WO2016092977A1 - Method and device for acquiring timing for molecule measurement - Google Patents

Method and device for acquiring timing for molecule measurement Download PDF

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Publication number
WO2016092977A1
WO2016092977A1 PCT/JP2015/080633 JP2015080633W WO2016092977A1 WO 2016092977 A1 WO2016092977 A1 WO 2016092977A1 JP 2015080633 W JP2015080633 W JP 2015080633W WO 2016092977 A1 WO2016092977 A1 WO 2016092977A1
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biological sample
timing
divided biological
molecular measurement
divided
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French (fr)
Japanese (ja)
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青広 前田
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富士フイルム株式会社
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • 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

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  • the present invention relates to a molecular measurement timing acquisition method and apparatus for acquiring the molecular measurement timing or pretreatment timing of a biological sample.
  • the amount and state of a biomolecule have been measured for a biological sample.
  • the effect of a drug can be assessed by observing how the phenotype changes after the drug is applied to the cell.
  • molecular measurement it is possible to infer the molecular mechanism that caused the phenotypic change.
  • the molecular measurement is meaningless even if it is performed before or after the change of the molecule caused by the drug occurs, and it is desirable to perform the measurement in the time zone when the change occurs.
  • Patent Document 1 discloses a system that performs cell image analysis in time series, but does not show a method for narrowing down the time zone of molecular measurement.
  • an object of the present invention is to provide a molecular measurement timing acquisition method and apparatus capable of acquiring the timing of molecular measurement or pretreatment effectively and efficiently.
  • the molecular measurement timing acquisition method of the present invention divides a biological sample to be measured to create a plurality of divided biological samples, and gives a stimulus to one divided biological sample among the plurality of divided biological samples Then, the divided biological sample is imaged a plurality of times over time, and the molecular measurement timing or molecular measurement of the divided biological sample other than the divided biological sample to be imaged is performed based on the variation in the feature amount of the captured images. It is characterized by acquiring the timing of preprocessing for
  • the molecular measurement timing acquisition method of the present invention it is preferable to notify the timing of molecular measurement or the timing of pre-processing by a notification device.
  • the same stimulus as the stimulus given to the divided biological sample to be imaged is given to the divided biological sample other than the divided biological sample to be imaged, It is preferable to perform molecular measurement based on the acquired timing of molecular measurement or to perform preprocessing based on the acquired timing of preprocessing.
  • the molecular measurement timing acquisition method of the present invention it is preferable to perform molecular measurement based on the acquired molecular measurement timing, or to perform preprocessing based on the acquired preprocessing timing.
  • the delay time is a time from when a molecular change in a cell, which is a biological sample, occurs until a feature amount of the image changes.
  • a plurality of delay times corresponding to the types of image feature values are set, and a plurality of delays are applied to a plurality of divided biological samples other than the divided biological sample to be imaged.
  • a stimulus is given to each of the plurality of divided biological samples at different timings, and the stimulus is applied, the feature amount of the image of the divided biological sample to be imaged changes. It is preferable to obtain the molecular measurement timing or preprocessing timing of the divided biological sample to which the delay time corresponding to the type is assigned.
  • the feature amount based on the outer shape of the cell in the image the feature amount based on the movement of the cell in the image, the feature amount based on the fluorescently labeled molecule in the image, and the image It is preferable that a delay time corresponding to at least two of the feature quantities based on the intracellular organelles is set.
  • the molecular measurement timing acquisition device of the present invention provides a plurality of divided biological samples over time from the time when stimulation is given to one divided biological sample among a plurality of divided biological samples obtained by dividing a biological sample to be measured. Based on fluctuations in the feature quantity of a plurality of images picked up by the image pickup unit and the plurality of images picked up by the image pickup unit, molecular measurement timing of a divided biological sample other than the divided biological sample to be imaged or preprocessing for molecular measurement And a timing acquisition unit for acquiring the timing.
  • the molecular measurement timing acquisition apparatus of the present invention preferably includes a notification unit that notifies the timing of molecular measurement or the timing of preprocessing.
  • the molecular measurement timing acquisition device of the present invention after the same stimulus as the stimulus given to the divided biological sample to be imaged is given to the divided biological sample other than the divided biological sample to be imaged, It is preferable to provide a molecular measurement unit that performs molecular measurement based on the acquired molecular measurement timing.
  • the molecular measurement timing acquisition device of the present invention after the same stimulus as the stimulus given to the divided biological sample to be imaged is given to the divided biological sample other than the divided biological sample to be imaged, It is preferable to provide a preprocessing unit that performs preprocessing based on the acquired preprocessing timing.
  • the timing acquisition unit determines when the feature amount of the image of the divided biological sample to be imaged fluctuates. It is preferable to acquire the timing of molecular measurement of a divided biological sample other than the divided biological sample to be imaged or the timing of preprocessing for molecular measurement.
  • the molecular measurement timing acquisition device of the present invention preferably includes a molecular measurement unit that performs molecular measurement based on the timing of the molecular measurement acquired by the timing acquisition unit.
  • the molecular measurement timing acquisition device of the present invention preferably includes a preprocessing unit that performs preprocessing based on the timing of the preprocessing acquired by the timing acquisition unit.
  • a biological sample to be measured is divided to create a plurality of divided biological samples, and stimulation is performed on one divided biological sample among the plurality of divided biological samples.
  • the divided biological sample is imaged a plurality of times over time from the time point given. Then, based on the variation in the feature amount of the plurality of captured images, the molecular measurement timing of the divided biological sample other than the divided biological sample to be imaged or the preprocessing timing for the molecular measurement is acquired. Therefore, the timing of molecular measurement or pretreatment can be acquired effectively and efficiently.
  • the phenotype of a biological sample changes after a stimulus is applied and then a molecular variation occurs due to the stimulus.
  • phenotypic changes during or immediately after molecular fluctuations are detected as image features, as in the present invention, and molecular measurement or preprocessing timing is based on this.
  • variation can be measured increases rather than the method in which a human acquires the timing of molecular measurement or pre-processing based on knowledge and intuition.
  • the molecular measurement in an extra time zone can be reduced or eliminated, the cost of performing the molecular measurement can be reduced.
  • the block diagram which shows schematic structure of the molecule
  • the flowchart for demonstrating the molecular measurement method using one Embodiment of the molecular measurement timing acquisition method of this invention Schematic diagram for explaining the process of creating a divided biological sample The figure which shows an example of the time-dependent change of the image feature-value acquired about the division
  • FIG. 1 is a block diagram showing a schematic configuration of the molecular measuring apparatus of the present embodiment.
  • the molecular measurement apparatus of the present embodiment includes a stimulation unit 10, an imaging unit 20, a control unit 30, a display unit 40, an input unit 50, a preprocessing unit 60, and a molecular measurement unit. 70.
  • a biological sample to be measured is divided to create a plurality of divided biological samples.
  • the biological sample before division is composed of, for example, a single cell line, and a divided biological sample is created by dispensing this single cell line into, for example, a plurality of the same containers by approximately the same number of cells.
  • the cell group of the biological sample is a cell group that can be regarded as substantially the same in evaluating the property using the result of the molecular measurement, and each of the divided biological samples created by dividing the cell group is divided.
  • a cell group is a cell that can be regarded as substantially the same.
  • pluripotent stem cells such as iPS (induced pluripotent stem cells) cells and ES (embryonic stem cells) cells
  • nerves derived from stem cells skin, myocardium or liver cells, or taken from the human body
  • skin, retina, heart muscle, blood cells, nerve or organ cells there are skin, retina, heart muscle, blood cells, nerve or organ cells.
  • the creation of the divided biological sample may be performed manually by the user, or may be performed automatically using, for example, an apparatus including a mechanism for sucking cells and a robot arm.
  • the stimulating unit 10 gives stimulation to each divided biological sample created as described above.
  • Examples of the stimulus given to each divided biological sample include addition of a drug whose physical mechanism is to be examined or physical shock.
  • the stimulus is not limited to these stimuli, and any stimulus may be used as long as it is a stimulus necessary for measuring and evaluating each divided biological sample.
  • the stimulation to each divided biological sample may be performed manually by the user, for example, by automatically providing a device for automatically adding a drug to a container containing the divided biological sample. May be stimulated.
  • the imaging unit 20 images one divided biological sample out of the plurality of divided biological samples created as described above a plurality of times over time.
  • the imaging unit 20 includes a microscope such as a phase contrast microscope, a differential interference microscope, a bright field microscope, or a fluorescence microscope.
  • These microscopes include an image sensor such as a CMOS (Complementary Metal-Oxide Semiconductor) sensor or a CCD (Charge-Coupled Device) sensor.
  • CMOS Complementary Metal-Oxide Semiconductor
  • CCD Charge-Coupled Device
  • the interval at which the divided biological sample is imaged by the imaging unit 20 can be arbitrarily set by the user, and is set according to the purpose of the evaluation. For example, when it is desired to evaluate the initial response of a divided biological sample to a drug by molecular measurement, a relatively short interval such as several hours to several tens of hours is set.
  • the initial response is the target molecule to which the drug binds directly and the variation in the position of the molecule close to the target molecule in its mechanism of action.
  • the molecular change resulting from the action of a drug is a change in the molecule that performs a process when the cancer cell dies, for example, in the case of an anticancer drug that kills the cancer cell.
  • a drug that promotes cell differentiation it is a molecular variation that performs a differentiation process.
  • a relatively short interval such as several hours to several tens of hours is set, while the action of a drug that takes a relatively long time to exhibit an effect.
  • relatively long intervals such as weeks to months are set.
  • the control unit 30 includes a CPU (Central Processing Unit) and the like, and controls the entire molecular measurement apparatus.
  • the control unit 30 of the present embodiment includes a timing acquisition unit 31.
  • the timing acquisition unit 31 receives an image captured with time in the imaging unit 20, and based on the variation in the feature amount of the input plurality of images, the divided biological sample other than the divided biological sample to be imaged.
  • the timing of molecular measurement or the timing of preprocessing for molecular measurement is acquired. A method for acquiring the timing of molecular measurement or the timing of preprocessing will be described in detail later.
  • control unit 30 provides information indicating that it is molecular measurement timing or information indicating that it is preprocessing timing based on the molecular measurement timing or preprocessing timing acquired by the timing acquisition unit 31. This is displayed on the display unit 40. Based on the information displayed on the display unit 40, the user can start molecular measurement or preprocessing of a divided biological sample other than the imaging target.
  • the control unit 30 controls the imaging operation of the imaging unit 20, or controls the stimulation operation when the stimulation unit 10 automatically stimulates the divided biological sample. It is.
  • the display unit 40 includes a display device such as a liquid crystal display.
  • the display unit 40 displays information indicating the timing of molecular measurement or preprocessing as described above. Moreover, you may make it display the image imaged by the imaging part 20.
  • the input unit 50 includes input devices such as a keyboard and a mouse, and accepts various setting inputs by the user. For example, the setting input of the imaging interval in the imaging part 20 mentioned above is received.
  • the pre-processing unit 60 performs pre-processing on the divided biological sample to be subjected to molecular measurement.
  • the preprocessing performed by the preprocessing unit 60 is processing performed before molecular measurement so that molecular measurement of a divided biological sample is possible.
  • chemical treatment is often added although it depends on the type of molecular measurement. For example, when performing molecular measurement using a cDNA (complementary ribonucleic acid) microarray, cells are dissolved, RNA (ribonucleic acid) is extracted, cRNA (complementary ribonucleic acid) and cDNA are synthesized, amplified, and fragmented. A process for performing hybridization is performed as a pretreatment.
  • the molecular measurement unit 70 performs molecular measurement for measuring the amount and state of biomolecules in the divided biological sample.
  • Molecular measurement methods include molecular measurement using a cDNA microarray, expression and state measurement using a next-generation sequencer, proteome measurement using a mass spectrometer, or metabolome measurement. Further, biomarker search may be performed. In this case, among the molecules whose amount has increased or decreased since the drug is added by the stimulating unit 10, those that correlate with the drug concentration are determined as biomarkers.
