。  [0149] With the above processing procedure, the presence or absence of cell division can be determined, and the parent-child relationship of cells can be known. By determining the parent-child relationship of cells at a plurality of times and accumulating them in the recording unit 302, it is possible to obtain connections between generations of individual cells, that is, a cell lineage. 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. Here, 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.
[0150] 最後に、細胞系譜表示手段としての表示部 304にて、認識した細胞領域 (細胞領 域）と系譜作成部 317により作成された細胞系譜を表示する。図 25は、時刻 t〜tに  [0150] Finally, the display unit 304 as a cell lineage display means displays the recognized cell region (cell region) and the cell lineage created by the lineage creation unit 317. Figure 25 shows the
1 3 ついての観察結果の表示の一例を示す説明図である。図 25に示すように、表示部 3 04が備える表示装置の画面 314は、 4つの表示領域 314c〜314fに区画され、表示 領域 314c〜314eのそれぞれには、検出対象時点である時点 tを含めた 3つの時点  It is explanatory drawing which shows an example of a display of the observation result about 1 3. As shown in FIG. 25, 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
3  Three
t , t , tの各細胞領域を示す画像情報が表示される。表示領域 314fには、各時点 Image information indicating each cell region of t, t, and t is displayed. In the display area 314f, each time point
1 2 3 one two Three
の各細胞領域の対応関係が木構造 (ツリー構造)で表示され、細胞分裂によって生じ た細胞の系譜情報が同時に表示される。  The correspondence of 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.
[0151] なお、画面 314上で、全ての情報を一度に表示できない場合は、操作者の指示や 表示部 304の能力に応じて表示する内容を選択する。ここで、細胞領域にはラベリン グ処理を適用し、領域毎に識別可能な色、輝度、線種、パターンを与え、ラベル画像 として表示する。ラベル画像の表示に代えて、同じ表示範囲の細胞赤外光画像或い は蛍光画像を表示しても、三者を切り替えられるようにしても良い。或いは、三者のう ち複数を連動して表示させても良いし、重ね合わせて表示しても良い。図 38に示す ように、ラベル画像の形状を実際の細胞の形状と対応させれば、さらに視認性が向 上する。 [0151] If not all information can be displayed on the screen 314 at one time, the contents to be displayed are selected according to the operator's instruction and the capability of the display unit 304. Here, 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. Instead of displaying the 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. Or 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.
[0152] 系譜情報は、時間を深さ方向とした木構造として表示し、木構造の節点はある時刻 での細胞領域に対応する。ラベル画像の色、輝度、輪郭線の属性、パターンのうち 少なくとも 1つを対応する細胞領域の表示と一致させ、両者の対応を視覚的に明確 化する。図 38は、両者のパターンを対応させた表示例を示している。  [0152] The genealogy information is displayed as a tree structure with time as the depth direction, and the nodes of the tree structure correspond to the cell region at a certain time. At least one of the color, brightness, contour attribute, and pattern of the label image is matched with the display of the corresponding cell region, and the correspondence between the two is visually clarified. FIG. 38 shows a display example in which both patterns are associated with each other.
[0153] マウス等の入力部 303を用いた操作者の指示により、両者の表示内容が同期して 強調表示されるようにすれば、表示内容の確認がより簡便となる。この場合、操作者 力 Sラベル画像における細胞領域又は木構造の接点のどちらか片方を選択した場合、 選択された対象を強調表示すると共に、他方の対応する表示物も強調表示する。さ らに、選択された細胞に対する系譜上の祖先 (親を含む)や子孫 (子を含む)も併せ て強調すれば、細胞の系譜を視覚的に把握することが容易となる。  [0153] If 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. In this case, when one of the cell area or the tree-structured contact in the operator force S label image is selected, the selected object is highlighted and the other corresponding display object is also highlighted. Furthermore, if the ancestors (including parents) and descendants (including children) of the selected cell are also emphasized, it becomes easier to visually grasp the cell lineage.
[0154] 図 26は、操作者が時刻 tにおける細胞領域 321を選択した例であり、選択された FIG. 26 shows an example in which the operator selects cell region 321 at time t.
2  2
領域 321の輪郭線幅を太くすることで強調表示すると共に、系譜上の祖先に当たる 時刻 tの細胞領域 322、系譜上の子孫に当たる時刻 tの細胞領域 323、及び系譜 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
1 3 13
表示において対応する節点と節点間の接続線が同期して強調される様子を表してい る。  In the display, the corresponding nodes and the connection lines between the nodes are highlighted in synchronization.
[0155] 以上、本実施の形態 3によれば、蛍光物質によって標識された複数の生細胞を経 時的に撮像し、個々の細胞を認識し、経時的な位置変化を追尾しつつ、細胞の分裂 を検出し、細胞の系譜情報を獲得することができる。すなわち、長期に細胞を培養し ていくうちに細胞分裂が発生すれば、これを検出することができ、また、長期に亘つて 細胞を培養していくうちに細胞分裂が発生しても、これを検出することで、細胞観察 の精度が向上する。特に、上述の第 1〜第 5の処理手順によれば、細胞分裂を精度 よく発見することができる。  [0155] As described above, according to the third embodiment, 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. In other words, if 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. By detecting, the accuracy of cell observation is improved. In particular, according to the first to fifth processing procedures described above, cell division can be detected with high accuracy.
[0156] (実施の形態 4)  [Embodiment 4]
本発明の実施の形態 4は、実施の形態 1〜3の機能を組み合わせた顕微鏡システ ムへの適用例を示す。本顕微鏡システムは、実施の形態 1と同様に、蛍光タンパクを 導入した複数の生細胞を培養したまま撮像し、個々の細胞の領域を認識し、経時的 な位置変化を追尾しつつ、個々の細胞の特徴を示す細胞パラメータを独立に計測し た上で、実施の形態 2と同様のアポトーシス検査、又は実施の形態 3と同様の細胞分 裂検出を行う。 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. As in the first embodiment, 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.
