JPWO2015004917A1 - Observation method, cell sheet manufacturing method, cell sheet manufacturing apparatus, and cell sheet observation apparatus - Google Patents

Abstract

An observation method, which includes a first observation stage for observing an observation object, and a second observation stage for observing the observation object based on information obtained by the first observation stage. The step of detecting a state of a cell included in the region has a step of determining whether to observe the cell based on the state of the cell detected in the first observation step. In the observation method, the first observation stage includes a step of determining whether or not the cells included in the observation region are in a predetermined state, and the second observation stage is a cell determined to be in the predetermined state. Or determining whether or not to observe the observation region based on the number of cells or the ratio to the number of cells in the observation region.

Description

  The present invention relates to an observation method, an observation apparatus, a cell sheet manufacturing method, and a cell sheet manufacturing apparatus.

One of the methods for observing a living body such as a cell with high accuracy without damaging the living body such as a cell is a method for generating an observation image by detecting CARS light (coherent anti-Stokes Raman scattering light) generated in an observation target (for example, a patent) Reference 1).
Japanese Patent Application Laid-Open No. 2005-062155

  CARS light is generated in a narrow region where excitation light is collected. For this reason, CARS observation has high resolution and composition (lipid or protein) selectivity. However, since it is an observation method using a non-linear process, the signal light is weak, and in exchange for high resolution, the area that can be observed per unit time is narrow, and observation takes time.

  In the first aspect of the present invention, the first observation stage includes a first observation stage for observing the observation object and a second observation stage for observing the observation object based on the information obtained by the first observation stage. Includes a step of detecting a state of a cell included in the observation region, and the second observation step is a step of determining whether to observe the cell based on the state of the cell detected in the first observation step. An observation method is provided.

  In the second aspect of the present invention, the first observation stage includes a first observation stage for observing the observation object, and a second observation stage for observing the observation object based on information obtained by the first observation stage. Has a step of detecting the state of the cells included in the observation region, and the second observation step processes the image data of the observed observation object based on the state of the cells detected in the first observation step. An observation method is provided that includes a step of determining whether or not.

  In a third aspect of the present invention, a cell sheet comprising a preparation stage for isolating cells and preparing a cell line, a culture stage for culturing the cell line on a cell sheet, and an observation stage for observing the cells by the above observation method A manufacturing method is provided.

  In the fourth aspect of the present invention, a cell sheet comprising: a preparation unit for isolating cells and preparing a cell line; a culture unit for culturing the cell line on a cell sheet; and an observation unit for observing the cells by the observation method. A manufacturing apparatus is provided.

  In the fifth aspect of the present invention, the first observation unit includes a first observation unit that observes the observation target, and a second observation unit that observes the observation target based on the information obtained by the first observation unit. Has a detection unit for detecting the state of the cells included in the observation region, and the second observation unit determines whether to observe the cells based on the state of the cells detected by the detection unit An observation device is provided.

  In the sixth aspect of the present invention, the first observation unit includes a first observation unit that observes the observation target, and a second observation unit that observes the observation target based on information obtained by the first observation unit. Has a detection unit for detecting the state of the cells included in the observation region, and the second observation unit processes the image data of the observed observation object based on the state of the cells detected by the first observation unit. An observation apparatus having a determination unit for determining whether or not is provided.

  The above summary of the present invention does not enumerate all necessary features of the present invention. Sub-combinations of these feature groups can also be an invention.

1 is a schematic diagram showing the structure of a laser microscope 101. FIG. 2 is a block diagram of a control system of the laser microscope 101. FIG. 3 is a flowchart showing an observation procedure using a laser microscope 101. It is a figure which shows the observation image by the detection of differential interference light. FIG. 6 is a diagram illustrating a process of determining a detection area by a second detection system 150. It is a figure which shows the observation image of the living cell by the detection of CARS light. It is a figure which shows the observation image of the dead cell by the detection of CARS light. 2 is a schematic diagram showing a structure of a laser microscope 102. FIG.

  Hereinafter, the present invention will be described through embodiments of the invention, but the following embodiments do not limit the invention according to the claims. In addition, not all the combinations of features described in the embodiments are essential for the solving means of the invention.

  FIG. 1 is a schematic diagram showing the structure of the laser microscope 101. The laser microscope 101 includes a stage 110, a laser device 120, an optical system 130, a first detection system 140, a second detection system 150, a control system 160, and a scanning system 180. The laser microscope 101 can be used for both observation using differential interference light and observation using CARS light detection.

  The stage 110 supports the sample 112 observed by the laser microscope 101. The sample 112 supported by the stage 110 is irradiated with laser light as irradiation light from one surface (the lower surface in the drawing in the drawing). In addition, an observation image is formed by detecting the exit light emitted by the linear optical phenomenon or the emergent light emitted by the nonlinear optical phenomenon from the side opposite to the side irradiated with the laser light with respect to the stage 110. Is done. In the present embodiment, differential interference light is used as the emitted light emitted by the linear optical phenomenon, and CARS light is used as the emitted light emitted by the nonlinear optical phenomenon.

The laser device 120 includes a plurality of laser light sources 122, 124, 126 and a combiner 128. The laser light source 122, 124, 126 generates separately a laser beam having different wavelengths lambda _d, lambda _P, the lambda _S together. In the present embodiment, a laser beam having a wavelength lambda _d of one laser light source 122 is generated, the observation by detection of differential interference light is used as illumination light.

The other laser light sources 124 and 126 generate pulse lasers used for observation by CARS light detection. The pulse lasers generated by the laser light sources 124 and 126 have wavelengths λ _P and λ _S. As the laser light sources 124 and 126, for example, a mode-locked picosecond Nd: YVO ₄ laser, a mode-locked picosecond yttrium laser, or the like can be used. One of the laser light sources 124 and 126 may be replaced with an optical parametric oscillator that converts the wavelength of the pulse laser generated by the other laser light sources 124 and 126.

