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WO2018225382A1 - Dispositif et procédé d'analyse d'état de cellule - Google Patents

Dispositif et procédé d'analyse d'état de cellule Download PDF

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Publication number
WO2018225382A1
WO2018225382A1 PCT/JP2018/015523 JP2018015523W WO2018225382A1 WO 2018225382 A1 WO2018225382 A1 WO 2018225382A1 JP 2018015523 W JP2018015523 W JP 2018015523W WO 2018225382 A1 WO2018225382 A1 WO 2018225382A1
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WIPO (PCT)
Prior art keywords
light
sample
cell
spheroid
objective lens
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PCT/JP2018/015523
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English (en)
Japanese (ja)
Inventor
直子 千田
賢太郎 大澤
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Hitachi High Tech Corp
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Hitachi High Technologies Corp
Hitachi High Tech Corp
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/06—Means for illuminating specimens
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/34—Microscope slides, e.g. mounting specimens on microscope slides
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G02B21/36—Microscopes arranged for photographic purposes or projection purposes or digital imaging or video purposes including associated control and data processing arrangements

Definitions

  • the present invention relates to an apparatus and method for analyzing the state of a cell, for example, a spheroid.
  • Spheroids are known to cause cell death such as necrosis mainly from the center because the nutrients and oxygen in the medium are difficult to reach the cells in the center during the culturing process.
  • cell death refers to necrosis (necrosis) and apoptosis (natural death).
  • necrosis cells expand, the cytoplasm changes, and the cell membrane ruptures.
  • apoptosis cells shrink and are phagocytosed by immune cells.
  • the spheroid outer shape does not change at this time.
  • spheroids that cause cell death inside are considered to be unsuitable for transplantation because the number of living cells is small and undesirable substances may be released from dead cells.
  • spheroids are mainly used for drug discovery, and cell death inside spheroids has not been a serious problem.
  • the spheroid evaluation method is not necessarily noninvasive, and a method of measuring the enzyme activity of living cells using a dye has been used for counting the number of living cells.
  • non-invasive evaluation techniques for spheroids corresponding to those quality evaluation items are required.
  • spheroids are verified by observation with a phase contrast microscope or tissue staining. Observation of cells with a phase contrast microscope is non-invasive, but the phase contrast microscope cannot measure the three-dimensional shape of a spheroid or evaluate cell death that occurs inside the spheroid. Although cell death can be evaluated by tissue staining, it is an invasive technique for fixing and embedding spheroids, and it takes time to judge the result, and the spheroid for transplantation itself cannot be evaluated. Therefore, it is essential to develop a technology capable of noninvasively evaluating cell death inside the spheroid.
  • those having a cell level resolution include an optical coherence tomography (OCT), a reflective confocal microscope, and the like. These are techniques capable of illuminating a sample and imaging the internal state using reflected light from within the sample.
  • OCT optical coherence tomography
  • a reflective confocal microscope and the like.
  • Patent Document 1 in the epi-illumination microscope, a reflection mirror is provided, the objective lens is driven in the z-axis direction, the focal position is variable, and the detection light reflected from the back surface of the sample is detected via the reflection surface. Describes how to perform three-dimensional observation of samples.
  • Patent Document 2 cells are placed in an observation container with a reflecting surface and the incident wavefront is deformed twice compared to conventional transmission observation by detecting light that has been transmitted twice through incident light, improving contrast. It describes how to make it.
  • 3D measurement with reflected light is considered effective as a non-invasive method for analyzing cell states such as cell death inside spheroids.
  • the sample assumed in Patent Document 1 has a spherical shape, it is not a biological sample, and the acquired data is not information inside the sample but relates to the outer peripheral shape of the sample. In such a case, even if there is defocused reflected light from the reflecting surface, the reflected light from the outer periphery of the sample to be detected is sufficiently large, so the problem of image degradation is not serious.
  • the measurement sample is a spheroid that is a cell agglomeration and the data to be acquired is information inside the sample, the defocused reflected light from the reflecting surface causes noise degradation as noise. This is due to the fact that the reflectance of cells is as small as 1 to 10 m%.