  • the biological sample S0 to be measured is dispensed into a plurality of the same containers by the substantially same number of cells to create divided biological samples S1 to S3 (S10).
  • the biological sample S0 to be measured is dispensed into a plurality of the same containers by the substantially same number of cells to create divided biological samples S1 to S3 (S10).
  • three first to third divided biological samples S1 to S3 are created.
  • the number of divided biological samples is not limited to this, and any number may be used as long as the number is two or more. .
  • the container of the first divided biological sample S1 is installed in the stimulation unit 10, and stimulation such as drug addition is given to the first divided biological sample S1 by the stimulation unit 10 (S12).
  • the time at which the stimulus is given to the first divided biological sample S1 is acquired by the timing acquisition unit 31 of the control unit 30.
  • the time when the stimulus is given to the first divided biological sample S ⁇ b> 1 may be input by the user using the input unit 50.
  • the stimulation unit 10 automatically gives stimulation
  • the stimulation time is output from the stimulation unit 10 to the timing acquisition unit 31 to stimulate the first divided biological sample S1. May be automatically acquired.
  • the container of the first divided biological sample S1 is installed in the imaging unit 20, and a plurality of images are captured over time by the imaging unit 20 (S14).
  • the images of the first divided biological sample S1 imaged by the imaging unit 20 are sequentially output to the timing acquisition unit 31 of the control unit 30. Then, the timing acquisition unit 31 calculates a feature amount from each input image.
  • Features include, for example, feature quantities based on the outline of cells in the image, feature quantities based on cell movement in the image, feature quantities based on fluorescently labeled molecules in the image, and intracellular organelles in the image. At least one feature amount among the feature amounts to be based is calculated. When calculating a plurality of feature amounts, the feature amounts may be weighted and added.
  • the feature amount based on the outer shape of the cell in the image for example, the feature amount of the cell size such as the average value of the area, diameter, or length of the plurality of cells included in the image, or the plurality of features included in the image There is a feature amount of the cell shape such as an average value of the circularity of the cell.
  • the feature amount based on the movement of cells in the image includes the moving speed and the average value of moving distances of a plurality of cells included in the image. Note that the moving speed and moving distance of the cells are calculated based on a plurality of images. Known methods can be used for the cell identification method and the tracking method of the movement trajectory.
  • the feature amount based on the fluorescently labeled molecule in the image for example, there is a feature amount based on the intensity of the fluorescence emitted from the fluorescently labeled molecule, that is, the average value or maximum value of the luminance of the image.
  • the feature amount based on the organelle in the image includes, for example, the moving distance and moving speed of mitochondria contained in the cell.
  • the moving speed and moving distance of mitochondria are also calculated based on a plurality of images.
  • Known methods can be used as a method for identifying mitochondria and a method for tracking the movement locus thereof.
  • the timing acquisition unit 31 sequentially calculates the feature amount of the image as described above, and acquires the temporal change of the image feature amount as shown in FIG. Then, for example, when the peak value as shown in FIG. 4 exists in the temporal change of the feature amount of the image, the timing acquisition unit 31 specifies the time of the peak value as the time of the variation point of the feature amount. Record (S18). In the case of the example illustrated in FIG. 4, the timing T ⁇ b> 1 and the time T ⁇ b> 2 are specified and recorded by the timing acquisition unit 31. Then, the timing acquisition unit 31 calculates time t1 from time T0 to time T1 when the first divided biological sample S1 is stimulated and time t2 from time T0 to time T2.
  • the second and third divided biological samples S2 and S3 are installed in the stimulating unit 10, and these divided biological samples are included in these divided biological samples.
  • the same stimulus as the stimulus applied to the first divided biological sample S1 is given by the stimulation unit 10 (S20).
  • the timing at which the stimulus is applied to the second and third divided biological samples S2 and S3 is acquired by the timing acquisition unit 31 of the control unit 30.
  • the time at which the stimulus is applied to the second and third divided biological samples S2 may be input by the user using the input unit 50, or may be automatically acquired.
  • the timing acquisition unit 31 measures the time from the time when the stimulus is given to the second and third divided biological samples S2 and S3, and the time t1 from time T0 to time T1 described above has elapsed. Whether or not is detected.
  • the control unit 30 causes the display unit 40 to display a message for prompting molecular measurement or preprocessing for the second divided biological sample S2 (S22). .
  • the user installs the second divided biological sample S2 in the preprocessing unit 60, and performs the molecular measurement on the second divided biological sample S2. Processing is performed (S24). Further, the pre-processed second divided biological sample S2 is installed in the molecular measurement unit 70, and the molecular measurement of the second divided biological sample S2 is performed (S26).
  • the display unit 40 displays a message to notify the user of the molecular measurement timing or the preprocessing timing.
  • the notification method is not limited thereto. Instead, for example, the light source may be turned on or notified by voice. In this case, the light source and the sound source correspond to the notification device or the notification unit of the present invention.
  • the timing acquisition unit 31 detects whether or not the time t2 from the time T0 to the time T2 described above has elapsed.
  • the control unit 30 causes the display unit 40 to display a message for prompting molecular measurement or preprocessing for the third divided biological sample S3 (S22). .
  • the user installs the third divided biological sample S3 in the preprocessing unit 60, and performs the molecular measurement on the third divided biological sample S3. Processing is performed (S24). Furthermore, the pre-processed third divided biological sample S3 is placed in the molecular measurement unit 70, and the molecular measurement of the third divided biological sample S3 is performed (S26).
  • the biological sample S0 to be measured is divided to create a plurality of divided biological samples S1 to S3, and one divided biological sample S1 among the plurality of divided biological samples is obtained. Then, the divided biological sample S1 is imaged a plurality of times over time from the time point when the stimulus is applied. Then, based on the variation in the feature amount of the plurality of captured images, the molecular measurement timing of the divided biological samples S2 and S3 other than the divided biological sample to be imaged or the preprocessing timing for the molecular measurement is acquired. Since it did in this way, the timing of molecular measurement or pre-processing can be acquired effectively and efficiently.
  • the containers of the first to third divided biological samples S1 to S3 may be manually transported by the user, or automatically transported using a robot arm, a transport table, a transport belt, or the like. You may do it.
  • segmentation biological sample S1 changed is acquired as the timing of the pre-processing of 2nd and 3rd division
  • the molecular variation of the stimulated cell occurs before the feature amount of the cell image varies, and it is more preferable to measure the molecular variation.
  • the biological sample S0 to be measured is dispensed into a plurality of the same containers by the substantially same number of cells, and divided biological samples S1 to S3 are created (S30).
  • the container of the first divided biological sample S1 is installed in the stimulating unit 10, and stimulation such as addition of a drug is given to the first divided biological sample S1 by the stimulating unit 10 (S32).
  • stimulation such as addition of a drug is given to the first divided biological sample S1 by the stimulating unit 10 (S32).
  • the time at which the stimulus is given to the first divided biological sample S1 is acquired by the timing acquisition unit 31 of the control unit 30.
  • the first divided biological sample S1 is installed in the imaging unit 20 as in the above embodiment, and a plurality of images are captured over time by the imaging unit 20 (S34). Then, the image of the first divided biological sample S1 captured by the imaging unit 20 is sequentially output to the timing acquisition unit 31 of the control unit 30, and the feature amount of the image is acquired by the timing acquisition unit 31 as in the above embodiment. It is calculated and its change with time is acquired (S36).
  • an elapsed time from the time T0 when the stimulus is given to the first divided biological sample S1 is a timing acquisition unit. 31. Then, the timing acquisition unit 31 detects whether or not the elapsed time from the time T0 has passed a preset delay time DT (S38).
  • This preset delay time is the time from the occurrence of molecular variation in the cell of the biological sample to the variation in the feature amount of the image, which is the time measured by conducting an experiment or the like in advance. .
  • the second and third divided biological samples S2 at the detection time T3. , S3 are installed in the stimulation unit 10, and the second and third divided biological samples S2, S3 are stimulated (S40).
  • control unit 30 causes the display unit 40 to display that the delay time DT has elapsed, and the user who sees the display manually stimulates the second and third divided biological samples S2 and S3.
  • the control unit 30 may control the stimulation unit 10 to automatically apply stimulation.
  • an image of the first divided biological sample S1 is obtained by the timing acquisition unit 31 as shown in FIG.
  • the timing acquisition unit 31 acquires the detection time T4 as the molecular measurement timing or the preprocessing timing, and the control unit 30 A message for prompting molecular measurement or preprocessing for the divided biological sample S2 is displayed on the display unit 40 (S44).
  • the variation point may be detected by detecting.
  • the time from when the stimulus is applied to the first divided biological sample S1 to the time when the molecular variation occurs and the time from when the stimulus is applied to the second divided biological sample S2 until the time when the molecular variation occurs.
  • the second divided biological sample S2 is stimulated with a delay time DT from the first divided biological sample S1, and therefore the first divided biological sample S1.
  • the variation point of the feature amount of the image is the time when the molecular variation occurs in the second divided biological sample S2.
  • the user installs the second divided biological sample S2 in the preprocessing unit 60, and performs the molecular measurement on the second divided biological sample S2. Processing is performed (S46). Further, the pre-processed second divided biological sample S2 is installed in the molecular measurement unit 70, and the molecular measurement of the second divided biological sample S2 is performed (S48).
  • the timing acquisition unit 31 determines the variation point of the feature amount of the image of the first divided biological sample S1. If it is detected again (S42, YES), the timing acquisition unit 31 acquires this detection time T5 as the molecular measurement timing or preprocessing timing of the third divided biological sample S3, and the control unit 30 A message prompting molecular measurement or preprocessing for the three divided biological samples S3 is displayed on the display unit 40 (S44).
  • the third divided biological sample S3 is stimulated with a delay time DT from the first divided biological sample S1.
  • the second variation point of the feature quantity of the image of one divided biological sample S1 is the time when the second molecular variation occurs in the third divided biological sample S3.
  • the user installs the third divided biological sample S3 in the preprocessing unit 60, and performs the molecular measurement on the third divided biological sample S3. Processing is performed (S46). Further, the pre-processed third divided biological sample S3 is installed in the molecular measurement unit 70, and the molecular measurement of the third divided biological sample S3 is performed (S48).
  • molecular variation can be measured more accurately.
  • the process for acquiring the molecular measurement timing or the preprocessing timing and the process for performing the molecular measurement can be performed in parallel, compared with the method for acquiring the molecular measurement timing or the preprocessing timing described above, The total processing period can be shortened.
  • the time from the first molecular variation to the variation in the image feature amount of the first divided biological sample S1 is the same as the time from the second molecular variation to the variation in the image feature amount.
  • the timing for applying the stimulus to the second and third divided biological samples S2 and S3 is both delayed by the delay time DT.
  • the time from the amount of change to the amount of change and the time from the second molecular change to the change in the image feature amount are not necessarily the same.
  • the feature amounts of different types of images may be calculated for the first time and the second time. In this case, the time from the molecular variation to the variation of the image feature amount is different for the first time and the second time. .
  • the time from the molecular change to the change of the image feature amount is relatively long, and the fluorescently labeled molecule
  • the time from molecular variation to image feature variation is often relatively short.
  • the first time calculates a feature amount based on a fluorescently labeled molecule or an intracellular organelle
  • the second time calculates a feature based on the outer shape of the cell or the movement of the cell.
  • a time when the feature amount based on the fluorescently labeled molecule or the organelle is changed is assigned as the delay time DT1
  • the third for the divided biological sample S3 a time when the feature amount based on the outer shape of the cell or the movement of the cell fluctuates may be assigned as the delay time DT2.
  • the second divided biological sample S2 is stimulated by delaying the delay time DT1 from the first divided biological sample S1, and the first divided biological sample S3 is stimulated.
  • Stimulation may be applied by delaying the sample S1 by the delay time DT2.
  • the variation point of the first image feature amount of the first divided biological sample S1 is acquired as the molecular measurement timing or the preprocessing timing of the second divided biological sample S2, and 2 of the first divided biological sample S1.
  • the variation point of the second image feature amount is acquired as the molecular measurement timing or the preprocessing timing of the third divided biological sample S3.