Of the two-wavelength picosecond pulses generated by the laser light sources 124 and 126, the pulse laser having the shorter wavelength λ _P is used as pump light in CARS light observation. Of the picosecond pulses generated by the laser light sources 124 and 126, the pulse laser having the longer wavelength λ _S is used as Stokes light in CARS light observation.

  Laser beams emitted from the laser light sources 122, 124, and 126 are incident on the combiner 128 through the polarizing plates 121 and 123, respectively. The polarizing plate 121 for pump light and Stokes light and the polarizing plate 123 for illumination light have polarization planes shifted by 45 degrees from each other. As a result, the pump light, Stokes light, and illumination light become linearly polarized light having polarization planes C and D that are shifted from each other by 45 °. Further, the polarizing plate 123 of the laser light source 126 is also used as a polarizer in the case of performing observation with differential interference light.

  Note that the direction of the polarization plane C and the polarization plane D may be either P-polarized light or S-polarized light with respect to the reflecting mirrors such as the reflecting mirror 192 and the galvano scanner 182. If priority is given to the detection sensitivity of CARS light, the polarization plane C is preferably incident on these as P-polarized light or S-polarized light.

  The combiner 128 integrates the laser beams generated by the plurality of laser light sources 122, 124, and 126 and emits them as irradiation light on a single optical path. Thereby, the irradiation light which combined pump light and Stokes light can be irradiated to the sample 112, and CARS light can be generated. In the combiner 128, it is preferable to use a dichroic mirror that can maintain the degree of polarization of the pump light and Stokes light used for CARS light observation and illumination light used for differential interference light observation.

  The laser device 120 may be provided with a delay optical path for the purpose of synchronizing the pump light generated by the laser light sources 122 and 124 and the Stokes light. The delay optical path can be formed by a plurality of reflecting mirrors whose intervals can be changed. In the laser device 120, the Stokes light may be broadened using a photonic crystal fiber.

  In the laser microscope 101, the scanning system 180 includes a galvano scanner 182 and a scan lens 184. The galvano scanner 182 includes a reflecting mirror that swings around two axes whose directions are different from each other, and two-dimensionally displaces the optical path of incident irradiation light in a direction intersecting the optical axis. The scan lens 184 focuses the irradiation light emitted from the galvano scanner 182 on a predetermined primary image plane 186. Thereby, the observation region of the sample 112 is scanned with the irradiation light emitted from the laser device 120, and the irradiation region can be irradiated with the irradiation region having a predetermined area.

  The optical system 130 includes a pair of first objective lenses 132 and 134 disposed on the laser device 120 side with respect to the stage 110, and a condenser lens 136 disposed on the opposite side of the objective lens 132 and 134 with respect to the stage 110. Have The objective lenses 132 and 134 focus the irradiation light irradiated toward the sample 112 within the observation region in the sample 112.

  On the incident end side of the optical system 130, a reflecting mirror 192 is disposed on the optical path of the irradiation light between the laser device 120 and the objective lens 132. The reflecting mirror 192 bends the optical path of the irradiation light to prevent the structure of the laser microscope 101 from becoming excessively high.

  As the reflecting mirror 192, a metal mirror that reflects incident light on the surface may be used instead of the total reflection prism. Thereby, the reflecting mirror 192 can alleviate the occurrence of a phase difference depending on the polarization direction.

  The optical system 130 includes a pair of birefringent prisms 172 and 174 disposed on the optical path of the irradiation light with the sample 112 supported by the stage 110 interposed therebetween. One birefringent prism 172 disposed upstream of the sample 112 in the propagation direction of the irradiation light is disposed between the pair of objective lenses 132 and 134. The other birefringent prism 174 disposed on the downstream side with respect to the sample 112 is disposed so as to sandwich the condenser lens 136 together with the sample 112.

  The upstream birefringent prism 172 separates the irradiation light irradiated toward the sample 112 into a pair of light beams whose polarizations are orthogonal to each other. The separated pair of light beams propagate in parallel in slightly spatially different positions and pass through the sample 112 very close, but different positions. The distance between the pair of polarized light in the sample 112 is called a shear amount, and is about 0.2 μm, for example.

  As the birefringent prisms 172 and 174, a Woraton prism capable of taking out incident linearly polarized light with an opening angle in a non-parallel manner can be used. Furthermore, a nomarski rhythm having the focal point of two divided polarized lights outside the prism can be preferably used.

  A pair of polarized lights that have passed through different positions in the sample 112 are combined into one light beam in the birefringent prism 174 downstream of the sample 112. However, since the sample 112 is transmitted through different positions, the combined polarized light propagates through different optical path lengths. For this reason, a phase difference is generated between the pair of polarized lights, and the intensity of the combined light beam is changed. In this way, an observation image that well reflects the shape of the sample 112 is obtained by detecting and imaging differential interference light that reflects the distribution of the optical characteristics of the sample 112.

  The birefringent prisms 172 and 174 are exclusively used for observation by detecting differential interference light. Therefore, when the laser microscope 101 is observed by detecting CARS light, the birefringent prisms 172 and 174 may be removed from the optical path of the irradiation light. On the other hand, when the birefringent prisms 172 and 174 are inserted into and removed from the optical path, the characteristics of the optical system 130 may change and the observation position on the sample 112 may be shifted. Therefore, when the observation positions of the observation by differential interference light and the observation by CARS light detection are made to coincide with each other with high accuracy, the observation by CARS light detection may be performed while the birefringent prisms 172 and 174 are attached. .

  A first detection system 140 and a second detection system 150 are arranged on the downstream side of the birefringent prism 174 along the propagation optical path of the irradiation light. The first detection system 140 is used to detect differential interference light among the detection light emitted from the sample 112. The second detection system 150 is used to detect CARS light out of the detection light emitted from the sample 112.