  • the penetration depth of the three-dimensional measuring device is usually reduced by imaging the spheroid using a three-dimensional measuring device and installing a reflecting surface on the opposite side of the objective lens with respect to the sample. It was found that the information inside the back of the spheroids that can not be reached can also be acquired. It has also been found that a high-quality image can be obtained by blocking the central portion of the light beam to suppress the defocus component from the reflecting surface that becomes noise. The present invention has been completed based on the above findings.
  • this disclosure A light source; An objective lens for condensing the light from the light source inside the sample; A light-shielding portion that shields the central portion of the light beam from the light source; A reflection surface installation place for installing a reflection surface that is provided on the opposite side of the objective lens with respect to the sample and reflects light whose light beam center is shielded by the light shielding portion; And a photodetector The photodetector can detect the reflected light reflected again by the reflecting surface, the signal light from the focal position irradiated with the light reflected by the reflecting surface,
  • the light-shielding unit relates to a three-dimensional measurement apparatus, wherein the light-shielding unit is configured to shield at least a part of the light reflected from the reflection surface and transmitted through the sample.
  • This disclosure also Irradiating the sample with light from a light source, A step of acquiring a plurality of sample tomographic images having different distances from the light irradiation position, wherein (a) the distance from the light irradiation position exceeds a light penetration depth limit, the light from the light source The central part of the light beam is shielded by the light shielding part, and the sampled tomographic image is acquired by reflecting the shielded light by the reflecting surface. (B) The distance from the light irradiation position is the distance to the light penetration depth limit.
  • the present invention relates to a cell state analysis method including a step of analyzing a cell state of a sample based on an acquired image.
  • the present invention provides an apparatus and method for analyzing a cell state.
  • the apparatus and method of the present disclosure can noninvasively analyze the state inside the back surface of a cell (for example, spheroid) that was not obtained from normal one-way observation. Therefore, it is useful for producing cells, particularly cells for use in regenerative medicine such as transplantation, for example, spheroids.
  • the present disclosure relates to a method for analyzing a cell state and a three-dimensional measurement apparatus that can be used for analysis of the cell state.
  • the sample to be analyzed is not particularly limited, and can be a sample containing cells.
  • cells of any form such as single cells, cell clusters, tissues, etc. can be analyzed.
  • the cell is a cell mass containing spheroids.
  • a spheroid refers to a three-dimensional cell aggregate in which cells aggregate and aggregate.
  • spheroids composed of stem cells, chondrocytes, hepatocytes, corneal cells, epidermal cells, cardiomyocytes, nerve cells, and progenitor cells thereof are used, but not limited thereto.
  • the origin of the cell is not particularly limited, and is, for example, an animal, preferably a mammalian cell. Specifically, a primate (human, monkey, chimpanzee, gorilla, etc.), an experimental animal (mouse, rat, etc.) ), Livestock animals (cattle, pigs, rabbits, etc.) and pet animals (dogs, cats, etc.).
  • FIG. 2 shows a stage until the spheroid is produced and collapsed.
  • FIG. 2 shows the cell structure observed from the side when the culture vessel 201 is in the depth direction of the figure.
  • stem cells were used as an example.
  • a spheroid becomes a three-dimensional organization through the following steps.
  • S202 Stem cells 202 aggregate and a part thereof adheres to the culture surface.
  • the present disclosure analyzes a cell state (for example, a cell state inside a spheroid) by performing imaging of a sample such as a spheroid using an optical instrument characterized by high resolution and analyzing the internal structure.
  • a cell state for example, a cell state inside a spheroid
  • an optical instrument characterized by high resolution and analyzing the internal structure.
  • the back side of the sample that has exceeded the depth of penetration without the reflective surface is imaged, and the light beam that irradiates the light from the light source
  • a light-shielding part that shields the center part, defocused reflected light from the reflective surface that becomes noise is suppressed and a clear image is acquired Analyze cells.
