Cell observation method and device therefor

Abstract

To provide a cell observation method and a device therefor which easily allow provision of an image obtained by reflecting ion permeability of a cell monolayer as well as provision of the image in real time.SOLUTION: A cell observation device comprises a working electrode and a counter electrode facing each other with a liquid culture medium therebetween, a semiconductor thin film provided to the working electrode, a light source for emitting monochromatic light to a sample surface of the semiconductor thin film, and an imaging sensor for imaging reflected light or transmitted light. A cell observation method comprises: arranging a cell monolayer along the sample surface, repeatedly turning on and off the voltage of the counter electrode to the working electrode, performing synchronization and imaging, obtaining an integrated image by a plurality of differential images of one set of images imaged for each ON / OFF, and obtaining an image corresponding to a position of a tight junction of the cell monolayer having ion permeability.SELECTED DRAWING: Figure 3

Description

[Technical field]

[0001] The present invention relates to a cell observation method and an apparatus therefor that provide images reflecting the barrier function of cells, and in particular to a cell observation method and an apparatus therefor that provide images reflecting ion permeability associated with the opening and closing of tight junctions in a cell monolayer. [Background technology]

[0002] Biological activity is maintained by the barrier function of cells. For example, cells in the digestive organs are responsible for taking in only nutrients into the blood and excluding harmful substances. In addition, biological cells such as skin cells and circulatory system cells also have their own barrier functions. In recent years, there has been a demand for evaluating the barrier function of biological cells in the research and development of various products, especially in the field of drug discovery. As a specific example, the extent to which drugs penetrate the cell barrier of the digestive organs and are absorbed by the human body is an important concern in drug discovery.

[0003] Meanwhile, transepithelial electrical resistance (TEER) measurement is widely known as a standard method for evaluating the barrier function of cells. This method measures the bulk of the entire cell, and is unable to determine the relationship between the measured electrical resistance change and the microscopic changes of the cell. On the other hand, it has been proposed to spatially map the structure of cells by labeling the cells and optically observing the labels. In addition, scanning ion conductance microscopy (SICM) measurements can be used to obtain images reflecting the barrier function of cells by scanning electrodes without labeling the cells.

[0004] For example, Patent Document 1 discloses a method of using SICM to obtain images of the structure and shape of a cell surface. A patch clamp nanopipette arranged perpendicular to a sample is used as a scanning probe, and the tip of the probe is brought close to the cell surface to keep the ionic current passing through the pipette constant, while maintaining a constant distance between the tip and the sample, to scan the cell surface and generate a surface image of the cell. In addition, the SICM pipette probe is used to measure membrane currents while mechanically stimulating the cell while simultaneously performing an electrophysiological technique using patch clamping, and intracellular Ca 2+ It is also said that imaging can be performed. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Special Publication No. 2009-545736 Summary of the Invention [Problem to be solved by the invention]

[0006] As described above, SICM measurement can obtain images that reflect the barrier function of cells, for example, images that reflect ion permeability associated with the opening and closing of tight junctions in a cell monolayer. However, since it is necessary to scan the pipette as an electrode, the device is complicated, and it takes several tens of minutes to obtain one image, making it difficult to capture the state of biological cells that changes over time in real time.

[0007] The present invention has been made in consideration of the above-mentioned circumstances, and an object of the present invention is to provide a cell observation method and apparatus therefor that does not require the procedure of labeling cells, can easily provide images that reflect the ion permeability of a cell monolayer, and can provide images in real time. [Means for solving the problem]

[0008] The method of the present invention is a cell observation method that provides an image reflecting the ion permeability of a cell monolayer, comprising: a working electrode and a counter electrode that face each other with a liquid culture medium therebetween; a semiconductor thin film provided along the surface of the working electrode that faces the counter electrode; a light source that irradiates monochromatic light onto the sample surface of the semiconductor thin film on the side opposite the working electrode; and an imager that images the irradiated light that is reflected by the sample surface or passes through the sample surface, and is characterized in that the cell monolayer is arranged along the sample surface, and a voltage of the counter electrode relative to the working electrode is repeatedly turned on and off, and the semiconductor thin film is imaged by the imager in synchronization with the on and off, to obtain an integrated image of multiple difference images of a set of images captured with each of the on / off, and the image corresponding to the position of the tight junction of the cell monolayer that provides the ion permeability is obtained.