  The first detection system 140 includes a polarizing plate 176, a condenser lens 142, a relay lens 144, and a photomultiplier tube 146. The polarizing plate 176 is used as an analyzer in observation using differential interference light. Thereby, a pair of polarized light incident through different optical paths causes interference, and becomes differential interference light having light and dark according to the shape of the sample 112 that is transparent to the irradiation light.

  The differential interference light is condensed by the condensing lens 142 and the relay lens 144, and is efficiently detected by the photomultiplier tube 146 to form an observation image. Unlike the phase contrast microscope, observation with differential interference light has no restriction on the numerical aperture of the illumination, so that the resolution in the direction perpendicular to the optical axis can be exhibited to the limit of the microscope objective lens. In addition, since the observation image obtained by the differential interference light has no halo in the outline, high resolution can be obtained.

  A reflecting mirror 194 is disposed between the condensing lens 142 and the relay lens 144 of the first detection system 140. The reflecting mirror 194 bends the optical path of the detection light to prevent the structure of the laser microscope 101 from becoming excessively high.

  The second detection system 150 includes a condenser lens 152, a relay lens 154, a photomultiplier tube 156, and a dichroic mirror 158. The dichroic mirror 158 branches the CARS light emitted from the sample 112 on the upstream side of the polarizing plate 176 of the first detection system 140 and introduces it into the photomultiplier tube 156 through the condenser lens 152 and the relay lens 154. Therefore, the CARS light is received by the photomultiplier tube 156 without reducing the light amount.

  The dichroic mirror 158 is used only when CARS light is detected. Therefore, when the differential interference light is observed, the dichroic mirror 158 may be extracted from the optical path of the detection light. The incident angle of the detection light emitted from the sample 112 with respect to the dichroic mirror 158 is preferably smaller than 45 °. Thereby, CARS light can be extracted efficiently.

  The control system 160 includes a control unit 162, a keyboard 164, a mouse 166, and a display unit 168. The control unit 162 can be formed by mounting a program that causes a general-purpose personal computer to execute a control procedure described later.

  The keyboard 164 and the mouse 166 are connected to the control unit 162 and operated when a user instruction is input to the control unit 162. The display unit 168 returns feedback to the user's operation using the keyboard 164 and the mouse 166, and displays the image or character string generated by the control unit 162 toward the user.

  The control unit 162 is connected to each of the laser device 120, the first detection system 140, the second detection system 150, and the scanning system 180 and controls each operation. Moreover, based on the detection results of the first detection system 140 and the second detection system 150, an image to be displayed on the display unit 168 is generated. In addition, the control unit 162 determines the state of the sample 112 based on the detection results of the first detection system 140 and the second detection system 150.

  The control system 160 controls the overall operation of the laser microscope 101 in accordance with instructions received from the user. Further, the control system 160 generates an observation image based on the detection results of the first detection system 140 and the second detection system 150. Further, the control system 160 may also output a character string or a code indicating a determination result reflecting the state of the sample 112.

  In the laser microscope 101 as described above, the polarization plane C in which the illumination light for observation by differential interference light and the pump light and Stokes light for observation by detection of CARS light intersect at 45 °. D. The dichroic mirror 158 disposed for the purpose of separating the CARS light emitted from the sample 112 is arranged so that the differential interference light is P-polarized light or S-polarized light. Therefore, the polarization characteristic of the interference film formed on the dichroic mirror 158 is reduced with respect to the wavelength of the differential interference light, and the observation accuracy of observation using the differential interference light is prevented from being lowered. For this purpose, the direction in which the dichroic mirror 158 and the reflecting mirror 192 are arranged may be different from that in FIG.

  The dichroic mirror 158 is used only when CARS light is detected. Therefore, when importance is attached to the image quality of observation using differential interference light, the dichroic mirror 158 may be provided so as to be extracted from the optical path of the detection light.

  FIG. 2 is a block diagram of a control system in the laser microscope 101. In the laser microscope 101, the control unit 162 includes a first determination unit 310, a storage unit 320, a scanning control unit 330, a detection control unit 340, and a second determination unit 350. The control unit 162 is coupled to the keyboard 164, the mouse 166, the display unit 168, the laser device 120, the first detection system 140, the second detection system, and the scanning system 180 through the input / output unit 163.

  The first determination unit 310 determines the state of the cell based on the detection image of the sample 112 generated by the detection control unit 340 based on the detection result of the first detection system 140 and the reference information referenced from the storage unit 320. It is determined whether or not In this embodiment, the predetermined state is a state in which the cell is dead. In this example, living cells are referred to as living cells, and dead cells are referred to as dead cells.

  That is, the first determination unit 310 determines whether the cell detected by the first detection system 140 is a live cell or a dead cell. For example, the first determination unit 310 acquires a detection image obtained from a known cell from the storage unit 320 as reference information, and evaluates the similarity between the detection image obtained from the sample 112 and the reference information. Cell viability may be determined.

  In addition, the first determination unit 310 acquires feature items extracted from a detection image obtained from a known cell from the storage unit 320 as reference information, and features of the detection image obtained from the sample 112 apply to the reference information. Whether the cell is alive or not may be determined depending on whether or not it is. The determination result by the first determination unit 310 is output to the outside through the display unit 168. Furthermore, the first determination unit 310 may leave part or all of the determination process to the user by displaying the reference information and the detected image toward the user through the display unit 168.

  Based on the state of the cells determined by the first determination unit 310, the second determination unit 350 determines whether or not the cells detected by the first detection system 140 are detected by the second detection system 150. The second determination unit 350 determines whether or not to observe the cells determined by the first determination unit 310 as being in a predetermined state, that is, dead cells. In the present embodiment, the second determination unit 350 determines the viability state of the cells that are not determined to be dead cells by the first determination unit 310.