  • the delay time corresponding to the type of the image feature amount as described above is set in advance as a table as shown in FIG. 8, and the user can select which type for the first divided biological sample S1.
  • the control unit 30 automatically obtains a delay time by referring to the table according to the setting and assigns it to the second and third divided biological samples S2 and S3. What should I do?

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Abstract

The purpose of the present invention is, in a molecule measurement timing acquisition method and a molecule measurement timing acquisition device both for acquiring a timing for a molecule measurement of a biological sample or a timing for a pretreatment of the molecule measurement, to acquire the timing for the molecule measurement or the timing for the pretreatment effectively and efficiently. A biological sample to be measured is divided into multiple divided biological samples; subsequently, a stimulus is applied to one divided biological sample among the multiple divided biological samples, and then the divided biological sample is imaged multiple times over time with starting from the time point of the application of the stimulus; and subsequently, on the basis of the change in a characteristic amount in the multiple images, a timing for a molecule measurement of each of the other divided biological samples than the imaged divided biological sample or a timing for a pretreatment for the molecule measurement is acquired.

Description

分子計測タイミング取得方法および装置Molecular measurement timing acquisition method and apparatus
　本発明は、生体試料の分子計測のタイミングまたは前処理のタイミングを取得する分子計測タイミング取得方法および装置に関するものである。 The present invention relates to a molecular measurement timing acquisition method and apparatus for acquiring the molecular measurement timing or pretreatment timing of a biological sample.
　従来、たとえば医薬品の作用機序解析やバイオマーカー探索などのために、生体試料を対象にして生体分子の量や状態を計測することが行われている。たとえば薬剤を細胞に与えた後で、表現型がどのように変化するかを観察することにより薬剤の効果が評価される。このとき分子計測も合わせて行うことにより、表現型の変化をもたらした分子機序を推測することができる。 Conventionally, for example, in order to analyze the mechanism of action of a drug or search for a biomarker, the amount and state of a biomolecule have been measured for a biological sample. For example, the effect of a drug can be assessed by observing how the phenotype changes after the drug is applied to the cell. At this time, by performing molecular measurement together, it is possible to infer the molecular mechanism that caused the phenotypic change.
　このとき、分子計測は、薬剤が引き起こした分子の変化が生じる前や終わった後で行っても意味がなく、変化が生じている時間帯で行うことが望ましい。 At this time, the molecular measurement is meaningless even if it is performed before or after the change of the molecule caused by the drug occurs, and it is desirable to perform the measurement in the time zone when the change occurs.
特開２０１２－１６３５３８号公報JP 2012-163538 A
　しかしながら、これまで、分子計測を行う時間帯は、人間が知識や勘に基づいて決めていた。または、時間帯を絞り込まずにひたすら計測を行う方法がとられてきた。なお、分子計測を行うためには試料を侵襲しなければならない場合が多く、その場合、一つの試料につき一つの時点でしか計測できない。 However, until now, the time period for molecular measurement has been determined by humans based on knowledge and intuition. Alternatively, a method has been used in which measurement is performed without narrowing down the time zone. In many cases, a sample must be invaded in order to perform molecular measurement. In such a case, measurement can be performed only at one time point per sample.
　従来技術では、計測のタイミングを外し、捉えたい分子変動を捉え損ねる場合がある。また、分子計測は比較的高コストであるのに、本来計測する必要のない時間帯についても行えば、ますますコストアップとなる問題がある。 In the conventional technology, there are cases where the measurement timing is missed and the molecular fluctuations to be captured are missed. In addition, although molecular measurement is relatively expensive, there is a problem that the cost is increased further if it is performed in a time zone where measurement is not necessarily performed.
　なお、特許文献１では細胞の画像解析を時系列で行うシステムが開示されているが、分子計測の時間帯を絞り込む方法は示されていない。 Note that Patent Document 1 discloses a system that performs cell image analysis in time series, but does not show a method for narrowing down the time zone of molecular measurement.
　本発明は、上記の問題に鑑み、効果的および効率的に分子計測または前処理のタイミングを取得することができる分子計測タイミング取得方法および装置を提供することを目的とする。 In view of the above problems, an object of the present invention is to provide a molecular measurement timing acquisition method and apparatus capable of acquiring the timing of molecular measurement or pretreatment effectively and efficiently.
　本発明の分子計測タイミング取得方法は、計測対象の生体試料を分割して複数の分割生体試料を作成し、その複数の分割生体試料のうちの１つの分割生体試料に対して刺激を与えた時点から、その分割生体試料を経時的に複数回撮像し、その撮像した複数枚の画像の特徴量の変動に基づいて、撮像対象の分割生体試料以外の分割生体試料の分子計測のタイミングまたは分子計測のための前処理のタイミングを取得することを特徴とする。 The molecular measurement timing acquisition method of the present invention divides a biological sample to be measured to create a plurality of divided biological samples, and gives a stimulus to one divided biological sample among the plurality of divided biological samples Then, the divided biological sample is imaged a plurality of times over time, and the molecular measurement timing or molecular measurement of the divided biological sample other than the divided biological sample to be imaged is performed based on the variation in the feature amount of the captured images. It is characterized by acquiring the timing of preprocessing for
　また、上記本発明の分子計測タイミング取得方法においては、分子計測のタイミングまたは前処理のタイミングを報知装置によって報知することが好ましい。 In the molecular measurement timing acquisition method of the present invention, it is preferable to notify the timing of molecular measurement or the timing of pre-processing by a notification device.
　また、上記本発明の分子計測タイミング取得方法においては、撮像対象の分割生体試料以外の分割生体試料に対して、撮像対象の分割生体試料に対して与えた刺激と同じ刺激を与えた後、上記取得した分子計測のタイミングに基づいて分子計測を行うまたは上記取得した前処理のタイミングに基づいて前処理を行うことが好ましい。 In the molecular measurement timing acquisition method of the present invention, the same stimulus as the stimulus given to the divided biological sample to be imaged is given to the divided biological sample other than the divided biological sample to be imaged, It is preferable to perform molecular measurement based on the acquired timing of molecular measurement or to perform preprocessing based on the acquired timing of preprocessing.
　また、上記本発明の分子計測タイミング取得方法においては、撮像対象の分割生体試料に対して刺激を与えた時点から、予め設定された遅延時間だけ経過した時点において、撮像対象の分割生体試料以外の分割生体試料に対して、撮像対象の分割生体試料に対して与えた刺激と同じ刺激を与え、その刺激を与えた後、撮像対象の分割生体試料の画像の特徴量が変動した時点を、撮像対象の分割生体試料以外の分割生体試料の分子計測のタイミングまたは分子計測のための前処理のタイミングとして取得することが好ましい。 In the molecular measurement timing acquisition method of the present invention described above, when a predetermined delay time elapses from the time when the divided biological sample to be imaged is stimulated, other than the divided biological sample to be imaged. The same stimulus as the stimulus given to the divided biological sample to be imaged is applied to the divided biological sample, and after the stimulus is applied, the time when the feature amount of the image of the divided biological sample to be imaged fluctuates is imaged It is preferable to obtain the timing of molecular measurement of a divided biological sample other than the target divided biological sample or the timing of preprocessing for molecular measurement.
　また、上記本発明の分子計測タイミング取得方法においては、上記取得した分子計測のタイミングに基づいて分子計測を行うまたは上記取得した前処理のタイミングに基づいて前処理を行うことが好ましい。 In the molecular measurement timing acquisition method of the present invention, it is preferable to perform molecular measurement based on the acquired molecular measurement timing, or to perform preprocessing based on the acquired preprocessing timing.
　また、上記本発明の分子計測タイミング取得方法においては、上記遅延時間は、生体試料である細胞内の分子変動が生じてから画像の特徴量の変動が生じるまでの時間とすることが好ましい。 In the molecular measurement timing acquisition method of the present invention, it is preferable that the delay time is a time from when a molecular change in a cell, which is a biological sample, occurs until a feature amount of the image changes.
　また、上記本発明の分子計測タイミング取得方法においては、画像の特徴量の種類に対応する遅延時間を複数設定し、撮像対象の分割生体試料以外の複数の分割生体試料に対して、複数の遅延時間をそれぞれ割り当てて、複数の分割生体試料に対してそれぞれ異なるタイミングで刺激を与え、その刺激を与えた後、撮像対象の分割生体試料の画像の特徴量が変動した際、その変動した特徴量の種類に対応した遅延時間が割り当てられた分割生体試料の分子計測のタイミングまたは前処理のタイミングを取得することが好ましい。 In the molecular measurement timing acquisition method of the present invention, a plurality of delay times corresponding to the types of image feature values are set, and a plurality of delays are applied to a plurality of divided biological samples other than the divided biological sample to be imaged. When time is allocated and a stimulus is given to each of the plurality of divided biological samples at different timings, and the stimulus is applied, the feature amount of the image of the divided biological sample to be imaged changes. It is preferable to obtain the molecular measurement timing or preprocessing timing of the divided biological sample to which the delay time corresponding to the type is assigned.
　また、上記本発明の分子計測タイミング取得方法においては、画像内の細胞の外形に基づく特徴量、画像内の細胞の動きに基づく特徴量、画像内の蛍光標識された分子に基づく特徴量および画像内の細胞内小器官に基づく特徴量のうちの少なくとも２つに対応した遅延時間が設定されることが好ましい。 In the molecular measurement timing acquisition method of the present invention, the feature amount based on the outer shape of the cell in the image, the feature amount based on the movement of the cell in the image, the feature amount based on the fluorescently labeled molecule in the image, and the image It is preferable that a delay time corresponding to at least two of the feature quantities based on the intracellular organelles is set.
　本発明の分子計測タイミング取得装置は、計測対象の生体試料を分割した複数の分割生体試料のうちの１つの分割生体試料に対して刺激を与えた時点から、その分割生体試料を経時的に複数回撮像する撮像部と、撮像部によって撮像された複数枚の画像の特徴量の変動に基づいて、撮像対象の分割生体試料以外の分割生体試料の分子計測のタイミングまたは分子計測のための前処理のタイミングを取得するタイミング取得部とを備えたことを特徴とする。 The molecular measurement timing acquisition device of the present invention provides a plurality of divided biological samples over time from the time when stimulation is given to one divided biological sample among a plurality of divided biological samples obtained by dividing a biological sample to be measured. Based on fluctuations in the feature quantity of a plurality of images picked up by the image pickup unit and the plurality of images picked up by the image pickup unit, molecular measurement timing of a divided biological sample other than the divided biological sample to be imaged or preprocessing for molecular measurement And a timing acquisition unit for acquiring the timing.
　また、上記本発明の分子計測タイミング取得装置においては、分子計測のタイミングまたは前処理のタイミングを報知する報知部を備えることが好ましい。 In addition, the molecular measurement timing acquisition apparatus of the present invention preferably includes a notification unit that notifies the timing of molecular measurement or the timing of preprocessing.
　また、上記本発明の分子計測タイミング取得装置においては、撮像対象の分割生体試料以外の分割生体試料に対して、撮像対象の分割生体試料に対して与えた刺激と同じ刺激が与えられた後、上記取得した分子計測のタイミングに基づいて分子計測を行う分子計測部を備えることが好ましい。 In the molecular measurement timing acquisition device of the present invention, after the same stimulus as the stimulus given to the divided biological sample to be imaged is given to the divided biological sample other than the divided biological sample to be imaged, It is preferable to provide a molecular measurement unit that performs molecular measurement based on the acquired molecular measurement timing.
　また、上記本発明の分子計測タイミング取得装置においては、撮像対象の分割生体試料以外の分割生体試料に対して、撮像対象の分割生体試料に対して与えた刺激と同じ刺激が与えられた後、上記取得した前処理のタイミングに基づいて前処理を行う前処理部を備えることが好ましい。 In the molecular measurement timing acquisition device of the present invention, after the same stimulus as the stimulus given to the divided biological sample to be imaged is given to the divided biological sample other than the divided biological sample to be imaged, It is preferable to provide a preprocessing unit that performs preprocessing based on the acquired preprocessing timing.