[0157] 図 27は、本実施の形態 4に係る顕微鏡システムの構成例を示す概略ブロック図で ある。図中、本装置を制御する制御部 301から培養系、撮像系各部への接続は特に 図示していない。実施の形態 4は、実施の形態 1と共通の構成として、あらカゝじめ蛍光 タンパクで標識した複数の生細胞 Cを保持したスライドガラス 102、観察窓 105、ステ ージ搬送機構 113等を含む培養部 101、撮像部 201 (対物光学系 205、結像光学 系 206、蛍光撮像部 207、赤外光照明部 208、ダイクロイツクミラー 209、結像光学系 210、赤外光撮像部 211等）、制御部 301、記録部 302、入力部 303、表示部 304、 前処理部 305、細胞認識部 306、パラメータ計測部 307、細胞追跡部 308、露出検 出部 309、撮像回数計数部 310、占有面積算出部 311、合焦検出部 312を備え、こ れらは実施の形態 1と同様に作用する。  FIG. 27 is a schematic block diagram showing a configuration example of the microscope system according to the fourth embodiment. In the figure, connections from the control unit 301 for controlling the apparatus to each part of the culture system and the imaging system are not shown. In 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. Incubation 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 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.
[0158] さらに、実施の形態 2と共通の構成として、励起光照明部 203'、ダイクロイツクミラー 204'、アポトーシス検出部 315を備え、これは実施の形態 2と同様に作用する。励起 光照明部 203'とダイクロイツクミラー 204'は実施の形態 1における照明部 203とダイ クロイツクミラー 204の機能を含んでいるため、アポトーシス検出を行わない場合にも 使用できる。アポトーシス検出機能を使用しない代わりに、実施の形態 1と同様の照 明部 203とダイクロイツクミラー 204を搭載しても良い。さらに、実施の形態 3と共通の 構成として、細胞分裂検出部 316及び系譜策西部 317を備え、これは実施の形態 3 と同様に作用する。  Furthermore, as a configuration common to the second 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.
[0159] 上記構成のうち、本実施の形態 4では、少なくとも、培養部 101、励起光照明部 20 3'、ダイクロイツクミラー 204'、対物光学系 205、結像光学系 206、蛍光撮像部 207、 赤外光照明部 208、ダイクロイツクミラー 209、結像光学系 210、赤外光撮像部 211 は顕微鏡 401の一部をなすように構成されて ヽるが、これ以外の機能を顕微鏡 401 に含めても良い。  [0159] Among the above configurations, in the fourth embodiment, at least the culture unit 101, the excitation light illumination unit 203 ', the dichroic mirror 204', the objective optical system 205, the imaging optical system 206, and the fluorescence imaging unit 207 The infrared illumination unit 208, the dichroic mirror 209, the imaging optical system 210, and the infrared light imaging unit 211 may be configured to be a part of the microscope 401, but the microscope 401 has other functions. May be included.
[0160] 本実施の形態 4も、実施の形態 1と同様に測定試料、すなわち標識を行った生細胞 Cの培養と撮像を経時的に行 ヽ、取得した蛍光画像と赤外光画像を記録部 302に記 録する。撮像した蛍光画像を前処理部 305及び細胞認識部 306で処理し、細胞領 域を認識する。処理手順は実施の形態 1と同様である。認識した細胞領域を記録部 302に記録する。アポトーシス検出を行う場合は、実施の形態 2と同様に細胞膜蛍光 画像を撮像し、これに基づいて細胞認識を行う。 [0160] In the fourth embodiment, as in the first 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. When apoptosis is detected, a cell membrane fluorescence image is taken as in the second embodiment, and cell recognition is performed based on this.
[0161] パラメータ計測部 307にお 、て、実施の形態 1で示した細胞領域の細胞パラメータ を計測する。必要な細胞パラメータは入力部 303からの指示によって変更できる。ァ ポトーシス検出又は細胞分裂検出を行う場合は、それぞれの処理に必要な細胞パラ メータを計測する。これらは実施の形態 2又は 3で述べた通りである。計測した細胞パ ラメータは記録部 302に記録する。  [0161] 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.
[0162] 細胞追跡部 308にお 、て、異なる時刻の細胞画像力も抽出された細胞領域の対応 付けを行う。処理手順は実施の形態 1と同様である。細胞領域の対応付け情報を、細 胞画像、細胞領域、パラメータ計測部 307において計測した細胞パラメータ、時刻情 報と関連付けて記録部 302に記録する。  [0162] 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.
[0163] さらに、アポトーシス検出部 315において、細胞のアポトーシスを検出する。アポト 一シス検出を行うかどうかは、少なくとも入力部 303からの指示によって選択でき、検 出結果を記録部 302に記録する。また、検出結果を表示部 304に表示しても良い。 さらに、細胞分裂検出部 316において、細胞分裂を検出する。細胞分裂検出を行う 力どうかは、少なくとも入力部 303からの指示によって選択でき、検出結果を記録部 3 02に記録する。また、検出結果を表示部 304に表示しても良い。したがって、本実施 の形態の入力部 303は、検出機能選択手段としても機能する。  [0163] Furthermore, 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.
[0164] なお、図 27ではアポトーシス検出部 315と細胞分裂検出部 316との両方を搭載し た構成を例示しているが、少なくとも一方を省略した構成であっても良ぐその場合、 処理速度の向上とコスト低減の効果が得られる。また、アポトーシス検出部 315と細 胞分裂検出部 316との少なくとも一方を搭載した構成において、入力部 303には公 開されな!、指示方法によって、機能を使用禁止にしてお!、ても良!、。  [0164] Although 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. In addition, in a configuration in which at least one of the apoptosis detection unit 315 and the cell division detection unit 316 is mounted, the input unit 303 is not open to the public! !
[0165] 以上、本実施の形態 4によれば、蛍光物質によって標識された複数の生細胞を経 時的に撮像し、個々の細胞を認識し、経時的な位置変化を追尾しつつ、細胞のアポ トーシス或いは細胞分裂を検出し、細胞の系譜情報を獲得する機能を、顕微鏡 401 をベースとした比較的安価で信頼性の高い装置として実現できる。 [0165] As described above, according to the fourth embodiment, 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. Apo 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.
[0166] 本発明は、上述した実施の形態に限らず、本発明の趣旨を逸脱しない範囲であれ ば、種々の変形が可能である。例えば、前述の細胞認識部 306、パラメータ計測部 3 07、細胞追跡部 308、アポトーシス検出部 315、細胞分裂検出部 316等の各部によ る処理手順は、あら力じめ用意された細胞観察プログラムを制御部 301などのマイク 口コンピュータで実行することにより実現するようにしてもよい。この細胞観察プロダラ ムは、インターネットなどのネットワークを介して配布することもできる。また、この細胞 観察プログラムは、ハードディスク、 FD、 CD-ROM, MO、 DVDなどのマイクロコン ピュータで読み取り可能な記録媒体に記録され、マイクロコンピュータによって記録 媒体から読み出されること〖こより実行することちできる。 [0166] The present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the spirit of the present invention. For example, 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. In addition, 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. .