  In the present embodiment, the second determination unit 350 observes cells for which the first determination unit 310 has not clarified whether the cell is a live cell or a dead cell. Accordingly, since the first determination unit 310 can detect a cell whose life or death is unknown by the second detection system 150, it is possible to determine the life or death of the cell without omission.

  Based on the detection image of the sample 112 generated by the detection control unit 340 based on the detection result of the second detection system 150 and the reference information referenced from the storage unit 320, the second determination unit 350 is a living cell. Determine if it is a dead cell. Here, the second determination unit 350 determines the determination based on the observation image generated by detecting the CARS light generated from the sample 112. Therefore, the determination by the second determination unit 350 has high accuracy based on the composition selectivity of CARS light observation.

  In the determination, for example, a detection image obtained from a known cell is acquired from the storage unit 320 as reference information, and the life and death of the cell is determined by evaluating the similarity between the detection image obtained from the sample 112 and the reference information. You may judge. In addition, the second determination unit 350 acquires the feature items extracted from the detection image obtained from the known cells from the storage unit 320 as reference information, and the feature of the detection image obtained from the sample 112 applies to the reference information. Whether the cell is alive or not may be determined depending on whether or not it is.

  Further, the second determination unit 350 compares the evaluation value calculated by image processing such as Fourier transform processing and differentiation processing on the detection image detected from the sample 112 with the evaluation value acquired from the storage unit 320, and is determined in advance. Whether the cells are alive or dead may be determined based on the threshold value. The determination result by the second determination unit 350 is output to the outside through the display unit 168. Furthermore, the second determination unit 350 may leave part or all of the determination process to the user by displaying the reference information and the detected image toward the user through the display unit 168.

  The storage unit 320 stores a control program that describes the control procedure of the control unit 162 itself. The control unit 162 reads the control program stored in the storage unit 320 and controls the operation of the laser microscope 101. In addition, the storage unit 320 stores the detection result detected by the first detection system 140 or the second detection system 150, and refers to it when the detection control unit 340 generates a detection image.

  Furthermore, the storage unit 320 stores reference information that is referred to when determining whether a cell serving as the sample 112 is alive or dead. The reference information may be a detection image detected from a cell whose life and death are known. Further, the reference information may be information representing a feature item of a detected image detected from a cell whose life and death are known. Furthermore, the reference information is information indicating an evaluation value calculated by performing image processing on a detected image detected from a cell whose life and death are known by Fourier transform processing, differentiation processing, and the like, a method for calculating the evaluation value, and the like. It may be.

  When instructed to use either the first detection system 140 or the second detection system 150, the scanning control unit 330 operates the scanning system 180 according to the detection system to be used. Thereby, the observation region of the sample 112 is scanned with the irradiation light, and the observation region predetermined in the sample 112 can be observed on one observation plane.

  The detection control unit 340 instructs the detection timing of the first detection system 140 and the second detection system 150 with reference to the scanning timing of the irradiation light and the light emission timing of the laser device 120. Further, the detection control unit 340 acquires the detection result of the first detection system 140 or the second detection system 150 together with the irradiation position of the irradiation light on the sample 112. Accordingly, the detection control unit 340 generates the observation image of the sample 112 by mapping the light intensity of the detection light to the light emission position in the sample 112.

  FIG. 3 is a flowchart showing a procedure for observing the sample 112 using the laser microscope 101. When observing the sample 112 using the laser microscope 101, first, the laser light source 126 and the first detection system 140 are enabled, and the sample 112 is observed with differential interference light (step S101).

  Thereby, the observation image can be generated by observing the sample 112 in a wide range and at high speed. For example, when an animal cell is observed, the observation image includes a plurality of cells. The resolution of the observation image obtained from the sample 112 depends on the resolution of the differential interference light. Therefore, from the observation image, a relatively large organelle such as a cell nucleus can be observed in addition to the cell membrane showing the outline of the cell.

  Therefore, in the control system 160, the first determination unit 310 performs the first dead cell determination based on the observation image obtained by the differential interference light (step S102). In the first dead cell determination, apparent dead cells are determined such that the cell membrane, nucleus, etc. have already been destroyed.

  Next, in the control system 160, the detection control unit 340 specifies an observation region by the second detection system 150 (step S103). At this stage, the cells determined to be dead cells in step S102 are excluded from the observation region by the second detection system 150.

  Next, the detection control unit 340 enables the laser light sources 122 and 124 and the second detection system 150 to perform observation by detecting CARS light (step S104). In the observation in step S104, the detection control unit 340 controls the scanning control unit 330 based on the observation region specified in step S103. Thereby, the cells that have already been determined to be dead cells in the first dead cell determination in step S102 are excluded from the observation target.

  That is, when scanning the CARS light on the observation target, it passes through without observing the place where the dead cell exists. For this reason, compared with the scanning speed when observing while acquiring image data, it is possible to increase the scanning speed of a place where dead cells exist. When passing through a place where dead cells exist, irradiation of CARS light to the place may be stopped. Therefore, the scanning time by the second detection system 150 is shorter than the case where all the observation target areas are observed by the second detection system 150 alone without using the first detection system 140. The time required for observation can be reduced. In addition, since the size of the detection target region by the second detection system 150 is smaller than that in the case where all the regions to be observed are observed only by the second detection system 150 without using the first detection system 140, The time required for observation by the two detection system 150 can be shortened. Therefore, the detection control unit 340 can quickly generate an observation image with high resolution using CARS light.

  Then, the 2nd determination part 350 performs a 2nd dead cell determination based on the observation image obtained by CARS light (step S105). Since the second dead cell determination is based on an observation image having information that can be detected up to individual molecular bonds, it is possible to accurately determine a cell to be observed. In this way, a wide region of the sample 112 can be determined with the observation image accuracy of CARS light having high composition selectivity. In addition, observation with CARS light has high resolution in the irradiation direction of irradiation light, that is, in the depth direction (depth direction) of the sample 112 that is the observation target. Therefore, the sample 112 can be observed with high resolution in the depth direction.