　また、上記本発明の分子計測タイミング取得装置においては、撮像対象の分割生体試料に対して刺激を与えた時点から、予め設定された遅延時間だけ経過した時点において、撮像対象の分割生体試料以外の分割生体試料に対して、撮像対象の分割生体試料に対して与えた刺激と同じ刺激を与える刺激部を備え、タイミング取得部が、撮像対象の分割生体試料の画像の特徴量が変動した時点を、撮像対象の分割生体試料以外の分割生体試料の分子計測のタイミングまたは分子計測のための前処理のタイミングとして取得することが好ましい。 In the molecular measurement timing acquisition device of the present invention described above, when a predetermined delay time elapses from the time when the divided biological sample to be imaged is stimulated, other than the divided biological sample to be imaged. A stimulating unit that applies the same stimulus as the stimulus given to the divided biological sample to be imaged to the divided biological sample, and the timing acquisition unit determines when the feature amount of the image of the divided biological sample to be imaged fluctuates. It is preferable to acquire the timing of molecular measurement of a divided biological sample other than the divided biological sample to be imaged or the timing of preprocessing for molecular measurement.
　また、上記本発明の分子計測タイミング取得装置においては、タイミング取得部によって取得された上記分子計測のタイミングに基づいて分子計測を行う分子計測部を備えることが好ましい。 The molecular measurement timing acquisition device of the present invention preferably includes a molecular measurement unit that performs molecular measurement based on the timing of the molecular measurement acquired by the timing acquisition unit.
　また、上記本発明の分子計測タイミング取得装置においては、タイミング取得部によって取得された上記前処理のタイミングに基づいて前処理を行う前処理部を備えることが好ましい。 The molecular measurement timing acquisition device of the present invention preferably includes a preprocessing unit that performs preprocessing based on the timing of the preprocessing acquired by the timing acquisition unit.
　本発明の分子計測タイミング取得方法および装置によれば、計測対象の生体試料を分割して複数の分割生体試料を作成し、その複数の分割生体試料のうちの１つの分割生体試料に対して刺激を与えた時点から、その分割生体試料を経時的に複数回撮像する。そして、その撮像した複数枚の画像の特徴量の変動に基づいて、撮像対象の分割生体試料以外の分割生体試料の分子計測のタイミングまたは分子計測のための前処理のタイミングを取得するようにしたので、効果的および効率的に分子計測または前処理のタイミングを取得することができる。 According to the molecular measurement timing acquisition method and apparatus of the present invention, a biological sample to be measured is divided to create a plurality of divided biological samples, and stimulation is performed on one divided biological sample among the plurality of divided biological samples. The divided biological sample is imaged a plurality of times over time from the time point given. Then, based on the variation in the feature amount of the plurality of captured images, the molecular measurement timing of the divided biological sample other than the divided biological sample to be imaged or the preprocessing timing for the molecular measurement is acquired. Therefore, the timing of molecular measurement or pretreatment can be acquired effectively and efficiently.
　すなわち、生体試料は、刺激が与えられた後、その刺激によって分子変動を生じた後に、表現型が変化する。分子変動を直接捉えることは困難であるが、本発明のように分子変動の発生中または発生直後の表現型の変化を画像の特徴量として捉え、これに基づいて分子計測または前処理のタイミングを取得することによって、人間が知識や勘に基づいて分子計測または前処理のタイミングを取得する方法よりも、分子変動が計測できる蓋然性が高まる。また、余計な時間帯での分子計測を削減ないし排除できるので、分子計測を行うコストを低減できる。 That is, the phenotype of a biological sample changes after a stimulus is applied and then a molecular variation occurs due to the stimulus. Although it is difficult to capture molecular fluctuations directly, phenotypic changes during or immediately after molecular fluctuations are detected as image features, as in the present invention, and molecular measurement or preprocessing timing is based on this. By acquiring, the probability that a molecule | numerator fluctuation | variation can be measured increases rather than the method in which a human acquires the timing of molecular measurement or pre-processing based on knowledge and intuition. In addition, since the molecular measurement in an extra time zone can be reduced or eliminated, the cost of performing the molecular measurement can be reduced.
本発明の分子計測タイミング取得装置の一実施形態を用いた分子計測装置の概略構成を示すブロック図The block diagram which shows schematic structure of the molecule | numerator measurement apparatus using one Embodiment of the molecule | numerator measurement timing acquisition apparatus of this invention. 本発明の分子計測タイミング取得方法の一実施形態を用いた分子計測方法を説明するためのフローチャートThe flowchart for demonstrating the molecular measurement method using one Embodiment of the molecular measurement timing acquisition method of this invention 分割生体試料の作成工程を説明するための模式図Schematic diagram for explaining the process of creating a divided biological sample 分割生体試料について取得された画像特徴量の経時変化の一例を示す図The figure which shows an example of the time-dependent change of the image feature-value acquired about the division | segmentation biological sample. 本発明の分子計測タイミング取得方法のその他の実施形態を用いた分子計測方法を説明するためのフローチャートThe flowchart for demonstrating the molecular measurement method using other embodiment of the molecular measurement timing acquisition method of this invention. 図５に示す分子計測方法を説明するためのタイミングチャートTiming chart for explaining the molecular measurement method shown in FIG. 図５に示す分子計測方法の変形例を説明するためのタイミングチャートTiming chart for explaining a modification of the molecular measurement method shown in FIG. 複数の画像特徴量と遅延時間とを対応づけたテーブルの一例を示す図The figure which shows an example of the table which matched several image feature-value and delay time
　以下、本発明の分子計測タイミング取得方法および装置の一実施形態を用いた分子計測装置について、図面を参照しながら詳細に説明する。図１は、本実施形態の分子計測装置の概略構成を示すブロック図である。 Hereinafter, a molecular measurement apparatus using an embodiment of the molecular measurement timing acquisition method and apparatus of the present invention will be described in detail with reference to the drawings. FIG. 1 is a block diagram showing a schematic configuration of the molecular measuring apparatus of the present embodiment.
　本実施形態の分子計測装置は、図１に示すように、刺激部１０と、撮像部２０と、制御部３０と、表示部４０と、入力部５０と、前処理部６０と、分子計測部７０とを備えている。 As shown in FIG. 1, the molecular measurement apparatus of the present embodiment includes a stimulation unit 10, an imaging unit 20, a control unit 30, a display unit 40, an input unit 50, a preprocessing unit 60, and a molecular measurement unit. 70.
　まず、本実施形態の分子計測装置を用いて分子計測を行う場合には、計測対象の生体試料を分割して複数の分割生体試料が作成される。分割前の生体試料は、たとえば単一の細胞株からなるものであり、この単一の細胞株をたとえば複数の同じ容器に略同じ細胞数だけ分注することによって分割生体試料が作成される。すなわち、生体試料の細胞群は、その分子計測の結果を用いて性質を評価する上で、実質的に同じとみなすことができる細胞群であり、これを分割して作成した各分割生体試料の細胞群も実質的に同じとみなすことができる細胞である。 First, when performing molecular measurement using the molecular measurement apparatus of this embodiment, a biological sample to be measured is divided to create a plurality of divided biological samples. The biological sample before division is composed of, for example, a single cell line, and a divided biological sample is created by dispensing this single cell line into, for example, a plurality of the same containers by approximately the same number of cells. That is, the cell group of the biological sample is a cell group that can be regarded as substantially the same in evaluating the property using the result of the molecular measurement, and each of the divided biological samples created by dividing the cell group is divided. A cell group is a cell that can be regarded as substantially the same.
　生体試料としては、たとえばｉＰＳ（induced pluripotent stem cells）細胞およびＥＳ（embryonic stem cells）細胞といった多能性幹細胞、幹細胞から分化誘導された神経、皮膚、心筋または肝臓の細胞、もしくは人体から取り出された皮膚、網膜、心筋、血球、神経または臓器の細胞などがある。 As biological samples, for example, pluripotent stem cells such as iPS (induced pluripotent stem cells) cells and ES (embryonic stem cells) cells, nerves derived from stem cells, skin, myocardium or liver cells, or taken from the human body There are skin, retina, heart muscle, blood cells, nerve or organ cells.
　なお、分割生体試料の作成は、ユーザが手動で行うようにしてもよいし、たとえば細胞を吸引する機構とロボットアームなどからなる装置を用いて自動で行うようにしてもよい。 Note that the creation of the divided biological sample may be performed manually by the user, or may be performed automatically using, for example, an apparatus including a mechanism for sucking cells and a robot arm.
　刺激部１０は、上述したようにして作成された各分割生体試料に対して刺激を与えるものである。各分割生体試料に対して与えられる刺激としては、たとえば作用機序を調べたい薬剤などの添加または物理的なショックなどがある。ただし、これらの刺激に限らず、各分割生体試料の分子計測を行って評価するために必要な刺激であれば、如何なる刺激でもよい。また、各分割生体試料への刺激は、ユーザが手動で行うようにしてもよいし、たとえば分割生体試料が収容された容器に対して自動的に薬剤を添加する装置などを設けることによって、自動的に刺激を与えるようにしてもよい。 The stimulating unit 10 gives stimulation to each divided biological sample created as described above. Examples of the stimulus given to each divided biological sample include addition of a drug whose physical mechanism is to be examined or physical shock. However, the stimulus is not limited to these stimuli, and any stimulus may be used as long as it is a stimulus necessary for measuring and evaluating each divided biological sample. Further, the stimulation to each divided biological sample may be performed manually by the user, for example, by automatically providing a device for automatically adding a drug to a container containing the divided biological sample. May be stimulated.
　撮像部２０は、上述したようにして作成された複数の分割生体試料のうちの１つの分割生体試料を経時的に複数回撮像するものである。具体的には、撮像部２０は、たとえば位相差顕微鏡、微分干渉顕微鏡、明視野顕微鏡または蛍光顕微鏡などの顕微鏡を備えたものである。これらの顕微鏡は、ＣＭＯＳ（Complementary Metal-Oxide Semiconductor）センサやＣＣＤ（Charge-Coupled Device）センサなどの撮像素子を備えている。 The imaging unit 20 images one divided biological sample out of the plurality of divided biological samples created as described above a plurality of times over time. Specifically, the imaging unit 20 includes a microscope such as a phase contrast microscope, a differential interference microscope, a bright field microscope, or a fluorescence microscope. These microscopes include an image sensor such as a CMOS (Complementary Metal-Oxide Semiconductor) sensor or a CCD (Charge-Coupled Device) sensor.
　なお、撮像部２０によって分割生体試料を撮像する間隔はユーザによって任意に設定することができ、評価の目的に応じてそれぞれ設定される。たとえば、分割生体試料の薬剤に対する初期応答を分子計測して評価したい場合には、数時間～数十時間といった比較的短い間隔が設定される。初期応答とは、薬が直接結合する標的分子と、その作用機序においてその標的分子に近いポジションの分子の変動のことである。 The interval at which the divided biological sample is imaged by the imaging unit 20 can be arbitrarily set by the user, and is set according to the purpose of the evaluation. For example, when it is desired to evaluate the initial response of a divided biological sample to a drug by molecular measurement, a relatively short interval such as several hours to several tens of hours is set. The initial response is the target molecule to which the drug binds directly and the variation in the position of the molecule close to the target molecule in its mechanism of action.
　一方、薬剤に対する初期応答とともに、薬剤が作用した結果の細胞の分子変化を評価したい場合には、数週間～数か月といった比較的長い間隔が設定される。薬剤が作用した結果の分子変化とは、たとえばがん細胞を殺す抗がん剤の場合、がん細胞が死ぬ際のプロセスを実行するような分子の変動のことである。また、細胞の分化を促す薬剤の場合、分化プロセスを実行するような分子の変動である。 On the other hand, when it is desired to evaluate the molecular response of the cell as a result of the action of the drug as well as the initial response to the drug, a relatively long interval of several weeks to several months is set. The molecular change resulting from the action of a drug is a change in the molecule that performs a process when the cancer cell dies, for example, in the case of an anticancer drug that kills the cancer cell. In addition, in the case of a drug that promotes cell differentiation, it is a molecular variation that performs a differentiation process.