産業上の利用可能性  Industrial applicability
[0167] 以上のように、本発明に係る細胞観察装置、細胞観察方法、顕微鏡システム、及び 細胞観察プログラムは、生細胞の特徴を示す細胞パラメータの経時的な計測に有用 であり、特に、培養中の細胞の観察に適している。 [0167] As described above, 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.

Claims

請求の範囲 The scope of the claims
[1] 細胞の特徴を示す細胞パラメータを経時的に計測する細胞観察装置であって、 赤外光線によって細胞を撮像する赤外光撮像手段を少なくとも含み細胞を撮像す る撮像手段と、  [1] A cell observation device that measures cell parameters indicating the characteristics of a cell over time, including at least an infrared light imaging unit that images cells with infrared rays, and an imaging unit that images cells.
該撮像手段で撮像した画像の細胞画像データから細胞を認識する細胞認識手段 と、  Cell recognition means for recognizing cells from cell image data of an image taken by the imaging means;
該細胞認識手段で認識した細胞の特徴を示す細胞パラメータを前記細胞画像デ ータに基づき計測する細胞パラメータ計測手段と、  Cell parameter measuring means for measuring cell parameters indicating the characteristics of the cells recognized by the cell recognition means based on the cell image data;
異なる時点で撮像された画像の前記細胞画像データから認識された異なる時点の 細胞同士の同一性を前記細胞パラメータに基づいて判別する細胞追跡手段と、 を備えることを特徴とする細胞観察装置。  A cell observation device comprising: cell tracking means for discriminating the identity of cells at different time points recognized from the cell image data of images taken at different time points based on the cell parameters.
[2] 前記撮像手段は、前記赤外光撮像手段によって前記細胞画像データを取得する ことを特徴とする請求項 1に記載の細胞観察装置。  2. The cell observation device according to claim 1, wherein the imaging unit acquires the cell image data by the infrared light imaging unit.
[3] 前記撮像手段は、細胞から発せられる光を撮像する細胞光撮像手段をさらに有し、 該細胞光撮像手段によって前記細胞画像データを取得することを特徴とする請求項[3] The imaging means further includes cell light imaging means for imaging light emitted from a cell, and the cell image data is acquired by the cell light imaging means.
1に記載の細胞観察装置。 The cell observation apparatus according to 1.
[4] 所定の時点における前記細胞画像データを取得したことを認識する撮像時点認識 手段と、 [4] An imaging time point recognition means for recognizing that the cell image data has been acquired at a predetermined time point;
該撮像時点認識手段で認識した結果を操作者に報知するための報知手段と、 をさらに備えることを特徴とする請求項 1〜3のいずれか 1つに記載の細胞観察装 置。  The cell observation device according to any one of claims 1 to 3, further comprising: notification means for notifying an operator of a result recognized by the imaging time point recognition means.
[5] 細胞を撮像した画像の中で細胞が占める度合 ヽを示す細胞占有値を算出する占 有面積算出手段と、  [5] An occupied area calculation means for calculating a cell occupation value indicating the degree of the cell occupies in the image of the cell,
前記細胞占有値が所定の値になったときに、該細胞占有値が該所定の値になった ことを操作者に知らせる報知手段と、  Informing means for notifying an operator that the cell occupancy value has reached the predetermined value when the cell occupancy value has reached a predetermined value;
をさらに備えることを特徴とする請求項 1〜3のいずれか 1つに記載の細胞観察装 置。  The cell observation device according to any one of claims 1 to 3, further comprising:
[6] 前記撮像手段は、固体撮像素子を備え、 細胞を収容する筐体を備えた細胞培養手段と、 [6] The imaging means includes a solid-state imaging device, A cell culture means comprising a housing for containing cells;
前記筐体と前記固体撮像素子との断熱を行う断熱手段と、をさらに備えることを特 徴とする請求項 1〜5のいずれ力 1つに記載の細胞観察装置。  6. The cell observation apparatus according to claim 1, further comprising a heat insulating means for performing heat insulation between the housing and the solid-state imaging device.
[7] 前記細胞パラメータ計測手段で計測した前記細胞パラメータのうち前記細胞追跡 手段で同一性があると判別した細胞の前記細胞パラメータ同士を関連付けて記録す る記録手段をさらに備えることを特徴とする請求項 1〜6のいずれか 1つに記載の細 胞観察装置。 [7] The apparatus further comprises a recording unit that records the cell parameters of the cells determined to be identical by the cell tracking unit among the cell parameters measured by the cell parameter measuring unit in association with each other. The cell observation device according to any one of claims 1 to 6.
[8] 前記細胞認識手段で認識した細胞を表す画像と複数時点の細胞につ!、ての前記 細胞パラメータとを関連付けて表示する細胞パラメータ表示手段をさらに備えることを 特徴とする請求項 1〜7のいずれか 1つに記載の細胞観察装置。  [8] The apparatus further comprises cell parameter display means for displaying an image representing a cell recognized by the cell recognition means and cells at a plurality of time points in association with the cell parameters. 8. The cell observation device according to any one of 7.
[9] 細胞のアポトーシスを前記細胞パラメータに基づいて検出するアポトーシス検出手 段をさらに備えることを特徴とする請求項 1〜8のいずれか 1つに記載の細胞観察装 置。  [9] The cell observation device according to any one of [1] to [8], further comprising an apoptosis detection unit that detects apoptosis of cells based on the cell parameters.
[10] 前記細胞認識手段で認識した細胞が細胞分裂の結果生じたものである力否かを前 記細胞パラメータに基づいて検出する細胞分裂検出手段をさらに備えることを特徴と する請求項 1〜9のいずれ力 1つに記載の細胞観察装置。  [10] The method of claim 1, further comprising cell division detection means for detecting whether or not the cell recognized by the cell recognition means is a force generated as a result of cell division based on the cell parameter. 9. The cell observation device according to any one of 9 above.