  At the time of detection by the second detection system 150 or at the time of determination by the second determination unit 350, the first observation image detected by the first detection system 140 and the second observation image detected by the second detection system 150 are simultaneously It may be displayed. In this case, for example, the first observation image is stored in the storage unit 320, and the stored first observation image is acquired and acquired upon detection by the second detection system 150 or determination by the second determination unit 350. A first observation image may be displayed. In addition, the detection by the first detection system 140 is continued even after the detection by the second detection system 150 is started, and the first observation image is detected at the time of detection by the second detection system 150 or the determination by the second determination unit 350. You may display a 2nd observation image. Further, by displaying an image showing the observation region by the second detection system 150 on the first observation image, it is possible to know which position of the first observation image the second detection system 150 is detecting. Also good.

  Thus, the sample 112 can be observed at high speed and with high accuracy using the laser microscope 101. In addition, when the sample 112 is a living cell, neither the observation with differential interference light nor the observation with CARS light damages the sample 112, so that the cell sheet before use can be observed.

  Furthermore, after finishing the above series of procedures, the observation image generated by the differential interference light and the observation image generated by the detection of the CARS light are synthesized, and the outline of the cell detected by the differential interference light and the CARS light It is possible to generate an observation image that displays the distribution of lipids and the like detected by the above. This facilitates the determination of the viability of individual cells.

  A step of detecting a spectrum of the cell nucleus in the second dead cell determination (step S105) may be provided for the cell nucleus detected in the first dead cell determination (step S102). Thereby, the viability of cells can be confirmed by different methods. Note that the spectrum of the cell nucleus can be measured by providing a polychromator in the second detection system 150 in place of the photomultiplier tube 156 or in addition to the photomultiplier tube 156, for example.

  When the observation process shown in FIG. 3 is performed, for example, when the first one of a plurality of cell sheets manufactured by the same process is observed, and as a result, the number or ratio of dead cells is smaller than a predetermined value For example, the cells determined to be dead cells by the first detection system 140 may be detected at the time of detection by the second detection system 150 at the time of observing each of the remaining plurality of cell sheets. Observation of either the first detection system 140 or the second detection system 150 may be omitted when observing the cell sheet.

  In addition, for the cell sheet for which it is determined that it is better to inspect carefully during the manufacturing process, the second detection system 150 again inspects the cells determined to be dead cells by the first detection system 140. Alternatively, the observation by the first detection system 140 may be omitted, and the observation process shown in FIG. 3 may be performed when observing another cell sheet. That is, the second determination unit 350 may determine that the second detection system 150 observes a cell that is determined to be a dead cell by the first determination unit 310.

  FIGS. 4 and 5 are diagrams illustrating the observation image generated based on the differential interference light in step S101. As shown, the observed sample 112 is a mixture of live and dead cells. As shown in a white circle in FIG. 4, it can be determined from the observation image that the living cell includes a clear cell nucleus. Therefore, the number of living cells contained in the sample 112 can be easily factored from the observation image.

  In addition, as shown by being surrounded by a white circle in FIG. 5, some cells include a plurality of small particles having high luminance. Such cells are presumed to be dead cells or dying cells. That is, the first determination unit 310 determines whether or not a cell is in a dying state, and determines that the cell is a dead cell when it is determined that the cell is in a dying state. Therefore, the cells determined to be dead cells in step S102 are excluded from the CARS light observation target in step S103. Moreover, the space | gap between cells is also excluded from the observation object by CARS light. The first determination unit 310 determines that the dying cell is a dead cell. Alternatively, the first determination unit 310 may determine that the dying cell is a living cell.

  Note that the cells that are determined to be alive or dead by the differential interference light are excluded from the objects to be observed by the CARS light. Therefore, the scanning time by the second detection system 150 is shortened as compared with the case where all regions to be observed are observed only by the second detection system 150 without using the first detection system 140. In addition, the area of the region observed with the CARS light is smaller than the case where all the regions to be observed are observed only with the second detection system 150 without using the first detection system 140. Therefore, the time required to complete the observation with CARS light can be shortened.

  FIG. 6 is a diagram illustrating an observation image generated by CARS light detection using the second detection system 150 of the laser microscope 101. The illustrated detection image was generated for a single observation plane perpendicular to the optical axis in a cell sheet formed by living cells.

  From the laser device 120 of the laser microscope 101, the sample 112 was irradiated with pump light having a wavelength of 1064 nm and Stokes light having a wavelength of 1550 nm. Thereby, in each of the cells included in the sample 112, CARS light having a wavelength of 820 nm is generated by the CH bond included in the lipid.

  Further, the sample 112 was repeatedly irradiated with the irradiation light while displacing the optical path by the scanning system 180, and the CARS light emitted from the square region having a side of 50 μm on the observation plane was detected by the photomultiplier tube 156. The detection control unit 340 generates an observation image by plotting the light intensity of the detected CARS light according to the scanning by the scanning system 180.

  The detection image generated in the detection control unit 340 has bright spots reflecting the distribution of lipids contained in the cell sheet. Further, a plurality of black spots appearing in the detection image correspond to the arrangement of cell nuclei not containing lipid. Therefore, for example, by counting these spots, the characteristics of lipid distribution in living cells can be detected. In this way, it is possible to accurately determine that the observation target is a living cell for each individual cell that cannot be determined by detection of differential interference light.

  In addition, when a Fourier transform is performed on a detection image detected from a living cell, it is characterized by a small number of high spatial frequency components. Therefore, a threshold value may be set in advance, and if the spatial frequency of a detected image detected from an unknown sample is lower than the threshold value, it may be determined that the sample cell is a living cell.