[0009] According to this feature, an image reflecting the ion permeability of the cell monolayer can be easily obtained.

[0010] In the above-mentioned invention, the difference image may be an image corresponding to a change in charge distribution of the semiconductor thin film based on a change in an electric field formed in the liquid medium near the sample surface during the on / off operation.

[0011] In the above-mentioned invention, the voltage may be controlled by providing a reference electrode in the liquid culture medium. With this feature, a stable voltage is applied, enabling dynamic observation.

[0012] In the above-mentioned invention, the imaging element may be provided to image the irradiated light passing through the sample surface, and the irradiated light may have a wavelength that passes through the semiconductor thin film and the working electrode. The wavelength may be a wavelength that can pass through the cell monolayer. With this feature, an image reflecting the ion permeability of the cell monolayer can be easily and stably obtained.

[0013] The device according to the present invention is a cell observation device that provides an image reflecting the ion permeability of a cell monolayer, and includes a working electrode and a counter electrode that face each other with a liquid culture medium therebetween, a semiconductor thin film provided along the surface of the working electrode that faces the counter electrode, a light source that irradiates monochromatic light onto the sample surface of the semiconductor thin film on the side opposite the working electrode, and an imager that images the irradiated light that is reflected by the sample surface or passes through the sample surface, and is characterized in that the cell monolayer is arranged along the sample surface, and a voltage of the counter electrode relative to the working electrode is repeatedly turned on and off, and an image of the semiconductor thin film is captured by the imager in synchronization with the on and off, to obtain an integrated image of multiple difference images of a set of images captured with each of the on / off, and the image corresponding to the position of the tight junction of the cell monolayer that provides the ion permeability is obtained.

[0014] According to this feature, an image reflecting the ion permeability of the cell monolayer can be easily obtained.

[0015] In the above-mentioned invention, the difference image may be an image corresponding to a change in charge distribution of the semiconductor thin film based on a change in an electric field formed in the liquid medium near the sample surface during the on / off operation.

[0016] In the above-mentioned invention, the voltage may be controlled by providing a reference electrode in the liquid culture medium. With this feature, a stable voltage is applied, enabling dynamic observation.

[0017] In the above-mentioned invention, the imaging element may be provided to image the irradiated light passing through the sample surface, and the irradiated light has a wavelength that passes through the semiconductor thin film and the working electrode. With this feature, an image reflecting the ion permeability of the cell monolayer can be easily and stably obtained. [Brief description of the drawings]

[0018] [Figure 1]FIG. 1 is a diagram showing a measurement cell of a cell observation device in one embodiment according to the present invention. [Diagram 2] FIG. 1 is a diagram showing the principle of change in irradiated light passing through an electric double layer capacitor. [Diagram 3] FIG. 1 is a block diagram of a cell observation device. [Figure 4] 1A is a diagram illustrating the principles of signal detection using a normal integration method and FIG. 1B is a diagram illustrating the principles of signal detection using a differential integration method. [Diagram 5] FIG. 2A is a top view and FIG. 2B is a side cross-sectional view of the measurement cell. [Figure 6] FIG. 1 is a diagram of the molecular structure of P3HT. [Figure 7] FIG. 13 is a diagram showing the modulation spectrum of a measurement cell using P3HT as the semiconductor thin film. [Figure 8] 4 is a timing chart of voltage application and image capture triggers. [Figure 9] (a) Optical microscopy images and modulation images taken during the formation of tight junctions in a Caco-2 cell monolayer, and (b) a graph of measured TEER values. [Figure 10] (a) Optical microscopy images and modulation images, and (b) graphs of TEER measurements taken during the process of tight junction opening following administration of capsaicin to a cell monolayer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] First, a cell observation method and an apparatus therefor according to one embodiment of the present invention will be described with reference to FIGS. 1 to 5. FIG.