[11] 細胞のアポトーシスを前記細胞パラメータに基づいて検出するアポトーシス検出手 段と、  [11] an apoptosis detection means for detecting cell apoptosis based on the cell parameters;
前記細胞認識手段で認識した細胞が細胞分裂の結果生じたものである力否かを前 記細胞パラメータに基づいて検出するための細胞分裂検出手段と、  Cell division detection means for detecting whether or not the cell recognized by the cell recognition means is a force generated as a result of cell division based on the cell parameters;
前記アポトーシス検出手段による検出を行うか否かと、前記細胞分裂検出手段によ る検出を行うか否かを、それぞれ選択する検出機能選択手段と、  Detection function selection means for selecting whether to perform detection by the apoptosis detection means and whether to perform detection by the cell division detection means;
をさらに備えることを特徴とする請求項 1〜8のいずれか 1つに記載の細胞観察装 置。  The cell observation device according to any one of claims 1 to 8, further comprising:
[12] 前記細胞分裂検出手段は、検出対象時点における細胞の面積を示す細胞パラメ ータと面積に関する閾値とを比較するとともに、該検出対象時点における細胞の輝度 を示す細胞パラメータと該検出対象時点に対して比較の対象となる時点における細 胞の輝度を示す細胞パラメータとを比較し、それぞれの比較結果に基づ ヽて該検出 対象時点における細胞が細胞分裂の結果生じたものである力否かを検出することを 特徴とする請求項 10又は 11に記載の細胞観察装置。 [12] The cell division detection means compares the 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 luminance of the cell at the detection target time point and the detection target time point. Cell parameters indicating the brightness of the cells at the time of comparison, and the detection based on the respective comparison results. 12. The cell observation device according to claim 10 or 11, wherein it is detected whether or not the force at a target time point is a result of cell division.
[13] 前記細胞分裂検出手段は、検出対象時点よりも時系列的に前の時点における細 胞の円形度を示す細胞パラメータの値を参照し、該検出対象時点における細胞が細 胞分裂の結果生じたものであるか否かを検出することを特徴とする請求項 10又は 11 に記載の細胞観察装置。  [13] The cell division detection means 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, and the cell at the detection target time point is a result of cell division. 12. The cell observation device according to claim 10, wherein it is detected whether or not it has occurred.
[14] 前記細胞分裂検出手段は、検出対象時点よりも時系列的に前の時点における細 胞の円形度を示す細胞パラメータの値を参照し、該円形度を示す細胞パラメータの 値が円形度に関する閾値と所定の関係を有している細胞を撮像した判定基準時点 を抽出し、該判定基準時点近傍の時点における細胞の円形度を示す細胞パラメータ 及び面積を示す細胞パラメータの値の増減傾向に基づ!/、て、該検出対象時点にお ける細胞が細胞分裂の結果生じたものである力否かを検出することを特徴とする請求 項 10又は 11に記載の細胞観察装置。  [14] 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 value of the cell parameter indicating the circularity is the circularity The reference time point at which a cell having a predetermined relationship with the threshold value is imaged is extracted, and the cell parameter indicating the circularity of the cell and the cell parameter value indicating the area tend to increase or decrease at a time point in the vicinity of the determination reference time point. 12. The cell observation device according to claim 10, wherein the cell observation device detects whether the cell at the detection target time point is a force generated as a result of cell division.
[15] 前記細胞分裂検出手段は、検出対象時点における細胞の細胞核の存在範囲の面 積と細胞質の存在範囲の面積とを比較した結果に基づいて、該検出対象時点にお ける細胞が細胞分裂の結果生じたものである力否かを検出することを特徴とする請求 項 10又は 11に記載の細胞観察装置。  [15] The cell division detection means determines whether the cell at the detection target time point is divided based on the result of comparing the area of the cell nucleus existence range and the area of the cytoplasm presence range at the detection target time point. The cell observation device according to claim 10 or 11, which detects whether or not the force is a result of the above.
[16] 前記細胞分裂検出手段は、検出対象時点における細胞の微小管の局在領域を求 め、細胞中の複数個所に局在しているか否かを検出することで、当該検出対象時点 における細胞が細胞分裂の結果生じたものである力否かを検出することを特徴とする 請求項 10又は 11に記載の細胞観察装置。  [16] The cell division detection means obtains a localized region of the microtubule of the cell at the detection target time point and detects whether it is localized at a plurality of locations in the cell. The cell observation apparatus according to claim 10 or 11, wherein it is detected whether or not a cell is a force resulting from cell division.
[17] 前記細胞分裂検出手段は、撮像された画像の細胞に対応する領域内で輝度に関 する閾値よりも高い輝度値を示す画素について各画素の輝度値を合計した輝度総 和を示す領域パラメータを、検出対象時点近傍の時点について求め、該検出対象時 点近傍の時点にっ 、ての前記輝度総和の変化の様子に基づ 、て、該検出対象時 点における細胞が細胞分裂の結果生じたものである力否かを検出することを特徴と する請求項 10又は 11に記載の細胞観察装置。  [17] The cell division detection means is an area that indicates a luminance sum obtained by summing the luminance values of the pixels with respect to a pixel that indicates a luminance value higher than a threshold value relating to luminance in an area corresponding to the cell of the captured image. The parameter is obtained for a time point near the detection target time point, and the cell at the detection target time point is the result of cell division based on the change in the luminance summation at the time point near the detection target time point. 12. The cell observation device according to claim 10 or 11, wherein it is detected whether or not a force is generated.
[18] 前記細胞分裂検出手段の検出結果に基づいて細胞系譜を作成する細胞系譜作 成手段をさらに備えることを特徴とする請求項 10〜 17の 、ずれか 1つに記載の細胞 観察装置。 [18] A cell lineage generating a cell lineage based on the detection result of the cell division detection means The cell observation device according to claim 10, further comprising a generating unit.
[19] 前記細胞系譜作成手段で作成した細胞系譜を木構造で表した画像として表示する 細胞系譜表示手段をさらに備えることを特徴とする請求項 18に記載の細胞観察装置  19. The cell observation device according to claim 18, further comprising a cell lineage display unit that displays the cell lineage created by the cell lineage creation unit as an image represented by a tree structure.
[20] 前記細胞系譜表示手段は、前記細胞認識手段で認識した細胞を表す画像と前記 細胞系譜を表す画像とを関連付けて表示することを特徴とする請求項 19に記載の 細胞観察装置。 20. The cell observation device according to claim 19, wherein the cell lineage display unit displays an image representing a cell recognized by the cell recognition unit and an image representing the cell lineage in association with each other.