  FIG. 7 is a diagram illustrating another observation image generated by CARS light detection using the second detection system 150 of the laser microscope 101. The illustrated detection image was generated for a single observation plane perpendicular to the optical axis in a cell sheet formed by necrotic dead cells.

  The observation conditions in the laser microscope 101 were the same as the case where the cell sheet of live cells shown in FIG. 6 was observed, including the wavelength of irradiation light. Therefore, the detection image shown in FIG. 8 reflects the lipid distribution in dead cells.

  From the detection image shown in the figure, the characteristics of a distribution in which bright spots generated by the presence of lipids are dispersed and locally concentrated can be seen. In this detected image, since the bright spot has a remarkably high brightness with respect to the surroundings, the brightness of the entire detected image is lowered and displayed so that the peak of each bright spot becomes clear.

  When the detected image detected from the dead cells as described above is subjected to Fourier transform, it is characterized by including a high component in the spatial frequency. Therefore, when a threshold value is set in advance and the spatial frequency of a detected image detected from an unknown sample exceeds the threshold value, it can be determined that the sample cell is a dead cell. In this way, it is possible to accurately determine that the observation target is a living cell for each individual cell that cannot be determined by detection of differential interference light.

  In the above example, the sample was irradiated with irradiation light that generates Raman scattered light in the molecules contained in the lipid. However, other types of molecules that can generate Raman scattered light may be selected as detection targets depending on the type of cells contained in the sample. Further, for example, while detecting the lipid distribution, an observation image in which the protein is detected may be generated, and noise may be removed by utilizing the exclusive distribution of the lipid and the protein.

  The index for determining the cell state based on the detection image is not limited to the above example. For example, as one of the cell determination indices, it can be exemplified that life / death is determined based on the feature that a living cell moves but a dead cell does not move.

  In this case, the same observation region of the cell serving as a sample is observed twice or more at time intervals, and an image obtained by observation can be compared to determine that a cell having no change is a dead cell. When using such a determination index based on cell movement, pay particular attention to whether or not the position of organelles such as mitochondria changes, and based on images that detect the distribution of lipids that are abundant in organelles. Thus, the movement of cells can be easily detected.

  Furthermore, matters that can be determined using the laser microscope 101 are not limited to cell life and death. For example, even for the same dead cell, it is possible to determine whether the dead cell is a necrotic death or an apoptotic death according to the characteristics of the detected image.

  The outline of apoptotic dead cells approximates the shape of a round circle when compared to living cells that were flat and elongated when viewed two-dimensionally. In addition, nuclear concentration occurs inside the cell. Furthermore, the cell membrane shrinks smaller than the living state. Furthermore, in apoptotic dead cells, several organelles in the cytoplasm are trapped in the lipid membrane, peeled off from other surrounding cells, and eventually become apoptotic endoplasmic reticulums divided into multiple.

  In this way, after observing a wide area with differential interference light capable of observing a wide range at high speed, and then observing a key point with high-resolution CARS light detection, a high-precision determination can be quickly made for a wide area of the sample 112. be able to.

  FIG. 8 is a diagram schematically showing the structure of another laser microscope 102. The laser microscope 102 has the same structure as the laser microscope 101 shown in FIG. Therefore, common elements are denoted by the same reference numerals, and redundant description is omitted.

  The laser microscope 102 differs from the laser microscope 101 in that it includes a third detection system 210. Similar to the second detection system 150, the third detection system 210 includes a dichroic mirror 218, a condenser lens 212, a relay lens 214, and a photomultiplier tube 216. However, the wavelength of light reflected by the dichroic mirror 218 is different from that of the dichroic mirror 158 of the second detection system 150.

  The dichroic mirror 218 in the third detection system 210 of the laser microscope 102 reflects a band including the third harmonic generated in the sample 112 by pump light or Stokes light emitted from either of the laser light sources 122 and 124. Therefore, in the third detection system 210, the photomultiplier tube 216 receives and detects the third harmonic collected by the condenser lens 212 and the relay lens 214.

The third harmonic is generated according to the third-order susceptibility χ ⁽³⁾ in the sample 112. Therefore, for example, when a cell is observed as the sample 112, a third harmonic is generated at the interface of the cell membrane, cell nucleus, or the like. Therefore, by selectively detecting the third harmonic in the third detection system 210, the contours of cells, nuclei, and the like can be detected with a resolution comparable to the observation by detection of CARS light.

  In addition, when the position of the cell membrane is known in advance by observation of differential interference light, only the relevant part is irradiated with a pulsed laser generated by the laser light sources 122 and 124 so that the outline of the cell is high-resolution. Can be detected. Therefore, even if the step of detecting the cell membrane by the third detection system 210 is added, the time required for the observation is greatly reduced as compared with the case where the entire observation region is scanned with a pulse laser.

  The dichroic mirror 218 is also used only when detecting the third harmonic. Therefore, when observing differential interference light, the dichroic mirror 218 may be extracted from the optical path of the detection light. The incident angle of the detection light emitted from the sample 112 with respect to the dichroic mirror 218 is preferably smaller than 45 °. Thereby, the third harmonic can be extracted efficiently.

  Further, the laser microscope 102 has a structure in which a third detection system 210 is provided in addition to the first detection system 140 and the second detection system 150. However, the third detection system 210 may be provided in place of either the first detection system 140 or the second detection system 150. Further, the first detection system 140, the second detection system 150, and the third detection system 210 may be formed as individual microscopes. Furthermore, the third detection system 210 may be formed using a detection system that detects stimulated Raman scattering light, which is one of the third-order nonlinear optical effects.

  In the above-described embodiment, dead cells are excluded from detection targets by the second detection system 150 by excluding cells determined to be dead cells by the first determination unit 310 from the observation region of the second detection system 150. Although an example has been shown, instead of this, dead cells may be excluded from detection targets of the second detection system 150 as follows.