　また、効き目が比較的早く現れる薬剤の作用機序を評価する場合には、数時間～数十時間といった比較的短い間隔が設定され、一方、効き目が現れるのに比較的時間がかかる薬剤の作用機序を評価する場合には、数週間～数か月といった比較的長い間隔が設定される。 In addition, when evaluating the mechanism of action of a drug that exhibits an effect relatively quickly, a relatively short interval such as several hours to several tens of hours is set, while the action of a drug that takes a relatively long time to exhibit an effect. When evaluating the mechanism, relatively long intervals such as weeks to months are set.
　制御部３０は、ＣＰＵ（Central Processing Unit）などを備えたものであり、分子計測装置全体を制御するものである。特に、本実施形態の制御部３０は、タイミング取得部３１を備えている。タイミング取得部３１は、撮像部２０において経時的に撮像された画像が入力され、その入力された複数枚の画像の特徴量の変動に基づいて、撮像対象の分割生体試料以外の分割生体試料の分子計測のタイミングまたは分子計測のための前処理のタイミングを取得するものである。なお、分子計測のタイミングまたは前処理のタイミングの取得方法については、後で詳述する。 The control unit 30 includes a CPU (Central Processing Unit) and the like, and controls the entire molecular measurement apparatus. In particular, the control unit 30 of the present embodiment includes a timing acquisition unit 31. The timing acquisition unit 31 receives an image captured with time in the imaging unit 20, and based on the variation in the feature amount of the input plurality of images, the divided biological sample other than the divided biological sample to be imaged. The timing of molecular measurement or the timing of preprocessing for molecular measurement is acquired. A method for acquiring the timing of molecular measurement or the timing of preprocessing will be described in detail later.
　また、制御部３０は、タイミング取得部３１によって取得された分子計測のタイミングまたは前処理のタイミングに基づいて、分子計測のタイミングであることを示す情報または前処理のタイミングであることを示す情報を表示部４０に表示させるものである。ユーザは、表示部４０に表示された情報を元に、撮像対象以外の分割生体試料の分子計測または前処理を開始することができる。 Further, the control unit 30 provides information indicating that it is molecular measurement timing or information indicating that it is preprocessing timing based on the molecular measurement timing or preprocessing timing acquired by the timing acquisition unit 31. This is displayed on the display unit 40. Based on the information displayed on the display unit 40, the user can start molecular measurement or preprocessing of a divided biological sample other than the imaging target.
　また、制御部３０は、撮像部２０の撮像動作を制御したり、また、刺激部１０が自動的に分割生体試料に刺激を与えるものである場合には、その刺激動作を制御したりするものである。 The control unit 30 controls the imaging operation of the imaging unit 20, or controls the stimulation operation when the stimulation unit 10 automatically stimulates the divided biological sample. It is.
　表示部４０は、液晶ディスプレイなどの表示デバイスを備えたものである。表示部４０は、上述したような分子計測または前処理のタイミングであることを示す情報を表示するものである。また、撮像部２０によって撮像された画像を表示するようにしてもよい。なお、本実施形態においては、表示部４０が本発明の報知装置または報知部に相当するものである。 The display unit 40 includes a display device such as a liquid crystal display. The display unit 40 displays information indicating the timing of molecular measurement or preprocessing as described above. Moreover, you may make it display the image imaged by the imaging part 20. FIG. In the present embodiment, the display unit 40 corresponds to the notification device or the notification unit of the present invention.
　入力部５０は、キーボードやマウスなどの入力デバイスを備えたものであり、ユーザによる種々の設定入力を受け付けるものである。たとえば、上述した撮像部２０における撮像間隔の設定入力を受け付けるものである。 The input unit 50 includes input devices such as a keyboard and a mouse, and accepts various setting inputs by the user. For example, the setting input of the imaging interval in the imaging part 20 mentioned above is received.
　前処理部６０は、分子計測対象の分割生体試料に対して前処理を施すものである。前処理部６０によって施される前処理とは、分割生体試料の分子計測が可能となるように分子計測の前に施される処理のことである。前処理としては、分子計測の種類などによって異なるが、化学的な処理を加える場合が多い。たとえばｃＤＮＡ（complementary deoxyribonucleic acid）マイクロアレイを用いた分子計測を行う場合には、細胞を溶かしてＲＮＡ（ribonucleic acid）を抽出し、ｃＲＮＡ（complementary ribonucleic acid）およびｃＤＮＡを合成および増幅し、断片化した後、ハイブリダイゼーションを行う処理が前処理として施される。 The pre-processing unit 60 performs pre-processing on the divided biological sample to be subjected to molecular measurement. The preprocessing performed by the preprocessing unit 60 is processing performed before molecular measurement so that molecular measurement of a divided biological sample is possible. As the pretreatment, chemical treatment is often added although it depends on the type of molecular measurement. For example, when performing molecular measurement using a cDNA (complementary ribonucleic acid) microarray, cells are dissolved, RNA (ribonucleic acid) is extracted, cRNA (complementary ribonucleic acid) and cDNA are synthesized, amplified, and fragmented. A process for performing hybridization is performed as a pretreatment.
　分子計測部７０は、分割生体試料における生体分子の量や状態を計測する分子計測を行うものである。分子計測の方法としては、ｃＤＮＡマイクロアレイを用いた分子計測、次世代シーケンサーを用いた発現および状態の計測、質量分析器を用いたプロテオーム計測、またはメタボローム計測などがある。また、バイオマーカー探索を行うようにしてもよい。この場合、刺激部１０で薬剤を添加した以降に量が増えたまたは量が減った分子のうち、薬剤濃度と相関するものをバイオマーカーとして決定する。 The molecular measurement unit 70 performs molecular measurement for measuring the amount and state of biomolecules in the divided biological sample. Molecular measurement methods include molecular measurement using a cDNA microarray, expression and state measurement using a next-generation sequencer, proteome measurement using a mass spectrometer, or metabolome measurement. Further, biomarker search may be performed. In this case, among the molecules whose amount has increased or decreased since the drug is added by the stimulating unit 10, those that correlate with the drug concentration are determined as biomarkers.
　次に、本実施形態の分子計測装置を用いた分子計測方法について、図２に示すフローチャート、図３および図４を参照しながら説明する。なお、本実施形態の分子計測装置は、分子計測のタイミングを取得する方法に特徴を有するものであるため、その点を中心に説明する。 Next, a molecular measurement method using the molecular measurement apparatus of the present embodiment will be described with reference to the flowchart shown in FIG. 2, FIG. 3 and FIG. In addition, since the molecular measurement apparatus of this embodiment has the characteristic in the method of acquiring the timing of molecular measurement, it demonstrates centering on the point.
　まず、図３に示すように、計測対象の生体試料Ｓ０が、複数の同じ容器に略同じ細胞数だけ分注されて分割生体試料Ｓ１～Ｓ３が作成される（Ｓ１０）。なお、本実施形態では、３つの第１～第３の分割生体試料Ｓ１～Ｓ３を作成するものとするが、分割生体試料の数はこれに限らず、２以上の数であればいくつでもよい。 First, as shown in FIG. 3, the biological sample S0 to be measured is dispensed into a plurality of the same containers by the substantially same number of cells to create divided biological samples S1 to S3 (S10). In the present embodiment, three first to third divided biological samples S1 to S3 are created. However, the number of divided biological samples is not limited to this, and any number may be used as long as the number is two or more. .
　次に、第１の分割生体試料Ｓ１の容器が刺激部１０に設置され、刺激部１０によって第１の分割生体試料Ｓ１に対して薬剤添加などの刺激が与えられる（Ｓ１２）。そして、この第１の分割生体試料Ｓ１に対して刺激が与えられた時刻が、制御部３０のタイミング取得部３１によって取得される。第１の分割生体試料Ｓ１に対して刺激が与えられた時刻は、ユーザが入力部５０を用いて入力するようにしてもよい。刺激部１０が自動的に刺激を与えるものである場合には、刺激を与えた時刻を刺激部１０からタイミング取得部３１に対して出力することによって、第１の分割生体試料Ｓ１に対して刺激が与えられた時刻を自動的に取得するようにしてもよい。 Next, the container of the first divided biological sample S1 is installed in the stimulation unit 10, and stimulation such as drug addition is given to the first divided biological sample S1 by the stimulation unit 10 (S12). The time at which the stimulus is given to the first divided biological sample S1 is acquired by the timing acquisition unit 31 of the control unit 30. The time when the stimulus is given to the first divided biological sample S <b> 1 may be input by the user using the input unit 50. When the stimulation unit 10 automatically gives stimulation, the stimulation time is output from the stimulation unit 10 to the timing acquisition unit 31 to stimulate the first divided biological sample S1. May be automatically acquired.
　次いで、第１の分割生体試料Ｓ１の容器が撮像部２０に設置され、撮像部２０によって経時的に複数枚の画像が撮像される（Ｓ１４）。 Next, the container of the first divided biological sample S1 is installed in the imaging unit 20, and a plurality of images are captured over time by the imaging unit 20 (S14).
　撮像部２０によって撮像された第１の分割生体試料Ｓ１の画像は、制御部３０のタイミング取得部３１に順次出力される。そして、タイミング取得部３１は、入力された各画像からそれぞれ特徴量を算出する。特徴量としては、たとえば画像内の細胞の外形に基づく特徴量、画像内の細胞の動きに基づく特徴量、画像内の蛍光標識された分子に基づく特徴量、および画像内の細胞内小器官に基づく特徴量のうちの少なくとも１つの特徴量が算出される。なお、複数の特徴量を算出する場合には、それらの特徴量に対して重み付けをして加算するようにしてもよい。 The images of the first divided biological sample S1 imaged by the imaging unit 20 are sequentially output to the timing acquisition unit 31 of the control unit 30. Then, the timing acquisition unit 31 calculates a feature amount from each input image. Features include, for example, feature quantities based on the outline of cells in the image, feature quantities based on cell movement in the image, feature quantities based on fluorescently labeled molecules in the image, and intracellular organelles in the image. At least one feature amount among the feature amounts to be based is calculated. When calculating a plurality of feature amounts, the feature amounts may be weighted and added.
　画像内の細胞の外形に基づく特徴量としては、たとえば画像内に含まれる複数の細胞の面積、径または長さの平均値などといった細胞の大きさの特徴量や、画像内に含まれる複数の細胞の円形度の平均値などといった細胞の形状の特徴量などがある。 As the feature amount based on the outer shape of the cell in the image, for example, the feature amount of the cell size such as the average value of the area, diameter, or length of the plurality of cells included in the image, or the plurality of features included in the image There is a feature amount of the cell shape such as an average value of the circularity of the cell.
　画像内の細胞の動きに基づく特徴量としては、画像内に含まれる複数の細胞の移動速度や移動距離の平均値などがある。なお、細胞の移動速度や移動距離は、複数枚の画像に基づいて算出されるものである。細胞の特定方法やその移動軌跡の追跡方法などは、公知な手法を用いることができる。 The feature amount based on the movement of cells in the image includes the moving speed and the average value of moving distances of a plurality of cells included in the image. Note that the moving speed and moving distance of the cells are calculated based on a plurality of images. Known methods can be used for the cell identification method and the tracking method of the movement trajectory.
　画像内の蛍光標識された分子に基づく特徴量としては、たとえば蛍光標識された分子から発せられた蛍光の強度に基づく特徴量があり、すなわち画像の輝度の平均値や最大値などがある。 As the feature amount based on the fluorescently labeled molecule in the image, for example, there is a feature amount based on the intensity of the fluorescence emitted from the fluorescently labeled molecule, that is, the average value or maximum value of the luminance of the image.
　また、画像内の細胞内小器官に基づく特徴量としては、たとえば細胞内に含まれるミトコンドリアの移動距離や移動速度などがある。なお、ミトコンドリアの移動速度や移動距離も、複数枚の画像に基づいて算出されるものである。ミトコンドリアの特定方法やその移動軌跡の追跡方法などは、公知な手法を用いることができる。 Further, the feature amount based on the organelle in the image includes, for example, the moving distance and moving speed of mitochondria contained in the cell. The moving speed and moving distance of mitochondria are also calculated based on a plurality of images. Known methods can be used as a method for identifying mitochondria and a method for tracking the movement locus thereof.