[0020] As shown in FIG. 1, a cell observation device 20 (see FIG. 3) includes a measurement cell 10 as a main part. The measurement cell 10 is formed on a transparent substrate 1 such as glass. A working electrode 2 is arranged on the substrate 1, and a semiconductor thin film 3 is arranged along the surface of the working electrode 2. A liquid medium 4 is then arranged on top of the working electrode 2. A cell monolayer 5 is arranged along the surface of the sample surface 3a that forms the upper surface of the semiconductor thin film 3. A counter electrode 6 is also arranged so as to face the working electrode 2 with the liquid medium 4 sandwiched between them. A reference electrode 7 is further provided in the liquid medium 4.

[0021] In the measurement cell 10 having such an arrangement, the principle of obtaining an image reflecting the ion permeability of the cell monolayer 5 for cell observation is as follows.

[0022] First, consider an electric double layer capacitor consisting of a working electrode 2, a counter electrode 6, a reference electrode 7, a semiconductor thin film 3, and a liquid medium 4. When a voltage is applied to the working electrode 2 with the potential of the liquid medium 4 measured by the reference electrode 7 as a reference, one of the positive and negative ions i inside the liquid medium 4 moves to the interface between the liquid medium 4 and the semiconductor thin film 3 (when a positive voltage is applied to the working electrode 2, the negative ions move), forming an electric double layer in which ion i accumulates on the liquid medium 4 side and charge e of the opposite sign to ion i accumulates on the semiconductor side.

[0023] Here, when the cell monolayer 5 exists on the surface of the semiconductor thin film 3 (the sample surface 3a opposite to the working electrode 2) and tight junctions are formed, the formation of an electric double layer is inhibited because the ions in the liquid medium 4 cannot penetrate the cell monolayer 5, and the charge e does not accumulate in the semiconductor thin film 3. On the other hand, when a part of the tight junction is open, the ion i penetrates only at the open part 5b, and the charge e accumulates in the semiconductor thin film 3. In this way, the charge e does not accumulate in the semiconductor thin film 3 at the closed part 5a of the tight junction, but the charge e accumulates in the semiconductor layer at the open part 5b of the tight junction. Therefore, if the charge distribution of the semiconductor thin film 3 can be visualized, an image with the opening and closing of the tight junction as contrast can be obtained. In other words, an image reflecting the ion permeability of the cell monolayer 5 is obtained. The charge distribution of the semiconductor can be visualized using modulation imaging, which will be described later.

[0024] The electric double layer accumulates a very high density of electric charges in a very narrow range of a few nm in thickness. Therefore, when even a thin substance (a few μm) as thin as the cell monolayer is present on the surface of the semiconductor thin film 3, the formation of the electric double layer is greatly inhibited. On the other hand, when the tight junction opens and a small gap is formed in the cell monolayer 5, an electric double layer is formed at the open portion 5b. In this way, the charge density is significantly different between the open portion 5a and the closed portion 5b of the tight junction, so that the opening and closing of the tight junction can be detected with high sensitivity. Furthermore, since cells can be cultured using the liquid medium 4 in contact with the semiconductor thin film 3, live cell imaging can also be performed in a culture environment.

[0025] As described above, in an electric double layer capacitor, the opening and closing of tight junctions induces a change in the charge density in the semiconductor layer, but it is usually not possible to visually confirm the charge distribution in the semiconductor thin film 3. Therefore, in this embodiment, the charge distribution in the semiconductor thin film 3 is visualized by a modulation imaging method. The measurement principle of modulation imaging will be described below.