[21] 請求項 1〜20のいずれか 1つに記載の細胞観察装置を備える顕微鏡システムであ つて、  [21] A microscope system comprising the cell observation device according to any one of claims 1 to 20,
細胞を拡大投影する結像光学系を備え、  An imaging optical system that magnifies and projects cells,
前記撮像手段は、前記結像光学系の結像面に拡大投影された細胞を撮像すること を特徴とする顕微鏡システム。  The microscope system characterized in that the imaging means images a cell that is enlarged and projected onto an imaging surface of the imaging optical system.
[22] 細胞の特徴を示す細胞パラメータを経時的に計測する細胞観察装置であって、 細胞を撮像した画像の細胞画像データから細胞を認識する細胞認識手段と、 該細胞認識手段で認識した細胞の特徴を示す細胞パラメータを前記細胞画像デ ータに基づき計測する細胞パラメータ計測手段と、 [22] A cell observation device that measures cell parameters indicating the characteristics of a cell over time, a cell recognition unit that recognizes a cell from cell image data of an image of the cell, and a cell that is recognized by the cell recognition unit Cell parameter measuring means for measuring cell parameters indicating the characteristics of the cell based on the cell image data;
前記細胞認識手段で認識した細胞が細胞分裂の結果生じたものである力否かを前 記細胞パラメータに基づいて検出する細胞分裂検出手段と、  Cell division detection means for detecting whether the cell recognized by the cell recognition means is a force generated as a result of cell division based on the cell parameter;
を備え、  With
前記細胞分裂検出手段は、検出対象時点における細胞の面積を示す細胞パラメ ータと面積に関する閾値とを比較するとともに、該検出対象時点における細胞の輝度 を示す細胞パラメータと該検出対象時点に対して比較の対象となる時点における細 胞の輝度を示す細胞パラメータとを比較し、それぞれの比較結果に基づ ヽて該検出 対象時点における細胞が細胞分裂の結果生じたものである力否かを検出することを 特徴とする細胞観察装置。  The cell division detection means compares the cell parameter indicating the area of the cell at the detection target time point with a threshold relating to the area, and compares the cell parameter indicating the luminance of the cell at the detection target time point and the detection target time point. Comparing the cell parameters that indicate the brightness of the cells at the time points to be compared, and detecting whether the cells at the time points to be detected are the result of cell division based on the comparison results. A cell observation apparatus characterized by:
[23] 細胞の特徴を示す細胞パラメータを経時的に計測する細胞観察装置であって、 細胞を撮像した画像の細胞画像データから細胞を認識する細胞認識手段と、 該細胞認識手段で認識した細胞の特徴を示す細胞パラメータを前記細胞画像デ ータに基づき計測する細胞パラメータ計測手段と、 [23] A cell observation device for measuring a cell parameter indicating characteristics of a cell over time, a cell recognition means for recognizing a cell from cell image data of an image of the cell, Cell parameter measuring means for measuring cell parameters indicating the characteristics of the cells recognized by the cell recognition means based on the cell image data;
前記細胞認識手段で認識した細胞が細胞分裂の結果生じたものである力否かを前 記細胞パラメータに基づいて検出する細胞分裂検出手段と、  Cell division detection means for detecting whether the cell recognized by the cell recognition means is a force generated as a result of cell division based on the cell parameter;
を備え、  With
前記細胞分裂検出手段は、検出対象時点よりも時系列的に前の時点における細 胞の円形度を示す細胞パラメータの値を参照し、該検出対象時点における細胞が細 胞分裂の結果生じたものであるか否かを検出することを特徴とする細胞観察装置。  The cell division detection means refers to a value of a cell parameter indicating the circularity of the cell at a time point chronologically before the detection target time point, and the cell at the detection target time point is a result of cell division. A cell observation apparatus for detecting whether or not
[24] 細胞の特徴を示す細胞パラメータを経時的に計測する細胞観察装置であって、 細胞を撮像した画像の細胞画像データから細胞を認識する細胞認識手段と、 該細胞認識手段で認識した細胞の特徴を示す細胞パラメータを前記細胞画像デ ータに基づき計測する細胞パラメータ計測手段と、  [24] A cell observation device for measuring cell parameters indicating the characteristics of a cell over time, a cell recognition means for recognizing a cell from cell image data of an image of the cell, 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;
前記細胞認識手段で認識した細胞が細胞分裂の結果生じたものである力否かを前 記細胞パラメータに基づいて検出する細胞分裂検出手段と、  Cell division detection means for detecting whether the cell recognized by the cell recognition means is a force generated as a result of cell division based on the cell parameter;
を備え、  With
前記細胞分裂検出手段は、検出対象時点よりも時系列的に前の時点における細 胞の円形度を示す細胞パラメータの値を参照し、該円形度を示す細胞パラメータの 値が円形度に関する閾値と所定の関係を有している細胞を撮像した判定基準時点 を抽出し、該判定基準時点近傍の時点における細胞の円形度を示す細胞パラメータ 及び面積を示す細胞パラメータの値の増減傾向に基づ!/、て、該検出対象時点にお ける細胞が細胞分裂の結果生じたものである力否かを検出することを特徴とする細胞 観察装置。  The cell division detection means refers to a cell parameter value indicating the circularity of the cell at a time point chronologically prior to the detection target time point, and the cell parameter value indicating the circularity is a threshold value related to the circularity. Based on the increasing / decreasing tendency of the cell parameter indicating the circularity of the cell and the cell parameter indicating the area at a time in the vicinity of the determination reference time point by extracting the determination reference time point at which the cells having the predetermined relationship are imaged! A cell observation device that detects whether or not the cell at the detection target time point is a result of cell division.
[25] 細胞の特徴を示す細胞パラメータを経時的に計測する細胞観察装置であって、 細胞を撮像した画像の細胞画像データから細胞を認識する細胞認識手段と、 該細胞認識手段で認識した細胞の特徴を示す細胞パラメータを前記細胞画像デ ータに基づき計測する細胞パラメータ計測手段と、  [25] A cell observation device for measuring cell parameters indicating the characteristics of a cell over time, a cell recognition means for recognizing a cell from cell image data of an image of the cell, 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;
前記細胞認識手段で認識した細胞が細胞分裂の結果生じたものである力否かを前 記細胞パラメータに基づいて検出する細胞分裂検出手段と、 を備え、 Cell division detection means for detecting whether the cell recognized by the cell recognition means is a force generated as a result of cell division based on the cell parameter; With
前記細胞分裂検出手段は、検出対象時点における細胞の細胞核の存在範囲の面 積と細胞質の存在範囲の面積とを比較した結果に基づいて、該検出対象時点にお ける細胞が細胞分裂の結果生じたものである力否かを検出することを特徴とする細胞 観察装置。  The cell division detection means generates a cell at the detection target time point as a result of cell division based on the result of comparing the area of the cell nucleus existence area and the area of the cytoplasm presence range at the detection target time point. A cell observation device characterized by detecting whether or not the force is a natural force.