　タイミング取得部３１は、上述したような画像の特徴量を順次算出し、図４に示すような画像特徴量の経時変化を取得する。そして、タイミング取得部３１は、たとえば画像の特徴量の経時変化において、図４に示すようなピーク値が存在する場合には、そのピーク値の時刻を特徴量の変動点の時刻として特定して記録する（Ｓ１８）。図４に示す例の場合、タイミング取得部３１によって時刻Ｔ１と時刻Ｔ２とが特定されて記録される。そして、タイミング取得部３１は、第１の分割生体試料Ｓ１に対して刺激を与えた時刻Ｔ０から時刻Ｔ１までの時間ｔ１と、時刻Ｔ０から時刻Ｔ２までの時間ｔ２とを算出する。 The timing acquisition unit 31 sequentially calculates the feature amount of the image as described above, and acquires the temporal change of the image feature amount as shown in FIG. Then, for example, when the peak value as shown in FIG. 4 exists in the temporal change of the feature amount of the image, the timing acquisition unit 31 specifies the time of the peak value as the time of the variation point of the feature amount. Record (S18). In the case of the example illustrated in FIG. 4, the timing T <b> 1 and the time T <b> 2 are specified and recorded by the timing acquisition unit 31. Then, the timing acquisition unit 31 calculates time t1 from time T0 to time T1 when the first divided biological sample S1 is stimulated and time t2 from time T0 to time T2.
　上記のようにして第１の分割生体試料Ｓ１の画像の撮像が終了した後、次に、第２および第３の分割生体試料Ｓ２，Ｓ３が刺激部１０に設置され、これらの分割生体試料に対して、第１の分割生体試料Ｓ１に対して施された刺激と同じ刺激が刺激部１０によって与えられる（Ｓ２０）。 After the imaging of the first divided biological sample S1 is completed as described above, next, the second and third divided biological samples S2 and S3 are installed in the stimulating unit 10, and these divided biological samples are included in these divided biological samples. On the other hand, the same stimulus as the stimulus applied to the first divided biological sample S1 is given by the stimulation unit 10 (S20).
　そして、第２および第３の分割生体試料Ｓ２，Ｓ３に対して刺激が与えられた時刻が制御部３０のタイミング取得部３１によって取得される。第２および第３の分割生体試料Ｓ２に対して刺激が与えられた時刻も、ユーザが入力部５０を用いて入力するようにしてもよいし、自動的に取得するようにしてもよい。 Then, the timing at which the stimulus is applied to the second and third divided biological samples S2 and S3 is acquired by the timing acquisition unit 31 of the control unit 30. The time at which the stimulus is applied to the second and third divided biological samples S2 may be input by the user using the input unit 50, or may be automatically acquired.
　次いで、タイミング取得部３１は、第２および第３の分割生体試料Ｓ２，Ｓ３に対して刺激が与えられた時刻からの時間を計測し、上述した時刻Ｔ０から時刻Ｔ１までの時間ｔ１が経過したか否かを検出する。タイミング取得部３１によって時間ｔ１が経過したことが検出された場合には、制御部３０は、第２の分割生体試料Ｓ２について分子計測または前処理を促すメッセージを表示部４０に表示させる（Ｓ２２）。 Next, the timing acquisition unit 31 measures the time from the time when the stimulus is given to the second and third divided biological samples S2 and S3, and the time t1 from time T0 to time T1 described above has elapsed. Whether or not is detected. When the timing acquisition unit 31 detects that the time t1 has elapsed, the control unit 30 causes the display unit 40 to display a message for prompting molecular measurement or preprocessing for the second divided biological sample S2 (S22). .
　ユーザは、表示部４０に上記メッセージが表示された場合には、第２の分割生体試料Ｓ２を前処理部６０に設置し、第２の分割生体試料Ｓ２に対して分子計測を行うための前処理を施す（Ｓ２４）。さらに、前処理の施された第２の分割生体試料Ｓ２を分子計測部７０に設置し、第２の分割生体試料Ｓ２の分子計測を行う（Ｓ２６）。なお、本実施形態においては、上述したように表示部４０にメッセージを表示させることによって、ユーザに対して分子計測タイミングまたは前処理タイミングを報知するようにしたが、報知の方法としてはこれに限らず、たとえば光源を点灯させたり、音声で知らせたりするようにしてもよい。この場合、光源や音源が本発明の報知装置または報知部に相当する。 When the above message is displayed on the display unit 40, the user installs the second divided biological sample S2 in the preprocessing unit 60, and performs the molecular measurement on the second divided biological sample S2. Processing is performed (S24). Further, the pre-processed second divided biological sample S2 is installed in the molecular measurement unit 70, and the molecular measurement of the second divided biological sample S2 is performed (S26). In the present embodiment, as described above, the display unit 40 displays a message to notify the user of the molecular measurement timing or the preprocessing timing. However, the notification method is not limited thereto. Instead, for example, the light source may be turned on or notified by voice. In this case, the light source and the sound source correspond to the notification device or the notification unit of the present invention.
　また、タイミング取得部３１は、上述した時刻Ｔ０から時刻Ｔ２までの時間ｔ２が経過したか否かを検出する。タイミング取得部３１によって時間ｔ２が経過したことが検出された場合には、制御部３０は、第３の分割生体試料Ｓ３について分子計測または前処理を促すメッセージを表示部４０に表示させる（Ｓ２２）。 Also, the timing acquisition unit 31 detects whether or not the time t2 from the time T0 to the time T2 described above has elapsed. When the timing acquisition unit 31 detects that the time t2 has elapsed, the control unit 30 causes the display unit 40 to display a message for prompting molecular measurement or preprocessing for the third divided biological sample S3 (S22). .
　ユーザは、表示部４０に上記メッセージが表示された場合には、第３の分割生体試料Ｓ３を前処理部６０に設置し、第３の分割生体試料Ｓ３に対して分子計測を行うための前処理を施す（Ｓ２４）。さらに、前処理の施された第３の分割生体試料Ｓ３を分子計測部７０に設置し、第３の分割生体試料Ｓ３の分子計測を行う（Ｓ２６）。 When the above message is displayed on the display unit 40, the user installs the third divided biological sample S3 in the preprocessing unit 60, and performs the molecular measurement on the third divided biological sample S3. Processing is performed (S24). Furthermore, the pre-processed third divided biological sample S3 is placed in the molecular measurement unit 70, and the molecular measurement of the third divided biological sample S3 is performed (S26).
　上記実施形態の分子計測装置によれば、計測対象の生体試料Ｓ０を分割して複数の分割生体試料Ｓ1～Ｓ３を作成し、その複数の分割生体試料のうちの１つの分割生体試料Ｓ１に対して刺激を与えた時点から、その分割生体試料Ｓ１を経時的に複数回撮像する。そして、その撮像した複数枚の画像の特徴量の変動に基づいて、撮像対象の分割生体試料以外の分割生体試料Ｓ２，Ｓ３の分子計測のタイミングまたは分子計測のための前処理のタイミングを取得するようにしたので、効果的および効率的に分子計測または前処理のタイミングを取得することができる。 According to the molecular measurement apparatus of the above embodiment, the biological sample S0 to be measured is divided to create a plurality of divided biological samples S1 to S3, and one divided biological sample S1 among the plurality of divided biological samples is obtained. Then, the divided biological sample S1 is imaged a plurality of times over time from the time point when the stimulus is applied. Then, based on the variation in the feature amount of the plurality of captured images, the molecular measurement timing of the divided biological samples S2 and S3 other than the divided biological sample to be imaged or the preprocessing timing for the molecular measurement is acquired. Since it did in this way, the timing of molecular measurement or pre-processing can be acquired effectively and efficiently.
　なお、第１～第３の分割生体試料Ｓ１～Ｓ３の容器の搬送については、ユーザが手動で行うようにしてもよいし、ロボットアームや搬送テーブルや搬送ベルトなどを用いて自動的に搬送するようにしてもよい。 The containers of the first to third divided biological samples S1 to S3 may be manually transported by the user, or automatically transported using a robot arm, a transport table, a transport belt, or the like. You may do it.
　また、上記実施形態では、第１の分割生体試料Ｓ１を撮像した画像の特徴量が変動した時点を第２および第３の分割生体試料Ｓ２，Ｓ３の前処理のタイミングまたは分子計測のタイミングとして取得するようにしたが、刺激された細胞の分子変動は、厳密には細胞の画像の特徴量が変動する前から生じており、その分子変動を計測することがより好ましい。 Moreover, in the said embodiment, the time of the feature-value variation of the image which imaged 1st division | segmentation biological sample S1 changed is acquired as the timing of the pre-processing of 2nd and 3rd division | segmentation biological sample S2, S3, or the timing of molecular measurement. However, strictly speaking, the molecular variation of the stimulated cell occurs before the feature amount of the cell image varies, and it is more preferable to measure the molecular variation.
　したがって、以下、このような画像の特徴量の変動の前に発生した分子変動を計測することができる分子計測タイミングまたは前処理タイミングの取得方法について、図５に示すフローチャートおよび図６を参照しながら説明する。なお、分子計測装置の概略構成は、図１に示す上記実施形態の概略構成と同様である。 Therefore, hereinafter, a molecular measurement timing or preprocessing timing acquisition method capable of measuring the molecular variation generated before the variation of the image feature amount will be described with reference to the flowchart shown in FIG. 5 and FIG. explain. The schematic configuration of the molecular measurement apparatus is the same as the schematic configuration of the above embodiment shown in FIG.
　まず、上記実施形態と同様に、計測対象の生体試料Ｓ０が、複数の同じ容器に略同じ細胞数だけ分注されて分割生体試料Ｓ１～Ｓ３が作成される（Ｓ３０）。 First, as in the above embodiment, the biological sample S0 to be measured is dispensed into a plurality of the same containers by the substantially same number of cells, and divided biological samples S1 to S3 are created (S30).
　次に、第１の分割生体試料Ｓ１の容器が刺激部１０に設置され、刺激部１０によって第１の分割生体試料Ｓ１に対して薬剤添加などの刺激が与えられる（Ｓ３２）。そして、この第１の分割生体試料Ｓ１に対して刺激が与えられた時刻が、制御部３０のタイミング取得部３１によって取得される。 Next, the container of the first divided biological sample S1 is installed in the stimulating unit 10, and stimulation such as addition of a drug is given to the first divided biological sample S1 by the stimulating unit 10 (S32). The time at which the stimulus is given to the first divided biological sample S1 is acquired by the timing acquisition unit 31 of the control unit 30.
　次いで、第１の分割生体試料Ｓ１については、上記実施形態と同様に、撮像部２０に設置され、撮像部２０によって経時的に複数枚の画像が撮像される（Ｓ３４）。そして、撮像部２０によって撮像された第１の分割生体試料Ｓ１の画像は、制御部３０のタイミング取得部３１に順次出力され、上記実施形態と同様に、タイミング取得部３１によって画像の特徴量が算出され、その経時変化が取得される（Ｓ３６）。 Next, the first divided biological sample S1 is installed in the imaging unit 20 as in the above embodiment, and a plurality of images are captured over time by the imaging unit 20 (S34). Then, the image of the first divided biological sample S1 captured by the imaging unit 20 is sequentially output to the timing acquisition unit 31 of the control unit 30, and the feature amount of the image is acquired by the timing acquisition unit 31 as in the above embodiment. It is calculated and its change with time is acquired (S36).
　一方、上述した第１の分割生体試料Ｓ１の画像の撮像および特徴量の算出に並行して、第１の分割生体試料Ｓ１に対して刺激が与えられた時刻Ｔ０からの経過時間がタイミング取得部３１によって計測される。そして、タイミング取得部３１は、時刻Ｔ０からの経過時間が予め設定された遅延時間ＤＴを経過したか否かを検出する（Ｓ３８）。この予め設定された遅延時間とは、生体試料の細胞内の分子変動が生じてからその画像の特徴量の変動が生じるまでの時間であり、予め実験などを行うことによって計測された時間である。 On the other hand, in parallel with the above-described imaging of the first divided biological sample S1 and calculation of the feature amount, an elapsed time from the time T0 when the stimulus is given to the first divided biological sample S1 is a timing acquisition unit. 31. Then, the timing acquisition unit 31 detects whether or not the elapsed time from the time T0 has passed a preset delay time DT (S38). This preset delay time is the time from the occurrence of molecular variation in the cell of the biological sample to the variation in the feature amount of the image, which is the time measured by conducting an experiment or the like in advance. .