[0026] As shown in FIG. 2(a), in a measurement cell 10 in which a cell monolayer 5 is not arranged, when light is irradiated onto the sample surface 3a of the semiconductor thin film 3 without applying a voltage, the irradiated light passes through the underside of the substrate 1. Here, as shown in FIG. 2(b), a voltage is applied to the working electrode 2 based on the liquid potential of the reference electrode 7. Then, an electric double layer is formed in which ions i are accumulated on the liquid medium 4 side and charges e of the opposite sign to ions i are accumulated on the semiconductor thin film 3 side. At this time, the semiconductor thin film 3 in which charges e are accumulated changes its light transmittance very slightly (about 0.01%). In the modulation imaging method, this change in light transmittance can be captured with high sensitivity to visualize the charge distribution of the semiconductor thin film 3. The voltage applied to the working electrode 2 is a positive voltage when the semiconductor thin film 3 is a P-type semiconductor, and a negative voltage when the semiconductor thin film 3 is an N-type semiconductor.

[0027] Therefore, in this embodiment, a cell observation device 20 is used, as shown in Fig. 3. The cell observation device 20 includes a light source 11 that generates light to be irradiated onto a measurement cell 10, a bandpass filter 12 that monochromatizes the irradiated light, a condenser lens 13 that focuses the irradiated light toward the measurement cell 10, an objective lens 14 that collects the irradiated light that has passed through the measurement cell 10, and a two-dimensional detector 15 that includes an image sensor that receives the focused irradiated light.

[0028] The cell observation device 20 also includes a potentiostat 21 connected to the working electrode 2, counter electrode 6, and reference electrode 7 in the measurement cell 10 to control the potentials of these electrodes, a lock-in amplifier 22 connected to the potentiostat 21, and a function generator 23 connected to these.The cell observation device 20 also includes an image calculation device 24 that acquires and processes signals from the two-dimensional detector 15, and a personal computer 25 that operates the image calculation device 24 and obtains its output.The measurement cell 10 is preferably attached to the cell observation device 20 while being placed in the microscope incubator 16.

[0029] The light source 11 may be a halogen lamp, a deuterium lamp, a xenon lamp, or the like, and the irradiated light from the light source 11 is monochromatized by a bandpass filter 12. A spectroscope may be used instead of the bandpass filter 12. The monochromatized irradiated light is light in the ultraviolet to near infrared range. The light source 11 may be an LED capable of utilizing monochromatic light of various wavelengths in the ultraviolet to near infrared range. The irradiated light passing through the measurement cell 10 is collected by an objective lens 14 and imaged on a two-dimensional detector 15, thereby capturing an image of the charge distribution on the surface of the semiconductor thin film 3 of the measurement cell 10. As described later, when observing the cell monolayer 5 with transmitted light, a wavelength that can transmit the working electrode 2, the semiconductor thin film 3, and the cell monolayer 5 is selected as the wavelength of the light source 11.

[0030] This photographing is performed with and without a voltage applied to the working electrode 2 of the measurement cell 10, and the difference between the two images is taken, and the contrast resulting from the change in the light transmittance of the semiconductor thin film 3 due to the charge accumulation appears as an image. Based on this, it is possible to visualize the spatial distribution of the charge accumulated in the semiconductor thin film 3.

[0031] However, as shown in Fig. 4(a), the change in the light transmittance and reflectance of the semiconductor thin film 3 due to the accumulation of electric charges is very small, about 0.01%, so high sensitivity is required for detection. On the other hand, the intensity of the light source 11 and the sensitivity of the two-dimensional detector 15 used in the cell observation device 20 fluctuate over time, and these fluctuations are larger than the 0.01% change in light transmittance. For this reason, even if the intensity of the irradiated light is integrated in the state where a voltage is applied to the working electrode 2 and in the state where it is not applied, and the difference between these images is taken, the change cannot be detected because it is canceled out by the temporal fluctuation.