[26] 細胞の特徴を示す細胞パラメータを経時的に計測する細胞観察装置であって、 細胞を撮像した画像の細胞画像データから細胞を認識する細胞認識手段と、 該細胞認識手段で認識した細胞の特徴を示す細胞パラメータを前記細胞画像デ ータに基づき計測する細胞パラメータ計測手段と、  [26] A cell observation device for measuring cell parameters indicating the characteristics of a cell over time, a cell recognition means for recognizing a cell from cell image data of an image of the cell, 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;
前記細胞認識手段で認識した細胞が細胞分裂の結果生じたものである力否かを前 記細胞パラメータに基づいて検出する細胞分裂検出手段と、  Cell division detection means for detecting whether the cell recognized by the cell recognition means is a force generated as a result of cell division based on the cell parameter;
を備え、  With
前記細胞分裂検出手段は、検出対象時点における細胞の微小管の局在領域を求 め、細胞中の複数個所に局在しているか否かを検出することで、該検出対象時点に おける細胞が細胞分裂の結果生じたものである力否かを検出することを特徴とする細 胞観察装置。  The cell division detection means obtains a localized region of the microtubule of the cell at the detection target time point and detects whether or not the cell at the detection target time point is localized at a plurality of locations in the cell. A cell observation device for detecting whether or not a force is a result of cell division.
[27] 細胞の特徴を示す細胞パラメータを経時的に計測する細胞観察装置であって、 細胞を撮像した画像の細胞画像データから細胞を認識する細胞認識手段と、 該細胞認識手段で認識した細胞の特徴を示す細胞パラメータを前記細胞画像デ ータに基づき計測する細胞パラメータ計測手段と、  [27] A cell observation device for measuring cell parameters indicating the characteristics of a cell over time, a cell recognition unit for recognizing a cell from cell image data of an image of the cell, and a cell recognized by the cell recognition unit Cell parameter measuring means for measuring cell parameters indicating the characteristics of the cell based on the cell image data;
前記細胞認識手段で認識した細胞が細胞分裂の結果生じたものである力否かを前 記細胞パラメータに基づいて検出する細胞分裂検出手段と、  Cell division detection means for detecting whether the cell recognized by the cell recognition means is a force generated as a result of cell division based on the cell parameter;
を備え、  With
前記細胞分裂検出手段は、撮像された画像の細胞に対応する領域内で輝度に関 する閾値よりも高い輝度値を示す画素について各画素の輝度値を合計した輝度総 和を示す領域パラメータを、検出対象時点近傍の時点について求め、該検出対象時 点近傍の時点にっ 、ての前記輝度総和の変化の様子に基づ 、て、該検出対象時 点における細胞が細胞分裂の結果生じたものである力否かを検出することを特徴と する細胞観察装置。 The cell division detection means includes a region parameter indicating a luminance sum obtained by summing the luminance values of each pixel for a pixel having a luminance value higher than a threshold value related to luminance in a region corresponding to the cell of the captured image. A time point in the vicinity of the detection target time point is obtained, and the cell at the detection target time point is generated as a result of cell division based on the change in the luminance summation at the time point near the detection target time point. It is characterized by detecting whether it is force or not Cell observation device.
[28] 細胞の特徴を示す細胞パラメータを経時的に計測する細胞観察装置で細胞の観 察を行う細胞観察プログラムであって、  [28] A cell observation program for observing cells with a cell observation device that measures cell parameters indicating cell characteristics over time,
前記細胞観察装置に、  In the cell observation device,
細胞を撮像した画像の細胞画像データから細胞を認識する細胞認識ステップと、 該細胞認識ステップで認識した細胞の特徴を示す細胞パラメータを前記細胞画像 データに基づ 、て計測する細胞パラメータ計測ステップと、  A cell recognition step for recognizing a cell from cell image data of an image obtained by imaging a cell; a cell parameter measurement step for measuring a cell parameter indicating the characteristics of the cell recognized in the cell recognition step based on the cell image data; ,
前記細胞認識ステップで認識した細胞が細胞分裂の結果生じたものであるか否か を前記細胞パラメータに基づ!、て検出する細胞分裂検出ステップと、  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; and
を実行させるステップを含み、  Including the step of executing
前記細胞分裂検出ステップは、検出対象時点における細胞の面積を示す細胞パラ メータと面積に関する閾値とを比較するとともに、該検出対象時点における細胞の輝 度を示す細胞パラメータと該検出対象時点に対して比較の対象となる時点における 細胞の輝度を示す細胞パラメータとを比較し、それぞれの比較結果に基づ 、て当該 検出対象時点における細胞が細胞分裂の結果生じたものである力否かを検出するス テツプであることを特徴とする細胞観察プログラム。  The cell division detection step compares a cell parameter indicating the area of the cell at the detection target time point with a threshold related to the area, and compares the cell parameter indicating the brightness of the cell at the detection target time point and the detection target time point. The cell parameter indicating the brightness of the cell at the time point to be compared is compared, and based on the result of each comparison, it is detected whether or not the cell at the time point to be detected is the result of cell division. A cell observation program characterized by being a step.