  For example, the first determination unit 310 determines whether the cells included in the observation region of the first detection system 140 are in a predetermined state, that is, dead cells, and the second determination unit 350 Based on the number of cells determined to be dead cells in the observation area or the ratio of dead cells to the total number of cells in the observation area of the first detection system 140, the first detection system 140 observes the number of dead cells. It is determined whether or not the observed area is observed by the second detection system 150.

  When the number or ratio of cells determined to be dead cells in the observation region of the first detection system 140 is greater than or equal to a predetermined value, the second determination unit 350 sets the observation region of the first detection system 140 to the second It is determined that the observation is not performed by the detection system 150. That is, the observation region including the cells determined to be dead cells is excluded from the observation target of the second detection system 150. The ratio of dead cells to the total number of cells when the observation region of the first detection system 140 is excluded from the observation target of the second detection system 150 is, for example, 30% or more, and more preferably 10% or more.

  In addition, the second determination unit 350 determines the observation region of the first detection system 140 from the observation target of the second detection system 150 when the number or ratio of cells determined to be dead cells is equal to or less than a predetermined value. Observe with the second detection system without exclusion. At this time, it may be determined that cells other than the cells determined to be dead cells among the cells in the observation region of the first detection system 140 are observed by the second detection system 150.

  Moreover, in the above-described example, when the CARS light is scanned over the observation area including the dead cell or the dead cell to be observed, the second detection system 150 passes without observation. However, instead of this, the second detection system 150 may be observed at a location or area where dead cells exist. In this case, data processing such as data processing for life / death determination and cell nucleus spectrum detection by the second determination unit 350 is not performed. As a result, the entire data processing time by the second detection system 150 is shortened as compared with the case where the data detected by the second detection system 150 is processed for all regions to be observed. Can be shortened. Image data obtained by observing a place or area where a dead cell exists is stored in the storage unit 320, and when performing a reexamination of a cell determined to be a dead cell, the image data is acquired from the storage unit 320. Data processing.

  When the second determination unit 350 observes an observation area where the number or ratio of dead cells is equal to or less than a predetermined value, and the second determination unit 350 determines whether the number or ratio of dead cells is equal to or less than a predetermined value On the other hand, the second determination unit 350 determines that the observation region is good, and if it is equal to or greater than a predetermined value, the second determination unit 350 determines that the observation region is defective.

  Furthermore, the cell observation method can be used in a production apparatus for producing a cell sheet. That is, a manufacturing apparatus including a preparation unit that isolates cells and prepares a cell line, a culture unit that cultures the cell line on a cell sheet, and a determination unit that determines whether the cell sheet is alive or dead by the cell observation method. Moreover, by using the cell observation method, a high-quality cell sheet with few dead cells or many living cells can be efficiently produced. In this production apparatus, the cell observation method may be applied to observation of cells in culture or may be used in other production stages.

  As mentioned above, although this invention was demonstrated using embodiment, the technical scope of this invention is not limited to the range as described in the said embodiment. It will be apparent to those skilled in the art that various modifications or improvements can be added to the above-described embodiment. It is apparent from the scope of the claims that the embodiments added with such changes or improvements can be included in the technical scope of the present invention.

  The execution order of each process such as operations, procedures, steps, and stages in the apparatus, system, program, and method shown in the claims, the description, and the drawings is particularly “before” or “prior”. It should be noted that they can be implemented in any order unless the output of the previous process is used in the subsequent process. Regarding the operation flow in the claims, the description, and the drawings, even if it is described using “first”, “next”, etc. for the sake of convenience, it means that it is essential to carry out in this order. is not.

101, 102 laser microscope, 110 stage, 112 sample, 120 laser device, 121, 123, 176 polarizing plate, 122, 124, 126 laser light source, 128 combiner, 130 optical system, 132, 134 objective lens, 136 condenser lens, 140 First detection system, 142, 152, 212 Condensing lens, 144, 154, 214 Relay lens, 146, 156, 216 Photomultiplier tube, 150 Second detection system, 158, 218 Dichroic mirror, 160 control system, 162 control Part, 163 input / output part, 164 keyboard, 166 mouse, 168 display part, 172, 174 birefringent prism, 180 scanning system, 182 galvano scanner, 184 scan lens, 186 primary image plane, 192, 194 reflector, 210 Three detection systems, 310 First determination unit, 320 Storage unit, 330 Scan control unit, 340 Detection control unit, 350 Second determination unit

Claims (42)