[0032] Therefore, as shown in FIG. 4(b), modulation imaging is used. Here, the voltage is switched (modulated) periodically on a time scale shorter than the fluctuations in the intensity of the light source 11 and the sensitivity of the two-dimensional detector 15, images are taken of the voltage ON and OFF states in each period, the difference is calculated, and the difference images are integrated. This makes it possible to remove the influence of the fluctuations. The pulse for switching the voltage ON / OFF may be a square wave or a sine wave as long as it is synchronized. In addition, taking into consideration the fact that the period is shortened with respect to the above-mentioned fluctuations in sensitivity and that the period is lengthened so as to follow the ions i of the liquid medium 4, the frequency of the voltage modulation is preferably set within the range of 10 to 100 Hz.

[0033] The voltage is modulated by inputting a trigger output from a function generator 23 to a potentiostat 21, which then outputs a modulated voltage based on the reference electrode 7 to the working electrode 2. By using the three-electrode system of the working electrode 2, counter electrode 6, and reference electrode 7 and the potentiostat 21, a stable voltage can be output to the working electrode 2 without being affected by fluctuations in the potential within the liquid medium 4, making it possible to handle dynamic observations.

[0034] The image is captured in synchronization with the modulation voltage by inputting a trigger output from the function generator 23 to the two-dimensional detector 15 capable of capturing images at a high frame rate. It is preferable that the two-dimensional detector 15 has a noise level as low as possible, a wide dynamic range, and high sensitivity to the wavelength of the irradiated light. Furthermore, a high-speed image processor 24 is used to obtain the difference between the images in the voltage-on state and the voltage-off state in parallel with the image capture, and an integration process is performed. This makes it possible to obtain images in real time. The integration time is determined according to the S / N ratio of the captured image, and is adjusted within a range of, for example, several seconds to about six minutes.

[0035] That is, an integrated image is obtained by taking multiple difference images of a pair of images taken with the on / off state, and an image corresponding to the positions of the open tight junctions 5b of the cell monolayer 5 that provide ion permeability is obtained. This makes it possible to obtain an image that reflects the ion permeability of the cell monolayer 5. In other words, this difference image is an image that corresponds to the change in charge distribution of the semiconductor thin film 3 based on the change in the electric field formed in the liquid medium 4 near the sample surface 3a with the on / off state.

[0036] A method for measuring TEER (transepithelial electrical resistance) simultaneously with imaging of the charge distribution in the semiconductor thin film 3 by modulation imaging will be described. When imaging the charge distribution, an alternating current flows between the working electrode 2 and the counter electrode 6 due to the modulation voltage applied to the measurement cell 10. This alternating current is detected by the potentiostat 21, and current-to-voltage conversion is performed and input to the lock-in amplifier 22. By inputting a trigger output from the function generator 23 to the lock-in amplifier 22, it is possible to detect only the current component synchronized with the modulation voltage. The TEER can be obtained by dividing the applied voltage by the detected current. In this manner, TEER can be measured simultaneously with imaging of the modulation image.

[0037] The structure of the measurement cell 10 forming the electric double layer capacitor will be described in more detail with reference to FIG.

[0038] The measurement cell 10 has a structure that allows cells to be cultured and can be observed by modulation imaging. For example, the structure of the measurement cell 10 is such that a container with a plastic side wall 9 is placed on a substrate 1 made of glass coated with a working electrode 2 and a semiconductor thin film 3, and is filled with a liquid medium 4. A counter electrode 6 and a reference electrode 7 are immersed in the liquid medium 4. The working electrode 2 is a thin metal thin film that is thin enough to conduct electricity and transmit light, and has a thickness of about 8 nm. A transparent electrode such as ITO may also be used as the working electrode 2. The counter electrode 6 is an inert metal such as gold or platinum coiled into a coil with a diameter of about 1 cm, and is structured to allow light to pass through the center of the coil while having a large surface area.