　タイミング取得部３１によって時刻Ｔ０からの経過時間が遅延時間ＤＴを経過したことが検出された場合には、図６に示すように、その検出時点Ｔ３において、第２および第３の分割生体試料Ｓ２，Ｓ３が刺激部１０に設置され、第２および第３の分割生体試料Ｓ２，Ｓ３に対して刺激が与えられる（Ｓ４０）。 When it is detected by the timing acquisition unit 31 that the elapsed time from the time T0 has passed the delay time DT, as shown in FIG. 6, the second and third divided biological samples S2 at the detection time T3. , S3 are installed in the stimulation unit 10, and the second and third divided biological samples S2, S3 are stimulated (S40).
　なお、この際、制御部３０が、遅延時間ＤＴが経過したことを表示部４０に表示させ、その表示を見たユーザが、第２および第３の分割生体試料Ｓ２，Ｓ３に手動で刺激を与えるようにしてもよいし、刺激部１０が自動的に刺激を与えるものである場合には、制御部３０が刺激部１０を制御することによって自動的に刺激を与えるようにしてもよい。 At this time, the control unit 30 causes the display unit 40 to display that the delay time DT has elapsed, and the user who sees the display manually stimulates the second and third divided biological samples S2 and S3. Alternatively, when the stimulation unit 10 automatically applies stimulation, the control unit 30 may control the stimulation unit 10 to automatically apply stimulation.
　次いで、上述したようにして第２および第３の分割生体試料Ｓ２，Ｓ３に対して刺激が与えられた後、図６に示すように、タイミング取得部３１によって第１の分割生体試料Ｓ１の画像の特徴量の変動点が検出された場合には（Ｓ４２，ＹＥＳ）、タイミング取得部３１は、この検出時点Ｔ４を分子計測タイミングまたは前処理のタイミングとして取得し、制御部３０は、第２の分割生体試料Ｓ２について分子計測または前処理を促すメッセージを表示部４０に表示させる（Ｓ４４）。なお、タイミング取得部３１によって特徴量の変動点を検出する方法としては、たとえば画像の特徴量が図４に示すように経時変化する場合には、特徴量が増加傾向から減少傾向に変わったことを検出することによって変動点を検出するようにすればよい。 Next, after the stimulus is applied to the second and third divided biological samples S2 and S3 as described above, an image of the first divided biological sample S1 is obtained by the timing acquisition unit 31 as shown in FIG. When the variation point of the feature amount is detected (S42, YES), the timing acquisition unit 31 acquires the detection time T4 as the molecular measurement timing or the preprocessing timing, and the control unit 30 A message for prompting molecular measurement or preprocessing for the divided biological sample S2 is displayed on the display unit 40 (S44). In addition, as a method of detecting the variation point of the feature amount by the timing acquisition unit 31, for example, when the feature amount of the image changes with time as shown in FIG. 4, the feature amount has changed from an increasing tendency to a decreasing tendency. The variation point may be detected by detecting.
　ここで、第１の分割生体試料Ｓ１に対して刺激を与えてから分子変動を発生するまでの時間と、第２の分割生体試料Ｓ２に対して刺激を与えてから分子変動を発生するまでの時間とが同じ時間ａであるとすると、第２の分割生体試料Ｓ２は、第１の分割生体試料Ｓ１よりも遅延時間ＤＴだけ遅れて刺激を与えられているので、第１の分割生体試料Ｓ１の画像の特徴量の変動点は、第２の分割生体試料Ｓ２において、分子変動が発生している時刻となる。 Here, the time from when the stimulus is applied to the first divided biological sample S1 to the time when the molecular variation occurs, and the time from when the stimulus is applied to the second divided biological sample S2 until the time when the molecular variation occurs. Assuming that the time is the same time a, the second divided biological sample S2 is stimulated with a delay time DT from the first divided biological sample S1, and therefore the first divided biological sample S1. The variation point of the feature amount of the image is the time when the molecular variation occurs in the second divided biological sample S2.
　ユーザは、表示部４０に上記メッセージが表示された場合には、第２の分割生体試料Ｓ２を前処理部６０に設置し、第２の分割生体試料Ｓ２に対して分子計測を行うための前処理を施す（Ｓ４６）。さらに、前処理の施された第２の分割生体試料Ｓ２を分子計測部７０に設置し、第２の分割生体試料Ｓ２の分子計測を行う（Ｓ４８）。 When the above message is displayed on the display unit 40, the user installs the second divided biological sample S2 in the preprocessing unit 60, and performs the molecular measurement on the second divided biological sample S2. Processing is performed (S46). Further, the pre-processed second divided biological sample S2 is installed in the molecular measurement unit 70, and the molecular measurement of the second divided biological sample S2 is performed (S48).
　さらに、第２の分割生体試料Ｓ２の分子計測タイミングまたは前処理タイミングを取得した後、図６に示すように、タイミング取得部３１によって第１の分割生体試料Ｓ１の画像の特徴量の変動点が再び検出された場合には（Ｓ４２，ＹＥＳ）、タイミング取得部３１は、この検出時点Ｔ５を第３の分割生体試料Ｓ３の分子計測タイミングまたは前処理のタイミングとして取得し、制御部３０は、第３の分割生体試料Ｓ３について分子計測または前処理を促すメッセージを表示部４０に表示させる（Ｓ４４）。 Further, after acquiring the molecular measurement timing or the preprocessing timing of the second divided biological sample S2, as shown in FIG. 6, the timing acquisition unit 31 determines the variation point of the feature amount of the image of the first divided biological sample S1. If it is detected again (S42, YES), the timing acquisition unit 31 acquires this detection time T5 as the molecular measurement timing or preprocessing timing of the third divided biological sample S3, and the control unit 30 A message prompting molecular measurement or preprocessing for the three divided biological samples S3 is displayed on the display unit 40 (S44).
　ここで、第１の分割生体試料Ｓ１に対して刺激を与えてから２回目の分子変動を発生するまでの時間と、第３の分割生体試料Ｓ３に対して刺激を与えてから２回目の分子変動を発生するまでの時間とが同じ時間ｂであるとすると、第３の分割生体試料Ｓ３は、第１の分割生体試料Ｓ１よりも遅延時間ＤＴだけ遅れて刺激を与えられているので、第１の分割生体試料Ｓ１の画像の特徴量の２回目の変動点は、第３の分割生体試料Ｓ３において、２回目の分子変動が発生している時刻となる。 Here, the time from when the stimulus is applied to the first divided biological sample S1 until the second molecular fluctuation occurs, and the second molecule after the stimulus is applied to the third divided biological sample S3. Assuming that the time until the fluctuation occurs is the same time b, the third divided biological sample S3 is stimulated with a delay time DT from the first divided biological sample S1. The second variation point of the feature quantity of the image of one divided biological sample S1 is the time when the second molecular variation occurs in the third divided biological sample S3.
　ユーザは、表示部４０に上記メッセージが表示された場合には、第３の分割生体試料Ｓ３を前処理部６０に設置し、第３の分割生体試料Ｓ３に対して分子計測を行うための前処理を施す（Ｓ４６）。さらに、前処理の施された第３の分割生体試料Ｓ３を分子計測部７０に設置し、第３の分割生体試料Ｓ３の分子計測を行う（Ｓ４８）。 When the above message is displayed on the display unit 40, the user installs the third divided biological sample S3 in the preprocessing unit 60, and performs the molecular measurement on the third divided biological sample S3. Processing is performed (S46). Further, the pre-processed third divided biological sample S3 is installed in the molecular measurement unit 70, and the molecular measurement of the third divided biological sample S3 is performed (S48).
　上記実施形態の分子計測タイミングまたは前処理タイミングの取得方法によれば、分子変動をより的確に計測することができる。また、分子計測タイミングまたは前処理タイミングを取得するための処理と、分子計測を行う処理を並行して行うことができるので、先に説明した分子計測タイミングまたは前処理タイミングの取得方法と比較すると、トータルの処理期間を短縮することができる。 According to the molecular measurement timing or preprocessing timing acquisition method of the above embodiment, molecular variation can be measured more accurately. In addition, since the process for acquiring the molecular measurement timing or the preprocessing timing and the process for performing the molecular measurement can be performed in parallel, compared with the method for acquiring the molecular measurement timing or the preprocessing timing described above, The total processing period can be shortened.
　なお、図６に示す例では、第１の分割生体試料Ｓ１の１回目の分子変動から画像特徴量の変動までの時間と、２回目の分子変動から画像特徴量の変動までの時間を同じ時間ＤＴとして、第２および第３の分割生体試料Ｓ２，Ｓ３に対して刺激を与えるタイミングをともに遅延時間ＤＴだけ遅らせるようにしたが、第１の分割生体試料Ｓ１の１回目の分子変動から画像特徴量の変動までの時間と、２回目の分子変動から画像特徴量の変動までの時間とは必ずしも同じでなくてもよい。たとえば１回目と２回目とで異なる種類の画像の特徴量を算出するようにしてもよく、その場合、１回目と２回目とで分子変動から画像特徴量の変動までの時間が異なることになる。 In the example shown in FIG. 6, the time from the first molecular variation to the variation in the image feature amount of the first divided biological sample S1 is the same as the time from the second molecular variation to the variation in the image feature amount. As the DT, the timing for applying the stimulus to the second and third divided biological samples S2 and S3 is both delayed by the delay time DT. The time from the amount of change to the amount of change and the time from the second molecular change to the change in the image feature amount are not necessarily the same. For example, the feature amounts of different types of images may be calculated for the first time and the second time. In this case, the time from the molecular variation to the variation of the image feature amount is different for the first time and the second time. .
　たとえば、画像の特徴量として、上述した細胞の外形や細胞の移動に基づく特徴量を算出する場合には、分子変動から画像特徴量の変動までの時間は相対的に長く、蛍光標識された分子または細胞内小器官に基づく特徴量を算出する場合には、分子変動から画像特徴量の変動までの時間は相対的に短くなる場合が多い。 For example, when calculating the feature amount based on the above-described cell outline or cell movement as the feature amount of the image, the time from the molecular change to the change of the image feature amount is relatively long, and the fluorescently labeled molecule Alternatively, when calculating feature quantities based on intracellular organelles, the time from molecular variation to image feature variation is often relatively short.
　したがって、たとえば第１の分割生体試料Ｓ１について、１回目は蛍光標識された分子または細胞内小器官に基づく特徴量を算出し、２回目は細胞の外形や細胞の移動に基づく特徴を算出する場合には、たとえば、図７に示すように第２の分割生体試料Ｓ２に対しては、蛍光標識された分子または細胞内小器官に基づく特徴量が変動する時間を遅延時間ＤＴ１として割り当て、第３の分割生体試料Ｓ３に対しては、細胞の外形や細胞の移動に基づく特徴量が変動する時間を遅延時間ＤＴ２として割り当てるようにしてもよい。 Therefore, for example, for the first divided biological sample S1, the first time calculates a feature amount based on a fluorescently labeled molecule or an intracellular organelle, and the second time calculates a feature based on the outer shape of the cell or the movement of the cell. For example, as shown in FIG. 7, for the second divided biological sample S2, a time when the feature amount based on the fluorescently labeled molecule or the organelle is changed is assigned as the delay time DT1, and the third For the divided biological sample S3, a time when the feature amount based on the outer shape of the cell or the movement of the cell fluctuates may be assigned as the delay time DT2.