  • One aspect is a method of analyzing a cell state.
  • a cell sample during culture or at the end of culture is irradiated with light from a light source, preferably through a culture vessel, and three-dimensional measurement is performed, that is, a tomographic image of the sample is acquired.
  • the measuring device may be an optical device having a high resolution in three dimensions, and includes a light source, a condensing optical system (objective lens) that irradiates light from the light source to cells, and a photodetector that detects light. It is.
  • non-invasive (non-destructive and non-staining) optical instruments are used.
  • OCT Optical Coherence Tomography
  • an optical instrument having a non-invasive and three-dimensional resolution such as a reflective confocal microscope or a multiphoton excitation microscope, can be used.
  • the synthesized light generated by splitting the light from the light source into signal light and reference light, irradiating the signal light to the cell, and combining the signal light reflected from the cell with the reference light
  • This is the principle of detecting.
  • signal light overlaps and is reflected from various depths of the cell, but the component that interferes with the reference light is limited to the signal light component from a specific depth position. Measurement with high resolution becomes possible.
  • an OCT having a high spatial resolution of about 10 microns or less
  • imaging of a cell unit inside a spheroid is possible from the acquired image.
  • the luminance is lower than that of a normal cell portion, and therefore the presence or absence and volume can be analyzed. Therefore, cell states such as cell death can be analyzed by measuring three-dimensionally. With this information, it can be determined whether the spheroid culture process is smooth or transplantable.
  • this cell state analysis method can be automated by existing image processing techniques. It is also possible to measure spheroids incorporated in an automatic culture apparatus and cultured in a culture container in the automatic culture apparatus using OCT.
  • a plurality of sample tomographic images having different distances from the light irradiation position are acquired. Then, the three-dimensional shape of the sample and the tomographic image are analyzed based on the plurality of tomographic images, and the cell state in the sample is analyzed.
  • the central part of the light beam from the light source is shielded by the light shielding part, and the shielded light is reflected by the reflecting surface.
  • a reflective surface is provided on the opposite side of the objective lens with respect to the measurement sample. Thereby, it is possible to image and measure the back side of the sample that has exceeded the limit of the penetration depth without the reflecting surface (that is, the portion of the sample that cannot be measured without the reflecting surface). Further, in the measurement using the reflection surface, it is possible to obtain a clear image by suppressing the defocused reflected light from the reflection surface that becomes noise by providing a light shielding portion.
  • the sample is collected on the sample without using the reflecting surface, and a sample tomographic image is acquired.
  • “do not use a reflective surface” means to acquire a sample tomographic image without installing the reflective surface in the apparatus, and control so that the focal position of light is within the sample with the reflective surface installed in the apparatus. By irradiating with light, this means both obtaining a sample tomographic image without being affected by the reflecting surface.
  • the state of the cell can be analyzed by analyzing the image acquired by the three-dimensional measurement. For example, if the luminance is lower than a certain value by analyzing the luminance of an image acquired by three-dimensional measurement, it can be estimated that a cell in a cell death state exists at that part. On the other hand, if the luminance is higher than a certain value, it can be assumed that there is no cell dead cell at that site. It is also possible to quantitatively analyze how many cells are in a dead state from the luminance value.
  • Image characteristics related to the cell state such as brightness values, vary depending on the type of cell used, the optical system used, the sample composition, etc., so a specific cell state that has been learned in advance, namely cell death.
  • the cell state can be analyzed from the image data of the presence or absence of, for example, luminance data.
  • the intensity of the signal light from the measurement sample attenuates as it travels through the sample due to the influence of scattering and the like.
  • the distance traveled from the surface of the spheroid to the measurement point is different depending on the measurement point even in the same plane image.
  • it is preferable to perform correction that is, the luminance in the tomographic image is affected by the three-dimensional shape of the spheroid and / or the distance from the light irradiation position to the measurement position, and the greater the degree of cell overlap in the three-dimensional shape, the greater the distance from the light irradiation position.