[0039] Such a cell structure allows irradiated light incident from above to pass through the center of the coil and the working electrode 2 and be detected by the two-dimensional detector 15. This makes it possible to visualize the charge accumulated in the semiconductor thin film 3 by modulation imaging. Furthermore, in order to enable observation while culturing cells in the measurement cell 10, a scaffold layer 8 made of laminin, collagen, or the like, which serves as a scaffold for cells, is formed on the surface of the semiconductor thin film 3 to a thickness of about several nm. This significantly improves the adhesiveness of cells to the surface of the semiconductor thin film 3, and can greatly improve the efficiency of cell culturing.

[0040] Even if the scaffold layer 8 is formed, cells may occasionally peel off from the surface of the semiconductor thin film 3. The ion permeability may change at the peeled part of the cells, which may affect the measured value of TEER. Such cell peeling tends to occur near the edge of the measurement cell 10, that is, the side wall 9, where the stress on the cell monolayer 5 is strong. Therefore, in order to prevent the influence of such cell peeling on the measured value, for example, the working electrode 2 may be formed into a disk shape with a diameter of about 1 cm when viewed from above, and placed in the center of the measurement cell with a sufficient distance from the side wall 9. By limiting the size of the working electrode to the center of the measurement cell 10, even if the cells peel off at the edge of the measurement cell 10, the influence on the measured value of TEER can be suppressed.

[0041] The cell observation device 20 is a device for observing the cell by the irradiated light that has passed through the sample surface 3a, but this "passage" may be transmission as described above or reflection. In other words, the irradiated light may be reflected by the sample surface 3a for observation.

[0042] [Demonstration test: Equipment details] Next, cells were cultured in the measurement cell 10, and an image reflecting the ion permeability of the cell monolayer 5 was obtained by modulation imaging, while the TEER was measured. The results will be described below.

[0043] The cell observation device 20 used in the demonstration test will be described. In the cell observation device 20, the light source 11 is an LED capable of emitting light with a central wavelength of 565 nm, and the central wavelength of the transmission band of the bandpass filter 12 is 550 nm and the half width is 10 nm. In addition, a CMOS camera having sensitivity at 300-1100 nm, a maximum frame rate of 100 fps, a data bit of 16 bits, and a pixel count of 4 million pixels is used as the two-dimensional detector 15. The image calculation device 24 is a self-made one using an FPGA. The rest is as described above.

[0044] The measurement cell 10 was fabricated as follows. First, Cr was vacuum-deposited to a thickness of 1 nm on a glass substrate 1 as an electrode adhesive layer, and Au was vacuum-deposited on top of that to a thickness of 8 nm as a working electrode 2. A polymer semiconductor poly(3-hexylthiphene-2,5-diyl) [P3HT] (see FIG. 6) was spin-coated on top of that to form a film of 3 nm, and a plastic side wall 9 was then installed.

[0045] Next, the cell was filled with a laminin-based cell scaffold coating material solution to form a scaffold layer 8 on the semiconductor layer, and the cell scaffold coating material solution was removed. Then, the inside of the container formed by the side wall 9 of the measurement cell 10 was filled with liquid medium 4, and human small intestine epithelial-like cells (Caco-2) were seeded and cultured in an incubator. For the liquid medium 4, first, a cell monolayer 5 was formed by using a growth medium that promotes cell growth, and then the medium was changed to a differentiation induction medium and cultured to promote the formation of tight junctions in the cell monolayer 5. After the culture, the measurement cell 10 was set in a cell observation device 20, and a reference electrode 7 made of an Ag / AgCl electrode and a counter electrode 6 made of a coiled gold wire were immersed in the liquid medium 4. The change in light transmittance when a voltage of 0.45 V was applied to the working electrode 2 of the prepared measurement cell 10 based on the reference electrode 7 was measured for each wavelength.