　そして、第２の分割生体試料Ｓ２に対しては、第１の分割生体試料Ｓ１よりも遅延時間ＤＴ１だけ遅らせて刺激を与え、第３の分割生体試料Ｓ３に対しては、第１の分割生体試料Ｓ１よりも遅延時間ＤＴ２だけ遅らせて刺激を与えるようにしてもよい。 Then, the second divided biological sample S2 is stimulated by delaying the delay time DT1 from the first divided biological sample S1, and the first divided biological sample S3 is stimulated. Stimulation may be applied by delaying the sample S1 by the delay time DT2.
　この場合、第１の分割生体試料Ｓ１の１回目の画像特徴量の変動点が、第２の分割生体試料Ｓ２の分子計測タイミングまたは前処理タイミングとして取得され、第１の分割生体試料Ｓ１の２回目の画像特徴量の変動点が、第３の分割生体試料Ｓ３の分子計測タイミングまたは前処理タイミングとして取得される。 In this case, the variation point of the first image feature amount of the first divided biological sample S1 is acquired as the molecular measurement timing or the preprocessing timing of the second divided biological sample S2, and 2 of the first divided biological sample S1. The variation point of the second image feature amount is acquired as the molecular measurement timing or the preprocessing timing of the third divided biological sample S3.
　なお、上述したような画像の特徴量の種類に応じた遅延時間は、図８に示すようなテーブルとして予め設定しておくようにし、ユーザが、第１の分割生体試料Ｓ１に対してどの種類の特徴量を算出するかを設定し、その設定に応じて制御部３０がテーブルを参照して遅延時間を自動的に取得し、第２および第３の分割生体試料Ｓ２，Ｓ３に対して割り当てるようにすればよい。 Note that the delay time corresponding to the type of the image feature amount as described above is set in advance as a table as shown in FIG. 8, and the user can select which type for the first divided biological sample S1. The control unit 30 automatically obtains a delay time by referring to the table according to the setting and assigns it to the second and third divided biological samples S2 and S3. What should I do?
１０　 刺激部
２０　 撮像部
３０　 制御部
３１　 タイミング取得部
４０　 表示部
５０　 入力部
６０　 前処理部
７０　 分子計測部 DESCRIPTION OF SYMBOLS 10 Stimulation part 20 Imaging part 30 Control part 31 Timing acquisition part 40 Display part 50 Input part 60 Pre-processing part 70 Molecular measurement part

Claims (15)

  1. 　計測対象の生体試料を分割して複数の分割生体試料を作成し、
    　該複数の分割生体試料のうちの１つの分割生体試料に対して刺激を与えた時点から、該分割生体試料を経時的に複数回撮像し、
    　該撮像した複数枚の画像の特徴量の変動に基づいて、前記撮像対象の分割生体試料以外の分割生体試料の分子計測のタイミングまたは分子計測のための前処理のタイミングを取得することを特徴とする分子計測タイミング取得方法。     Divide the biological sample to be measured to create multiple divided biological samples,
    From the time when a stimulus is given to one of the plurality of divided biological samples, the divided biological sample is imaged multiple times over time,
    Obtaining the timing of molecular measurement of a divided biological sample other than the divided biological sample to be imaged or the timing of pre-processing for molecular measurement based on the variation in the feature amount of the plurality of captured images. To obtain molecular measurement timing.
  2. 　前記分子計測のタイミングまたは前記前処理のタイミングを報知装置によって報知する請求項１記載の分子計測タイミング取得方法。 The molecular measurement timing acquisition method according to claim 1, wherein a notification device notifies the timing of the molecular measurement or the timing of the preprocessing.
  3. 　前記撮像対象の分割生体試料以外の分割生体試料に対して、前記撮像対象の分割生体試料に対して与えた刺激と同じ刺激を与えた後、
    　前記取得した分子計測のタイミングに基づいて分子計測を行うまたは前記取得した前処理のタイミングに基づいて前処理を行う請求項１または２記載の分子計測タイミング取得方法。     After giving the same stimulus as the stimulus given to the divided biological sample other than the divided biological sample of the imaging target to the divided biological sample of the imaging target,
    The molecular measurement timing acquisition method according to claim 1, wherein molecular measurement is performed based on the acquired molecular measurement timing or preprocessing is performed based on the acquired preprocessing timing.
  4. 　前記撮像対象の分割生体試料に対して前記刺激を与えた時点から、予め設定された遅延時間だけ経過した時点において、前記撮像対象の分割生体試料以外の分割生体試料に対して、前記撮像対象の分割生体試料に対して与えた刺激と同じ刺激を与え、
    　該刺激を与えた後、前記撮像対象の分割生体試料の画像の特徴量が変動した時点を、前記撮像対象の分割生体試料以外の分割生体試料の分子計測のタイミングまたは分子計測のための前処理のタイミングとして取得する請求項１または２記載の分子計測タイミング取得方法。     When a predetermined delay time elapses from the time when the stimulus is applied to the divided biological sample to be imaged, the divided biological sample other than the divided biological sample to be imaged is compared with that of the imaging target. Give the same stimulus to the divided biological sample,
    After applying the stimulus, the time point when the feature amount of the image of the divided biological sample to be imaged fluctuates, and the timing of molecular measurement of a divided biological sample other than the divided biological sample to be imaged or preprocessing for molecular measurement The molecular measurement timing acquisition method according to claim 1, wherein the acquisition timing is acquired as the timing of the above.
  5. 　前記取得した分子計測のタイミングに基づいて分子計測を行うまたは前記取得した前処理のタイミングに基づいて前処理を行う請求項４記載の分子計測タイミング取得方法。 5. The molecular measurement timing acquisition method according to claim 4, wherein molecular measurement is performed based on the acquired molecular measurement timing or preprocessing is performed based on the acquired preprocessing timing.
  6. 　前記遅延時間が、前記生体試料である細胞内の分子変動が生じてから前記画像の特徴量の変動が生じるまでの時間である請求項４または５記載の分子計測タイミング取得方法。 6. The molecular measurement timing acquisition method according to claim 4 or 5, wherein the delay time is a time from when a molecular variation occurs in a cell, which is the biological sample, to a variation in the feature amount of the image.
  7. 　前記画像の特徴量の種類に対応する前記遅延時間を複数設定し、
    　前記撮像対象の分割生体試料以外の複数の分割生体試料に対して、前記複数の遅延時間をそれぞれ割り当てて、前記複数の分割生体試料に対してそれぞれ異なるタイミングで前記刺激を与え、
    　該刺激を与えた後、前記撮像対象の分割生体試料の画像の特徴量が変動した際、該変動した前記特徴量の種類に対応した前記遅延時間が割り当てられた前記分割生体試料の前記分子計測のタイミングまたは前記前処理のタイミングを取得する請求項４から６いずれか１項記載の分子計測タイミング取得方法。     A plurality of the delay times corresponding to the types of feature quantities of the image,
    Assigning each of the plurality of delay times to a plurality of divided biological samples other than the divided biological sample to be imaged, and applying the stimulation at different timings to the plurality of divided biological samples,
    After the stimulation, when the feature amount of the image of the divided biological sample to be imaged changes, the molecular measurement of the divided biological sample to which the delay time corresponding to the type of the changed feature amount is assigned. The molecular measurement timing acquisition method according to any one of claims 4 to 6, wherein the timing of said step or the timing of said preprocessing is acquired.
  8. 　前記画像内の細胞の外形に基づく前記特徴量、前記画像内の細胞の動きに基づく前記特徴量、前記画像内の蛍光標識された分子に基づく前記特徴量および前記画像内の細胞内小器官に基づく前記特徴量のうちの少なくとも２つに対応した前記遅延時間がそれぞれ設定されている請求項７記載の分子計測タイミング取得方法。 The feature amount based on the outer shape of the cell in the image, the feature amount based on the movement of the cell in the image, the feature amount based on the fluorescently labeled molecule in the image, and the intracellular organelle in the image The molecular measurement timing acquisition method according to claim 7, wherein the delay times corresponding to at least two of the feature quantities are set.
  9. 　計測対象の生体試料を分割した複数の分割生体試料のうちの１つの分割生体試料に対して刺激を与えた時点から、該分割生体試料を経時的に複数回撮像する撮像部と、
    　該撮像部によって撮像された複数枚の画像の特徴量の変動に基づいて、前記撮像対象の分割生体試料以外の分割生体試料の分子計測のタイミングまたは分子計測のための前処理のタイミングを取得するタイミング取得部とを備えたことを特徴とする分子計測タイミング取得装置。     An imaging unit that images the divided biological sample a plurality of times over time from the time when a stimulus is given to one divided biological sample among the plurality of divided biological samples obtained by dividing the biological sample to be measured;
    Based on fluctuations in feature quantities of a plurality of images captured by the imaging unit, the timing of molecular measurement of a divided biological sample other than the divided biological sample to be imaged or the timing of preprocessing for molecular measurement is acquired. A molecular measurement timing acquisition device comprising a timing acquisition unit.
  10. 　前記分子計測のタイミングまたは前記前処理のタイミングを報知する報知部を備えた請求項９記載の分子計測タイミング取得装置。 The molecular measurement timing acquisition device according to claim 9, further comprising a notification unit that notifies the timing of the molecular measurement or the timing of the preprocessing.
  11. 　前記撮像対象の分割生体試料以外の分割生体試料に対して、前記撮像対象の分割生体試料に対して与えた刺激と同じ刺激が与えられた後、前記取得した分子計測のタイミングに基づいて分子計測を行う分子計測部を備えた請求項９または１０記載の分子計測タイミング取得装置。 After the same stimulus as the stimulus given to the divided biological sample to be imaged is given to the divided biological sample other than the divided biological sample to be imaged, the molecular measurement is performed based on the timing of the acquired molecular measurement. The molecular measurement timing acquisition apparatus of Claim 9 or 10 provided with the molecular measurement part which performs.
  12. 　前記撮像対象の分割生体試料以外の分割生体試料に対して、前記撮像対象の分割生体試料に対して与えた刺激と同じ刺激が与えられた後、前記取得した前処理のタイミングに基づいて前処理を行う前処理部を備えた請求項９または１０記載の分子計測タイミング取得装置。 After the same stimulus as the stimulus given to the divided biological sample to be imaged is given to the divided biological sample other than the divided biological sample to be imaged, preprocessing is performed based on the timing of the acquired preprocessing. The molecular measurement timing acquisition apparatus of Claim 9 or 10 provided with the pre-processing part which performs.
  13. 　前記撮像対象の分割生体試料に対して前記刺激を与えた時点から、予め設定された遅延時間だけ経過した時点において、前記撮像対象の分割生体試料以外の分割生体試料に対して、前記撮像対象の分割生体試料に対して与えた刺激と同じ刺激を与える刺激部を備え、
    　前記タイミング取得部が、前記撮像対象の分割生体試料の画像の特徴量が変動した時点を、前記撮像対象の分割生体試料以外の分割生体試料の分子計測のタイミングまたは分子計測のための前処理のタイミングとして取得する請求項９または１０記載の分子計測タイミング取得装置。     When a predetermined delay time elapses from the time when the stimulus is applied to the divided biological sample to be imaged, the divided biological sample other than the divided biological sample to be imaged is compared with that of the imaging target. A stimulation unit that gives the same stimulus as the stimulus given to the divided biological sample is provided,
    The timing acquisition unit uses the timing of the molecular measurement of the divided biological sample other than the divided biological sample to be imaged or the preprocessing for the molecular measurement as the characteristic amount of the image of the divided biological sample to be imaged changes. The molecular measurement timing acquisition apparatus according to claim 9 or 10, which is acquired as timing.
  14. 　前記タイミング取得部によって取得された分子計測のタイミングに基づいて分子計測を行う分子計測部を備えた請求項１３記載の分子計測タイミング取得装置。 The molecular measurement timing acquisition apparatus according to claim 13, further comprising a molecular measurement unit that performs molecular measurement based on the molecular measurement timing acquired by the timing acquisition unit.
  15. 　前記タイミング取得部によって取得された前記前処理のタイミングに基づいて前処理を行う前処理部を備えた請求項１３または１４記載の分子計測タイミング取得装置。 The molecular measurement timing acquisition device according to claim 13 or 14, further comprising a preprocessing unit that performs preprocessing based on the timing of the preprocessing acquired by the timing acquisition unit.
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