  • the problem peculiar to spheroids can be solved by correcting the luminance in consideration of such signal intensity attenuation. Since signal strength attenuation varies depending on the type of cells used, the optical system used, the sample composition, etc., the luminance from the signal strength attenuation data related to the specific cell state inside the spheroids learned in advance. Can be corrected. Such correction may be automatically performed by the apparatus to be used, or may be manually performed by a practitioner.
  • This device A light source; An objective lens for condensing the light from the light source inside the sample; A light-shielding portion that shields the central portion of the light beam from the light source; A reflection surface installation place for installing a reflection surface that is provided on the opposite side of the objective lens with respect to the sample and reflects light whose light beam center is shielded by the light shielding portion; A light detector, and the light detector is capable of detecting reflected light that is reflected again by the reflecting surface from the focal position irradiated with the light reflected by the reflecting surface.
  • the light-shielding part shields at least a part of the light reflected from the reflecting surface and transmitted through the sample.
  • the light source, the objective lens, and the photodetector are optical devices having high resolution in the three dimensions as described above, preferably non-invasive (non-destructive and non-staining) optical devices.
  • the light source, objective lens and photodetector are configured as an optical coherence tomography (OCT).
  • the reflection surface can be of any shape as long as it has a shape, size and material capable of reflecting light.
  • the reflecting surface preferably has a reflectance that does not damage the sample.
  • the reflective surface may be installed in a place where the reflective surface is installed, or installed in advance. Also good.
  • the reflective surface is provided in a container containing a sample, and the reflective surface of the container is installed in the reflective surface installation site by arranging the container in the apparatus.
  • the reflection surface is previously installed in the reflection surface installation site of the three-dimensional measurement apparatus.
  • the reflective surface is installed at the reflective surface installation site during use.
  • the reflective surface can be removable.
  • the light-shielding portion can also be of any shape as long as it has a shape, size and material that can shield light. In addition, it is preferable to install so that the ratio of the area of the light shielding portion to the area of the objective lens is 9/25 or less.
  • the light shielding portion may be formed integrally with the objective lens, or may be provided between the objective lens and the light source and exchangeable.
  • the above apparatus can collect light on a sample using a reflection surface and a light-shielding portion and perform three-dimensional measurement, but preferably has a function of collecting light on a sample without using a reflection surface.
  • the apparatus further includes a drive unit that drives the objective lens at least in the optical axis direction and scans the focal position in the sample.
  • a drive unit that drives the objective lens at least in the optical axis direction and scans the focal position in the sample.
  • the objective lens is driven so that the focal position of light reflected from the reflective surface is within the sample (for example, FIG. 1).
  • the objective lens is driven so that the focal position of the light from the light source is within the sample (for example, FIG. 9).
  • the above device can be used to analyze the cell state. That is, the sample can be a cell-containing one, such as a spheroid.
  • Another aspect is a cell analysis device that noninvasively optically analyzes a cell state inside a spheroid.
  • This device analyzes a light source, a condensing optical system (objective lens) that irradiates cells with light from the light source, a photodetector that detects light from the cells, and an image based on information obtained from the photodetector.
  • An analysis unit The light source, the condensing optical system, and the photodetector are optical devices having high resolution in the three dimensions as described above, and preferably non-invasive (non-destructive and non-staining) optical devices.
  • the light source, collection optics and photodetector are configured as an optical coherence tomography (OCT).
  • OCT optical coherence tomography
  • the analysis unit includes an image acquisition unit that acquires a plurality of tomographic images having different distances from the light irradiation position (for example, a vertical distance from the culture surface), and an image analysis unit that analyzes the plurality of tomographic images. It is.
  • the image acquisition unit blocks the light beam central part of the light from the light source by the light blocking unit, and A sample tomographic image is acquired by reflecting on the reflecting surface.
  • the sample tomographic image is acquired without using the reflecting surface until the distance from the light irradiation position reaches the light penetration depth limit.
  • the analysis unit can be configured to analyze a cell state inside the spheroid (for example, presence or absence of cell death) from the three-dimensional shape of the spheroid and the tomographic image.