[0046] As shown in FIG. 7, the P3HT used in this embodiment shows a large change in light transmittance around a wavelength of 550 nm, so that the observation can be performed with the highest sensitivity by performing modulation imaging using light with a wavelength of 550 nm as the irradiating light. In this embodiment, as described above, monochromatic light with a wavelength of 550 nm was obtained by combining an LED capable of irradiating light with a central wavelength of 565 nm and a bandpass filter with a central wavelength of 550 nm and a half-width of 10 nm in the transmission band. In this way, it is preferable that the wavelength of the monochromatic light used as the irradiating light is a wavelength that shows a relatively large change in light transmittance due to a change in the charge distribution of the semiconductor thin film 3.

[0047] [Demonstration test: Imaging] Modulation imaging was performed on the measurement cell 10 in which Caco-2 cells were cultured, as follows. The measurement cell 10 was set between the condenser lens 13 and the objective lens 14, and the working electrode 2, the reference electrode 7, and the counter electrode 6 were connected to the potentiostat 21. A microscope incubator 16 was placed between the condenser lens 13 and the objective lens 14, and the temperature and CO2 concentration were kept suitable for cell culture by placing the measurement cell 10 in it. This enabled the observation of live cells in a culture environment for about one week continuously. The condenser lens 13 and the objective lens 14 were adjusted to focus on the sample surface 3a of the semiconductor thin film 3 of the measurement cell 10, and monochromatic light with a wavelength of 550 nm was irradiated from above the measurement cell 10.

[0048] As shown in FIG. 8, the voltage modulation and the photographing were synchronized. In detail, a trigger output from the function generator 23 was input to the potentiostat 21, and the potentiostat 21 output a modulated voltage based on the reference electrode 7 to the working electrode 2. The modulated voltage was a square wave in which voltages of 0.45V and 0V were alternately output at a repetition period of 45Hz. A trigger signal with a repetition period twice as long as the above (90Hz) was input to the CMOS camera, and photographing was performed in each state in which 0.45V was applied (ON state) and in which 0V was applied (OFF state). In parallel with the photographing, a high-speed image calculation device 24 was used to perform an integration process of the difference between the 0.45V image and the 0V image photographed at each period. As a result, an integrated image of the difference was obtained.

[0049] FIG. 9(a) shows the results of modulation imaging of the formation of tight junctions in the cell monolayer 5 of Caco-2 cells. FIG. 9(b) shows the TEER measured at the same time. The measurement was started immediately after the Caco-2 cells were cultured in a growth medium in a microscope incubator 16 and then replaced with a differentiation-inducing medium. With a normal optical microscope, no change in contrast was observed over time, but with the modulation image, a change in contrast was observed in which the white area spread over time. In addition, an increase in resistance value corresponding to the contrast change in the modulation image was observed in the TEER. In the modulation image, the dark areas correspond to high charge density, and the light areas correspond to low charge density. Therefore, the change in contrast to white means that as the tight junctions are formed, the ion permeability of the cell monolayer 5 decreases, making it difficult for an electric double layer to be formed (electric charge is difficult to accumulate in the semiconductor thin film 3). The increase in the resistance value of the TEER can also be explained without contradiction by the decrease in the ion permeability of the cell monolayer membrane.

[0050] As shown in FIG. 10, capsaicin, which has the effect of opening tight junctions, was administered to a cell monolayer 5 of Caco-2 cells in which tight junctions had been formed, and the changes were measured. The modulation image is shown in FIG. 10(a), and the measurement result of TEER is shown in FIG. 10(b). When 2 mM capsaicin was administered 6.3 hours after the start of observation, a mottled contrast appeared at 7.5 hours, and then the overall color became darker. In addition, a decrease in the resistance value corresponding to the contrast change in the modulation image was observed in the TEER. The darkening of the contrast color means that the tight junctions have opened, increasing the ion permeability of the cell monolayer 5, making it easier for an electric double layer to be formed (electric charge is more easily accumulated in the semiconductor layer). The decrease in the resistance value of the TEER can also be explained without contradiction by the increase in the ion permeability of the cell monolayer 5.