[29] 細胞の特徴を示す細胞パラメータを経時的に計測する細胞観察装置で細胞の観 察を行う細胞観察プログラムであって、 [29] A cell observation program for observing cells with a cell observation device that measures cell parameters indicating cell characteristics over time,
前記細胞観察装置に、  In the cell observation device,
細胞を撮像した画像の細胞画像データから細胞を認識する細胞認識ステップと、 該細胞認識ステップで認識した細胞の特徴を示す細胞パラメータを前記細胞画像 データに基づ 、て計測する細胞パラメータ計測ステップと、  A cell recognition step for recognizing a cell from cell image data of an image obtained by imaging a cell; a cell parameter measurement step for measuring a cell parameter indicating the characteristics of the cell recognized in the cell recognition step based on the cell image data; ,
前記細胞認識ステップで認識した細胞が細胞分裂の結果生じたものであるか否か を前記細胞パラメータに基づ!、て検出する細胞分裂検出ステップと、  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; and
を実行させるステップを含み、  Including the step of executing
前記細胞分裂検出ステップは、検出対象時点よりも時系列的に前の時点における 細胞の円形度を示す細胞パラメータの値を参照し、該検出対象時点における細胞が 細胞分裂の結果生じたものであるか否かを検出するステップであることを特徴とする 細胞観察プログラム。 The cell division detection step refers to the value of a cell parameter indicating the circularity of the cell at a time point chronologically before the detection target time point, and the cell at the detection target time point is a result of cell division. It is a step for detecting whether or not Cell observation program.
[30] 細胞の特徴を示す細胞パラメータを経時的に計測する細胞観察装置で細胞の観 察を行う細胞観察プログラムであって、  [30] A cell observation program for observing cells with a cell observation device that measures cell parameters indicating cell characteristics over time,
前記細胞観察装置に、  In the cell observation device,
細胞を撮像した画像の細胞画像データから細胞を認識する細胞認識ステップと、 該細胞認識ステップで認識した細胞の特徴を示す細胞パラメータを前記細胞画像 データに基づ 、て計測する細胞パラメータ計測ステップと、  A cell recognition step for recognizing a cell from cell image data of an image obtained by imaging a cell; a cell parameter measurement step for measuring a cell parameter indicating the characteristics of the cell recognized in the cell recognition step based on the cell image data; ,
前記細胞認識ステップで認識した細胞が細胞分裂の結果生じたものであるか否か を前記細胞パラメータに基づ!、て検出する細胞分裂検出ステップと、  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; and
を実行させるステップを含み、  Including the step of executing
前記細胞分裂検出ステップは、検出対象時点よりも時系列的に前の時点における 細胞の円形度を示す細胞パラメータの値を参照し、該円形度を示す細胞パラメータ の値が円形度に関する閾値と所定の関係を有している細胞を撮像した判定基準時 点を抽出し、該判定基準時点近傍の時点における細胞の円形度を示す細胞パラメ ータ及び面積を示す細胞パラメータの値の増減傾向に基づ 、て、該検出対象時点 における細胞が細胞分裂の結果生じたものである力否かを検出するステップであるこ とを特徴とする細胞観察プログラム。  The cell division detection step refers to a cell parameter value indicating the circularity of the cell at a time point chronologically before the detection target time point, and the cell parameter value indicating the circularity is a predetermined threshold value for the circularity. Is extracted based on the tendency of increase / decrease in the cell parameter indicating the circularity of the cell and the cell parameter indicating the area at a time near the determination reference time. A cell observation program characterized in that it is a step of detecting whether or not the cell at the detection target time point is a result of cell division.
[31] 細胞の特徴を示す細胞パラメータを経時的に計測する細胞観察装置で細胞の観 察を行う細胞観察プログラムであって、 [31] A cell observation program for observing cells with a cell observation device that measures cell parameters indicating cell characteristics over time,
前記細胞観察装置に、  In the cell observation device,
細胞を撮像した画像の細胞画像データから細胞を認識する細胞認識ステップと、 該細胞認識ステップで認識した細胞の特徴を示す細胞パラメータを前記細胞画像 データに基づ 、て計測する細胞パラメータ計測ステップと、  A cell recognition step for recognizing a cell from cell image data of an image obtained by imaging a cell; a cell parameter measurement step for measuring a cell parameter indicating the characteristics of the cell recognized in the cell recognition step based on the cell image data; ,
前記細胞認識ステップで認識した細胞が細胞分裂の結果生じたものであるか否か を前記細胞パラメータに基づ!、て検出する細胞分裂検出ステップと、  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; and
を実行させるステップを含み、  Including the step of executing
前記細胞分裂検出ステップは、検出対象時点における細胞の細胞核の存在範囲 の面積と細胞質の存在範囲の面積とを比較した結果に基づ 、て、該検出対象時点 における細胞が細胞分裂の結果生じたものである力否かを検出するステップであるこ とを特徴とする細胞観察プログラム。 In the cell division detection step, the detection target time point is determined based on the result of comparison between the area of the cell nucleus existing in the detection target time point and the area of the cytoplasm existing range. A cell observation program characterized in that it is a step for detecting whether or not a cell in the cell has a force resulting from cell division.
[32] 細胞の特徴を示す細胞パラメータを経時的に計測する細胞観察装置で細胞の観 察を行う細胞観察プログラムであって、  [32] A cell observation program for observing cells with a cell observation device that measures cell parameters indicating cell characteristics over time,
前記細胞観察装置に、  In the cell observation device,
細胞を撮像した画像の細胞画像データから細胞を認識する細胞認識ステップと、 該細胞認識ステップで認識した細胞の特徴を示す細胞パラメータを前記細胞画像 データに基づ 、て計測する細胞パラメータ計測ステップと、  A cell recognition step for recognizing a cell from cell image data of an image obtained by imaging a cell; a cell parameter measurement step for measuring a cell parameter indicating the characteristics of the cell recognized in the cell recognition step based on the cell image data; ,
前記細胞認識ステップで認識した細胞が細胞分裂の結果生じたものであるか否か を前記細胞パラメータに基づ!、て検出する細胞分裂検出ステップと、  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; and
を実行させるステップを含み、  Including the step of executing
前記細胞分裂検出ステップは、検出対象時点における細胞の微小管の局在領域 を求めて、細胞中の複数個所に局在しているか否かを検出することで、該検出対象 時点における細胞が細胞分裂の結果生じたものである力否かを検出するステップで あることを特徴とする細胞観察プログラム。  The cell division detection step obtains a localized region of the microtubule of the cell at the detection target time point, and detects whether or not the cell at the detection target time point is localized at a plurality of locations in the cell. A cell observation program characterized by being a step of detecting whether or not a force is a result of division.