  • the analysis unit may further include a measurement unit that measures the outer shape of the spheroid, thereby measuring the outer shape and / or volume of the spheroid and assisting in the analysis of the cell state inside the spheroid.
  • the device of the present disclosure may include an output device or may be connected to an external output device.
  • the output device can be any output device known in the art and includes, for example, an image and / or data display device, an alarm device, a printer, and the like.
  • the analysis unit may be configured to display an image showing a part determined to be cell death and a volume ratio of the spheroid volume to the cell death part on the display device. Further, at least one of issuing an alarm from the alarm device based on the analysis data or outputting a signal based on the analysis data to the cell culture device or another external device may be performed.
  • the apparatus of the present disclosure preferably further includes a storage unit that stores data related to a specific cell state.
  • a specific cell state a specific cell state inside the spheroid, ie, presence or absence of cell death
  • the cell state can be quickly and easily analyzed by comparison with the stored data.
  • Another aspect is a cell culture device for culturing spheroids, (1) a culture unit for culturing spheroids, and (2) an analysis unit for analyzing the internal cell state of the spheroids using light, (3) a control unit that controls culture and analysis of the spheroids.
  • the culture part of the cell culture device is not particularly limited as long as it can culture cells and form spheroids, and those skilled in the art will be able to select appropriate ones according to the type of target cells and the intended use of the spheroids.
  • a culture part can be comprised.
  • the culture unit includes a temperature-controlled room, a culture container disposed in the temperature-controlled room for culturing spheroids, a cell bottle coupled to the culture container and supplying a cell solution, and a culture medium coupled to the culture container.
  • a medium bottle to be supplied and a waste bottle for storing a medium that is coupled to the culture container and discarded from the culture container can be provided.
  • the analysis unit of the cell culture device includes a light source, a condensing optical system (objective lens) that irradiates the cell with light from the light source, a photodetector that detects light from the cell, and information acquired from the photodetector. And an image analysis unit that analyzes an image based on the image.
  • the image analysis unit includes an image acquisition unit that acquires a plurality of tomographic images having different distances from the light irradiation position (for example, a vertical distance from the culture surface), and an image analysis unit that analyzes the plurality of tomographic images. Is.
  • the image acquisition unit blocks the light beam central part of the light from the light source by the light blocking unit, and A sample tomographic image is acquired by reflecting on the reflecting surface.
  • the sample tomographic image is acquired without using the reflecting surface until the distance from the light irradiation position reaches the light penetration depth limit.
  • the analysis unit can be configured to analyze a cell state inside the spheroid (for example, presence or absence of cell death) from the three-dimensional shape of the spheroid and the tomographic image.
  • the analysis unit may further include a measurement unit that measures the outer shape of the spheroid, thereby measuring the outer shape and / or volume of the spheroid and assisting in the analysis of the cell state inside the spheroid.
  • the control unit of the cell culture device supplies the cell solution, supplies the medium, discards the medium, cultures the cells, installs the reflective surface, scans the focal position of the light, irradiates light, detects the light, acquires the image, Control of at least one of analysis, luminance measurement, and cell state analysis.
  • the control unit is configured to control spheroid culture in the culture unit based on an output from the analysis unit.
  • the control unit can use any control means known in the art, and can be a computer, for example.
  • the cell culture device of the present disclosure may further include an output device.
  • the output device displays the measured information, issues an alarm based on the measured information, outputs to an external device, or And / or feedback to the control unit or the input unit.
  • the alarm includes both a notification of abnormality and a notification of normality.
  • the functions described above may be configured by hardware or software.
  • Still another embodiment is a cell state analysis device that receives data from a cell culture device (cell culture unit) for culturing spheroids and analyzes the cell state inside the spheroids cultured in the cell culture device.
  • the cell culture device and the cell state analysis device may be integrated, or may be connected to each other in a geographically separated position by a network.
  • FIG. 3 shows an example of an automatic culture apparatus incorporating an OCT (optical coherence tomography).