[0051] From these findings, it is believed that the change in the barrier function of living cells can be visualized by the cell observation device 20. In particular, the appearance of the mottled pattern immediately after the administration of capsaicin is believed to reflect the microscopic dynamics of tight junctions that cannot be determined by measuring TEER alone, and this embodiment makes it possible to investigate such dynamics.

[0052] Although the embodiments of the present invention and modifications based thereon have been described above, the present invention is not necessarily limited thereto, and a person skilled in the art will be able to find various alternative embodiments and modifications without departing from the spirit of the present invention or the scope of the appended claims. [Explanation of symbols]

[0053] 2 working electrode 3. Semiconductor thin films 4. Liquid medium 5. Cell Monolayer 6 Counter Electrode 7 Reference electrode 10 Measuring Cell 11 Light source 14 Objective Lens 15 Two-dimensional detector 16 Microscope Incubator 21 Potentiostat 23 Function Generator

Claims

1. A cell observation method that provides an image reflecting the ion permeability of a cell monolayer, comprising: A working electrode and a counter electrode facing each other with a liquid medium therebetween; a semiconductor thin film provided along a surface of the working electrode opposite the counter electrode; a light source that irradiates a monochromatic irradiation light onto a sample surface of the semiconductor thin film on the opposite side to the working electrode; an imaging element that images the illumination light reflected by the sample surface or passing through the sample surface; A cell observation method characterized in that the cell monolayer is placed along the sample surface, the voltage of the counter electrode relative to the working electrode is repeatedly turned on and off, and the semiconductor thin film is imaged by the imaging element in synchronization with the on and off, thereby obtaining an integrated image of multiple difference images of a set of images taken with each of the on / off, and obtaining the image corresponding to the position of the tight junctions of the cell monolayer that provide the ion permeability.

2. The cell observation method according to claim 1, characterized in that the difference image is an image corresponding to a change in charge distribution of the semiconductor thin film based on a change in the electric field formed in the liquid medium near the sample surface when the light is turned on / off.

3. 2. The cell observation method according to claim 1, wherein the voltage is controlled by providing a reference electrode in the liquid culture medium.

4. A cell observation method according to any one of claims 1 to 3, characterized in that the imaging element is configured to image the irradiated light passing through the sample surface, the irradiated light having a wavelength that passes through the semiconductor thin film and the working electrode.

5. 5. The cell observation method according to claim 4, wherein the wavelength is a wavelength that can be transmitted through the cell monolayer.

6. A cell observation device that provides an image reflecting the ion permeability of a cell monolayer, A working electrode and a counter electrode facing each other with a liquid medium therebetween; a semiconductor thin film provided along a surface of the working electrode opposite the counter electrode; a light source that irradiates a monochromatic irradiation light onto a sample surface of the semiconductor thin film on the opposite side to the working electrode; an imaging element that images the illumination light reflected by the sample surface or passing through the sample surface; A cell observation device characterized in that the cell monolayer is placed along the sample surface, the voltage of the counter electrode relative to the working electrode is repeatedly turned on and off, and the semiconductor thin film is imaged by the imaging element in synchronization with the on and off, thereby obtaining an integrated image of multiple difference images of a set of images taken with each of the on / off, and obtaining the image corresponding to the position of the tight junction of the cell monolayer that provides the ion permeability.

7. The cell observation device according to claim 6, characterized in that the difference image is an image corresponding to a change in charge distribution of the semiconductor thin film based on a change in the electric field formed in the liquid medium near the sample surface when the light is turned on / off.

8. 7. The cell observation device according to claim 6, further comprising a reference electrode provided in the liquid culture medium, for controlling the voltage.

9. The cell observation device according to any one of claims 6 to 8, characterized in that the imaging element is configured to image the irradiated light passing through the sample surface, the irradiated light having a wavelength that passes through the semiconductor thin film and the working electrode.

Images