[33] 細胞の特徴を示す細胞パラメータを経時的に計測する細胞観察装置で細胞の観 察を行う細胞観察プログラムであって、 [33] A cell observation program for observing cells with a cell observation device that measures cell parameters indicating cell characteristics over time,
前記細胞観察装置に、  In the cell observation device,
細胞を撮像した画像の細胞画像データから細胞を認識する細胞認識ステップと、 該細胞認識ステップで認識した細胞の特徴を示す細胞パラメータを前記細胞画像 データに基づ 、て計測する細胞パラメータ計測ステップと、  A cell recognition step for recognizing a cell from cell image data of an image obtained by imaging a cell; a cell parameter measurement step for measuring a cell parameter indicating the characteristics of the cell recognized in the cell recognition step based on the cell image data; ,
前記細胞認識ステップで認識した細胞が細胞分裂の結果生じたものであるか否か を前記細胞パラメータに基づ!、て検出する細胞分裂検出ステップと、  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; and
を実行させるステップを含み、  Including the step of executing
前記細胞分裂検出ステップは、撮像された画像の細胞に対応する領域内で輝度に 関する閾値よりも高い輝度値を示す画素について各画素の輝度値を合計した輝度 総和を示す領域パラメータを、検出対象時点近傍の時点について求め、該検出対象 時点近傍の時点にっ 、ての前記輝度総和の変化の様子に基づ 、て、該検出対象 時点における細胞が細胞分裂の結果生じたものである力否かを検出するステップで あることを特徴とする細胞観察プログラム。 In the cell division detection step, a region parameter indicating a luminance sum obtained by summing the luminance values of each pixel for a pixel having a luminance value higher than a threshold value regarding luminance in an area corresponding to the cell of the captured image is detected. The detection target is obtained based on the state of the change in the luminance summation at the time near the detection target time. A cell observation program characterized in that it is a step for detecting whether or not a cell at a time point is a result of cell division.
[34] 細胞を培養する培養手段と、赤外光線により細胞を撮像する赤外光撮像手段を少 なくとも含み前記培養手段に収容されている細胞を撮像する撮像手段と、を備えた 細胞観察装置で細胞の特徴を示す細胞パラメータを経時的に計測する細胞観察方 法であって、  [34] Cell observation comprising: a culture means for culturing cells; and an imaging means for imaging cells contained in the culture means, including at least an infrared light imaging means for imaging cells with infrared light. A cell observation method in which cell parameters indicating the characteristics of cells are measured over time with an apparatus,
前記培養手段で細胞を培養しながら、培養中の細胞を前記撮像手段によって撮像 して細胞の画像を取得する培養細胞撮像工程と、  A cultured cell imaging step of obtaining an image of a cell by imaging the cell in culture by the imaging means while culturing the cell by the culture means;
該培養細胞撮像工程で取得した画像の細胞画像データ力ゝら撮像された細胞を認 識する細胞認識工程と、  A cell recognition step for recognizing a captured cell from the cell image data of the image obtained in the cultured cell imaging step;
該細胞認識工程で認識した細胞の特徴を示す細胞パラメータを前記細胞画像デ ータに基づいて計測する細胞パラメータ計測工程と、  A cell parameter measurement step of measuring a cell parameter indicating the characteristics of the cell recognized in the cell recognition step based on the cell image data;
を含み、前記培養細胞撮像工程、前記細胞認識工程及び前記細胞パラメータ計 測工程を、培養期間中の複数時点で行い、  And performing the culture cell imaging step, the cell recognition step and the cell parameter measurement step at a plurality of time points during the culture period,
撮像した複数時点の画像力も認識した細胞同士の同一性を前記細胞パラメータに 基づ 、て判別する細胞追跡工程と、  A cell tracking step of discriminating the identity of cells that have also recognized image power at multiple time points based on the cell parameters;
をさらに含み、  Further including
前記培養手段で細胞を培養して ヽる最中に、該培養手段に収容されて ヽる細胞を 前記赤外光撮像手段によって撮像して観察することを特徴とする細胞観察方法。  A cell observation method comprising observing images of cells contained in the culture means by imaging with the infrared light imaging means while the cells are cultured by the culture means.
[35] 細胞を培養する培養手段と、赤外光線により細胞を撮像する赤外光撮像手段を少 なくとも含み前記培養手段に収容されている細胞を撮像する撮像手段と、を備えた 細胞観察装置で細胞の特徴を示す細胞パラメータを経時的に計測する細胞観察方 法であって、 [35] Cell observation comprising: a culture means for culturing cells; and an imaging means for imaging cells contained in the culture means, including at least an infrared light imaging means for imaging cells with infrared light A cell observation method in which cell parameters indicating the characteristics of cells are measured over time with an apparatus,
前記培養手段で細胞を培養しながら、培養中の細胞を培養期間中の複数時点で 前記撮像手段によって撮像して細胞の画像を取得する培養細胞撮像工程と、 該培養細胞撮像工程で取得した各時点の画像の細胞画像データから撮像された 細胞を認識する細胞認識工程と、  While culturing cells with the culture means, a cultured cell imaging step in which cells in culture are imaged by the imaging means at a plurality of time points during the culture period to obtain an image of the cells, and each acquired in the cultured cell imaging step A cell recognition process for recognizing a cell imaged from cell image data of an image at a time point;
該細胞認識工程で認識した各時点の細胞の特徴を示す細胞パラメータを前記細 胞画像データに基づいて計測する細胞パラメータ計測工程と、 撮像した複数時点の画像力も認識した細胞同士の同一性を前記細胞パラメータに 基づ 、て判別する細胞追跡工程と、 Cell parameters indicating the characteristics of cells at each time point recognized in the cell recognition step A cell parameter measuring step for measuring based on the cell image data, a cell tracking step for determining the identity of the cells that have also recognized the image power at a plurality of time points based on the cell parameter,
を含み、  Including
前記培養手段で細胞を培養して ヽる最中に、該培養手段に収容されて ヽる細胞を 前記赤外光撮像手段によって撮像して観察することを特徴とする細胞観察方法。 前記培養細胞撮像工程、前記細胞認識工程、前記細胞パラメータ計測工程、及び 細胞追跡工程のうちの少なくともいずれ力 1つの工程の前に、前記培養手段に収容 されている細胞を赤外光線によって撮像することを特徴とする請求項 34又は 35に記 載の細胞観察方法。  A cell observation method comprising observing images of cells contained in the culture means by imaging with the infrared light imaging means while the cells are cultured by the culture means. Prior to at least one of the cultured cell imaging step, the cell recognition step, the cell parameter measurement step, and the cell tracking step, the cells contained in the culture means are imaged with infrared rays. 36. The cell observation method according to claim 34 or 35, wherein:
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