  • the automatic culture apparatus 301 of FIG. 3 has a temperature-controlled room 302 for cell culture.
  • An imaging unit 303 is installed in the temperature-controlled room.
  • a computer 306 including an analysis unit 304 and a storage unit 305 and an output device 307 are installed outside the temperature-controlled room.
  • the output device 307 includes, for example, an image display device that displays various types of information to an operator, an alarm device that issues an alarm by voice, a printer, and the like.
  • data can be transmitted to an external storage device or information terminal via a network or the like.
  • an instruction can be sent to the control unit 308 via various interfaces.
  • the automatic culture apparatus is controlled by the control unit 308.
  • Cell culture is performed in a plurality of culture vessels 314 installed inside the temperature-controlled room 302.
  • the necessary cell solution is supplied from the cell bottle 309 through the medium flow path 312.
  • the medium is supplied from the medium bottle 310 to the culture container 314 through the medium channel 312.
  • the unnecessary medium used for the culture is discarded into the waste liquid bottle 311 through the waste liquid channel 313.
  • Cell analysis of spheroids can be performed by measurement using an imaging unit 303 that images spheroids from the outside of the culture vessel.
  • an OCT optical coherence tomography
  • the entire configuration of the non-invasive three-dimensional measurement is made up of an imaging unit 303 that images the spheroid, an analysis unit 304 that analyzes the captured image and analyzes the state of cells inside the spheroid, and information necessary for the analysis in advance
  • an output device here, assuming an image monitor
  • the automatic culture apparatus of FIG. 3 may include an amino acid analysis unit (not shown) including an amino acid analyzer.
  • the old medium that becomes waste liquid when replacing the medium is discarded from the culture vessel 314 through the waste liquid channel 313 to the waste liquid bottle 311, but part of the culture supernatant flows to the culture supernatant analysis branched from the waste liquid channel 313.
  • the amino acid concentration in the supernatant can be analyzed by being transported through a path (not shown) to the amino acid analysis unit.
  • the cell state is analyzed by the analysis unit 304 and fed back to the control unit 308 of the automatic culture apparatus for determining the culture end timing and evaluating the quality of the cultured tissue.
  • the cell state is displayed on the output device 307, and the operator analyzes the cell state to determine the culture end timing and evaluate the quality of the cultured tissue.
  • the operator inputs to an input unit (not shown) in order to operate the control unit 308 and the computer 306 of the automatic culture apparatus as necessary.
  • the input unit may be configured to be able to input an instruction from a remote place via a network.
  • the analysis unit 304 is implemented as software that operates on a general-purpose computer 306 as a method for realizing the analysis unit 304, but may be configured by hardware.
  • FIG. 3 shows an example in which the computer 306, the control unit 308, and the like are arranged close to or integrated with the automatic culture apparatus 301.
  • the positions of the computer 306, the control unit 308, etc. are not limited to this.
  • wired or wireless networks it is also within the scope of the present invention to connect these via a network via the output device 307 and place them at a remote location.
  • OCT is used as the imaging unit 303.
  • FIG. 4 shows a basic configuration example of an OCT (optical coherence tomography) that is the imaging unit 303.
  • the OCT includes a light source 401, a beam splitter 402, an objective lens 403, a reference light mirror 404, and a detector 405.
  • the light from the light source 401 is branched into the signal light 407 and the reference light 408, and the signal light 407 is irradiated onto the sample spheroid 406.
  • the detector 405 detects the interference light 409 generated by combining the signal light reflected from the cell with the reference light. Thereby, the structure of the cell is visualized.
  • an interference optical system that generates three or more interference lights having different phase relationships may be provided.
  • the imaging unit may have a configuration other than OCT, and for example, a reflective confocal microscope can be used.
  • An example of the basic configuration of a reflective confocal microscope is shown in FIG. It comprises a light source 501, a beam splitter 502, an objective lens 503, a galvano mirror 504, and a detector 505.
  • the spheroid 507 is irradiated with light from the light source 501 via the beam splitter 502 and the galvanometer mirror 504.