A first observation stage for observing an observation object;
A second observation stage for observing the observation object based on the information obtained by the first observation stage,
The first observation step includes a step of detecting a state of a cell included in the observation region,
The second observation step includes a step of determining whether to observe the cell based on the state of the cell detected in the first observation step. The first observation step includes a step of determining whether or not the cells included in the observation region are in a predetermined state,
The second observation step includes a step of determining whether or not to observe the observation region based on the number of cells determined to be in the predetermined state or a ratio to the number of cells in the observation region. The observation method according to claim 1.   In the second observation step, when the number of cells determined to be in the predetermined state or the ratio to the number of cells in the observation region is greater than or equal to a predetermined value, the observation region is excluded from the observation target. The observation method according to claim 2.   In the second observation step, when the number of cells determined to be in the predetermined state or a ratio to the number of cells in the observation region is equal to or less than a predetermined value, the observation region is an observation target. The observation method according to Item 3.   5. The observation method according to claim 4, wherein in the second observation step, cells determined to be in the predetermined state among cells in the observation region are excluded from the observation target.   5. The observation method according to claim 4, wherein in the second observation stage, data relating to a cell determined to be in the predetermined state among cells in the observation region is not processed. The first observation step includes a step of determining whether or not the cells included in the observation region are in a predetermined state,
The observation method according to claim 1, wherein the second observation step includes a step of determining whether or not to observe a cell determined to be in the predetermined state.   The said 2nd observation step determines whether the cell which was not determined to be the said predetermined state among the cells in the said observation area | region is the said predetermined state. Observation method described in 1.   The observation method according to any one of claims 2 to 8, wherein the second observation step includes a step of observing a cell in which it was not possible to determine whether or not the predetermined state has occurred in the first observation step. .   The observation method according to claim 2, wherein the predetermined state is a state in which a cell is dead.   The first observation step determines whether or not the cell is in a dying state, and if it is determined that the cell is in a dying state, the cell is not determined to be a dead cell. The observation method according to any one of the above.   The first observation step determines whether or not the cell is in a dying state, and determines that the cell is a dead cell when it is determined that the cell is in a dying state. The observation method according to any one of the above.   The said 1st observation stage uses the detection image obtained from the known cell as reference information, and detects the state of a cell by comparing the detection image of the said observation object with the said reference information. Observation method according to item.   The observation method according to any one of claims 1 to 13, wherein in the first observation step, a state of the cell is detected based on a shape of the cell.   The observation method according to any one of claims 1 to 14, wherein in the first observation step, the state of the cell is detected based on whether or not at least one of a cell membrane and a cell nucleus of the cell is destroyed.   The observation method according to any one of claims 1 to 15, wherein the first observation step includes a step of observing a distribution of a refractive index interface and a nonlinear susceptibility interface in the observation target. The first observation step includes a step of identifying a nucleus of a cell included in the observation target,
The observation method according to any one of claims 1 to 16, wherein the second observation step includes a step of observing the cell nucleus detected in the first observation step.   The observation method according to claim 17, wherein the second observation step includes a step of detecting a spectrum of the cell nucleus detected in the first observation step.   The observation method according to any one of claims 1 to 18, wherein the first observation stage has a larger observation area per unit time than the second observation stage.   The said 2nd observation stage has a stage which observes with a higher resolution about the depth direction of an observation object compared with the said 1st observation stage. Observation method. The first observation stage includes
The observation method according to any one of claims 1 to 20, further comprising: a first detection step of detecting emitted light emitted from the observation object irradiated with irradiation light by a linear optical phenomenon.   The observation method according to claim 21, wherein the first observation stage includes any of differential interference observation and third harmonic detection. The second observation stage includes
The observation method according to any one of claims 1 to 22, further comprising: a second detection step of detecting emitted light emitted from the observation object irradiated with irradiation light by a nonlinear optical phenomenon.   The observation method according to claim 23, wherein the second observation step includes any one of coherent anti-Stokes Raman scattering light detection and stimulated Raman scattering light detection.   The observation method according to any one of claims 1 to 24, wherein the first observation step and the second observation step include a step of irradiating the observation target with common irradiation light.   The observation method according to any one of claims 1 to 25, wherein the second observation step includes a step of specifying a second observation region in the first observation region observed in the first observation step.   The observation method according to any one of claims 1 to 26, wherein in the second observation step, the observation object is irradiated with irradiation light having a wavelength longer than 350 nm.   The observation according to any one of claims 1 to 27, wherein the second observation step includes a step of detecting the Raman scattered light whose optical path has been changed by an optical path changing member inserted into an optical path of the Raman scattered light. Method.   The observation method according to any one of claims 1 to 28, wherein the first observation step and the second observation step use an optical system at least partially in common.   30. The observation according to claim 29, wherein the second observation step includes a step of detecting a part of the Raman scattered light branched by an optical path branching member inserted in an optical path of Raman scattered light emitted from the observation target. Method.   The observation method according to any one of claims 1 to 30, further comprising a display step of displaying the first detection image detected in the first observation step and the second detection image detected in the second observation step. . A storage step of storing the first detected image;
32. The observation method according to claim 31, wherein in the display step, the first detection image stored in the storage step is acquired, and the acquired first detection image is displayed. The first observation stage continues imaging of the observation target even after the second observation stage starts,
32. The observation method according to claim 31, wherein in the display step, a detection image of the observation target imaged when the second observation step is being performed is displayed.   The observation method according to any one of claims 31 to 33, wherein in the display step, an image indicating an observation region in the second observation step is displayed on the first detection image.   The observation method according to any one of claims 1 to 34, wherein the observation target is a cell sheet including a plurality of cells. A first observation stage for observing an observation object;
A second observation stage for observing the observation object based on the information obtained by the first observation stage,
The first observation step includes a step of detecting a state of a cell included in the observation region,
The second observation step includes a step of determining whether to process the observed data to be observed based on the state of the cells detected in the first observation step. A preparation stage of isolating cells and preparing a cell line;
Culturing the cell line in a cell sheet;
A cell sheet manufacturing method comprising: an observation step of observing the cells by the observation method according to any one of claims 1 to 36.   The cell sheet manufacturing method according to claim 37, wherein the observation step includes a determination step of determining whether the cell sheet is good or bad. A preparation unit for isolating cells and preparing a cell line;
A culture unit for culturing the cell line on a cell sheet;
A cell sheet manufacturing apparatus comprising: an observation unit that observes the cells by the observation method according to any one of claims 1 to 36.   The cell sheet manufacturing apparatus according to claim 39, wherein the observation unit includes a determination unit that determines whether the cell sheet is good or bad. A first observation unit for observing an observation object;
A second observation unit for observing the observation object based on the information obtained by the first observation unit;
The first observation unit includes a detection unit that detects a state of a cell included in the observation region,
The second observation unit is an observation apparatus including a determination unit that determines whether to observe the cell based on the state of the cell detected by the detection unit. A first observation unit for observing an observation object;
A second observation unit for observing the observation object based on the information obtained by the first observation unit;
The first observation unit includes a detection unit that detects a state of a cell included in the observation region,
The observation device having a determination unit that determines whether to process the observed data of the observation target based on the state of the cell detected by the first observation unit.