  • the reflected light from the cell is detected by the detector 505 through the pinhole portion 506 that blocks the reflected light component from other than the focal position.
  • FIG. 6 shows the measurement image of spheroids.
  • the left side of FIG. 6 is an image of a perspective view of a spheroid. Define xyz axes as shown here.
  • the right side is an image of the xy image at different z positions obtained from the OCT.
  • FIG. 7 shows the presence or absence of cell death inside the spheroid and an image diagram of an OCT image.
  • A is a necrotic spheroid (having the exact live cell region 701)
  • B is a partially necrotic spheroid (having the cell death region 702)
  • B is OCT due to necrosis.
  • the brightness of the image is partially reduced.
  • Fig. 8 shows a schematic flow diagram of the analysis of spheroid measurement by OCT and the cell state inside the spheroid.
  • the OCT imaging unit 303 installed inside the temperature-controlled room 302 captures an XZ tomographic image of the spheroid (S101).
  • the Z thickness of the spheroid is estimated from the curvature of the spheroid contour in the captured area even when the XZ image cannot capture the entire spheroid due to the penetration depth limit. it can.
  • the diameter of the XY plane may be measured using a two-dimensional measuring means such as a phase contrast microscope, and this may be regarded as the Z thickness. At this stage, the size of the light shielding portion may be determined.
  • C is a value less than the penetration depth limit.
  • the penetration depth limit is considered to be about 200-300 ⁇ m.
  • the measurement mode that does not use the reflective surface is shown in FIG. This corresponds to normal OCT measurement, and an area located closer to the light source than the sample back surface 909 reaching the penetration depth limit can be imaged.
  • the measurement mode using the reflective surface is shown in FIG.
  • the objective lens 104 is moved closer to the container bottom surface 101 than in the case of FIG.
  • the light from the light source passes through the objective lens 104 and is once reflected by the reflecting surface 107, and then irradiated to the focal position 108 in the sample.
  • the reflected light from the sample is reflected again by the reflecting surface 107, and then the objective lens. Pass through 104 and be detected.
  • the reflective surface 107 is preferably parallel to the culture bottom surface 101, and is preferably made of an appropriate material that increases the reflectivity and does not damage the spheroid to be measured, and has been subjected to a surface treatment.
  • the light from the light source that passes through the objective lens 104 is once reflected by the reflecting surface 107, then passes through the sample and returns to the objective lens, that is, the defocus component from the reflecting surface is noise. Therefore, by providing the light shielding part 105 that shields the central part of the light beam, the defocus component from the reflecting surface is suppressed, and a high-quality image is acquired.
  • the spheroid OCT image is displayed by combining the results of the measurement mode that does not use the reflective surface and the measurement mode that uses the reflective surface (S107), and the cell state, for example, the presence or absence of necrosis, is determined by image analysis. Whether it is appropriate or not is determined (S108). If the results are displayed in real time, the status of the cells can be monitored. In addition, remote operation is possible by transmitting to an external device via a network. Alternatively, when the analysis result satisfies a specific condition, an alarm can be issued by sound or video.
  • spheroids are imaged using a three-dimensional measuring device, and by installing a reflective surface on the opposite side of the objective lens with respect to the sample, in addition to normal three-dimensional measurement, Usually, the information inside the back surface of the spheroid that does not reach the penetration depth of the 3D measuring device is also acquired. Further, by shielding the central portion of the light beam, it is possible to acquire a high-quality image by suppressing the defocus component from the reflection surface that becomes noise. Further, by displaying the cell state such as the presence or absence of the cell death region inside the spheroid or storing it in the storage device, the operator can be informed of information regarding the state of the spheroid.
  • FIG. 10 shows images of spheroids obtained when the light shielding part is used and when it is not used.
  • D is an image of a spheroid image taken without shading. Without light shielding, the reflectivity from the cells is as small as 1 to 10 m%, so the defocus component from the reflecting surface becomes noise, making analysis inside the spheroid difficult.
  • E is an image of a spheroid image when shielded from light. By suppressing the defocus component from the reflecting surface, an image inside the spheroid can be clearly captured.
  • FIG. 11 shows a change in z resolution depending on the size of the light shielding portion. This is an example of a simulation result in which the Z resolution of the OCT is plotted against the ratio of the light shielding portion diameter to the aperture diameter (beam diameter) of the objective lens when an objective lens having a numerical aperture of 0.52 is used.
  • the ratio of the diameter of the light shielding portion to the aperture diameter of the objective lens is 0.6 or more, the resolution is significantly reduced.
  • the ratio of the area of the light shielding portion to the aperture area of the objective lens is preferably 9/25 or less.
  • the present invention is not limited to the above-described embodiment, and includes various modifications.
  • a part of the configuration of one embodiment can be replaced with the configuration of another embodiment, and the configuration of another embodiment can be added to the configuration of one embodiment.

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Abstract

L'invention concerne un procédé d'analyse d'état de cellule caractérisé en ce qu'il comprend : une étape d'irradiation de lumière à partir d'une source de lumière sur un échantillon ; une étape d'acquisition d'une pluralité d'images d'échantillon tomographique de différentes distances à partir d'une position d'irradiation de lumière et dans laquelle (a) pour des distances à partir de la position d'irradiation de lumière dépassant la limite de profondeur de pénétration de la lumière, la partie centrale du faisceau lumineux de la lumière provenant de la source de lumière est bloquée par une partie de blocage de lumière et des images d'échantillon tomographique sont acquises par réflexion de la lumière bloquée à partir d'une surface de réflexion et (b) pour des distances de la position d'irradiation de lumière jusqu'à la limite de profondeur de pénétration de la lumière, des images d'échantillon tomographique sont acquises sans utiliser la surface de réflexion ; et une étape pour analyser l'état de cellule de l'échantillon sur la base des images acquises.
PCT/JP2018/015523 2017-06-09 2018-04-13 Dispositif et procédé d'analyse d'état de cellule Ceased WO2018225382A1 (fr)

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JPH09218016A (ja) * 1995-12-08 1997-08-19 Kagaku Gijutsu Shinko Jigyodan 光干渉法による測定対象物の屈折率と厚さの同時測定方法及びそのための装置
JPH10325795A (ja) * 1996-08-04 1998-12-08 Matsushita Electric Ind Co Ltd 媒質の測定方法および測定装置
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JP2014048300A (ja) * 2012-08-29 2014-03-17 Hitachi Media Electoronics Co Ltd 光学装置
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JP6325858B2 (ja) * 2014-03-20 2018-05-16 株式会社Ｓｃｒｅｅｎホールディングス 薬効評価方法および画像処理装置
JP6437364B2 (ja) * 2015-03-30 2018-12-12 株式会社Ｓｃｒｅｅｎホールディングス 信号処理方法および画像処理装置
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Publication number Priority date Publication date Assignee Title
JPH09218016A (ja) * 1995-12-08 1997-08-19 Kagaku Gijutsu Shinko Jigyodan 光干渉法による測定対象物の屈折率と厚さの同時測定方法及びそのための装置
JPH10325795A (ja) * 1996-08-04 1998-12-08 Matsushita Electric Ind Co Ltd 媒質の測定方法および測定装置
JP2001141652A (ja) * 1999-11-18 2001-05-25 Japan Science & Technology Corp 光干渉法による測定対象物の屈折率と厚さの同時測定方法及びそのための装置
JP2014048300A (ja) * 2012-08-29 2014-03-17 Hitachi Media Electoronics Co Ltd 光学装置
JP2017215546A (ja) * 2016-06-02 2017-12-07 レーザーテック株式会社 共焦点顕微鏡

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2025164733A1 (fr) * 2024-01-31 2025-08-07 株式会社Racthera Procédé de détermination de région de tissu rétinien normal dans agrégat cellulaire et procédé de production de tissu rétinien implantable chez des animaux

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