JP7012556B2 - Cell observation device and cell observation method - Google Patents

Description

The present invention relates to a cell observation device and a cell observation method. More specifically, the present invention is used in observing the activity of various cultured cells (in the present specification, "cultured cells" are appropriately referred to as "cultured cells"). The present invention relates to a suitable cell observation apparatus and cell observation method.

Conventionally, as a method for observing the dynamic function of cells, an electrophysiological method for measuring an electric current or a voltage using an electrode has been known.

By the way, it is pointed out that these electrophysiological methods have problems such as limited number of measurement points, non-invasiveness because they come into contact with cells using electrodes, and skill in operation and handling. It had been.

The method of non-invasively observing the activity of cells and the like is an extremely important method in biological research and drug development.

On the other hand, in order to improve the problems pointed out in the above-mentioned electrophysiological method, specifically, to improve the invasiveness, and to increase the number of measurement points, for the membrane potential and specific ions. A method of measuring light using a function-sensitive dye whose fluorescence or the like changes is used.

However, the method of measuring light using this function-sensitive dye has a problem that the function-sensitive dye itself has some toxicity, and also has a problem that the price of the measuring device for measuring the light is expensive. there were.

As far as the cultured cardiomyocytes (in the present specification, the "cultured cardiomyocytes" are appropriately referred to as "cultured cardiomyocytes"), the unstained tissue by high-definition moving images is used. A system that automatically detects motion is manufactured and sold by Sony Imaging Products & Solutions Co., Ltd.

The above system complements the shortcomings of the above two methods, such as satisfying non-invasiveness, but the observation target is limited to cultured cardiomyocytes and the price is extremely high. There was a problem.

Since the prior art that the applicant of the present application knows at the time of filing the patent application is not an invention related to an invention known in the literature, there is no prior art document information to be described in the specification of the present application.

The present invention has various problems as described above of the prior art, that is, an invasive method requiring electrodes and staining, and a complicated and expensive device is required to satisfy non-invasiveness. It was made in view of the problem of being present, and its purpose is to refer to cultured myocardial cells and cultured stem cells (in the present specification, "cultured stem cells" to "cultured stem cells". It is possible to non-invasively observe the dynamic functions of various cells such as), and to provide an inexpensive and simple cell observation device and cell observation method. be.

In order to achieve the above object, the present invention is to observe cells non-contactly using, for example, visible light or near-infrared light without staining the cells with a dye.

Further, the present invention is configured as an inexpensive and simple device or method in order to promote research and application on cells.

Here, the present invention applies the academic knowledge that changes in light scattering of cells derived from cell activity can be observed by measuring using simple reflected light, and is based on the conventional technique described above. It solves the problems in measurement.

That is, according to the present invention, the activity of cells can be easily observed at low cost, without contact with cells, without staining and without invasiveness.

Here, to explain the above-mentioned academic findings, the academic findings are based on various academic studies investigating the relationship between cell activity and light scattering.

In such academic research, in order to efficiently and quantitatively measure and evaluate light scattering, an optical sensor that transmits polarized illumination light through a cell sample and detects polarization by transmitted light is used. The outline of the results of the above-mentioned academic research is as follows.

That is, according to academic research, it is known that the activity of biological cells is accompanied by the inflow and outflow of ions, and a considerable amount of water molecules also flow in and out at the same time as the inflow and outflow of ions.

The water molecule contracts and expands the intercellular space (intercellular space) between cells, so that light scattering changes. This change is minute, and generally, it is so small that it cannot be detected by a single activity, but it can be optically detected by performing averaging by repeated addition or the like.

Here, as an example of easy detection, it is known that continuous stimulation of brain slice specimens can be sufficiently detected without averaging.

In many academic studies, it has been shown that when cells are activated, light scattering changes in a decreasing direction, and the reason is thought to be due to the expansion of cell gaps (for example, the journal name "Journal name". NeuroImage 18 (2003) ”, pp. 214-230, title“ The relationship between changes in intrinsic optical signals and cell selling in rat spin s.

As a basic experimental step leading to the present invention, the inventor of the present application uses cultured myocardial cells as cells to be observed, and uses experimental equipment composed of a microscope, a stable light source, a polarizing plate, a low noise image sensor, and the like. This is an experiment with transmitted light similar to that conducted during the academic research.

As a result of the basic experiment, the inventor of the present application was able to detect in cultured cardiomyocytes by a single measurement, and was able to reproduce the results of academic research.

However, the inventor of the present application influences influences such as "a container for culturing cells (in the present specification, a" container for culturing cells "is appropriately referred to as a" culture container ")" in an experiment using transmitted light, particularly a resin. When producing a culture vessel by the molding process, the deterioration of the measurement signal due to the distortion (turning property) of the polarization transmission characteristics based on the resin molding process and the large noise due to the fluctuation of the water surface due to the vibration are large, and the application is intended for simple measurement. I found that it cannot be used as it is.

Based on the above-mentioned findings, the inventor of the present invention attempts to measure with reflected light, compares the measured signal with reflected light with the measured signal with the above-mentioned experiment with transmitted light, and finds conditions suitable for measurement. It has reached.

That is, the inventor of the present application observes the activity of cultured cells and the like non-invasively by experimentally optimizing the arrangement of the light source and the photodetector and constructing the system with a simple circuit of inexpensive parts. It led to the invention of the method of doing.

That is, the cell observation device according to the present invention is a cell observation device for observing the dynamic function of cells, in which light irradiation means for irradiating cells with light and scattering from the cells irradiated with light from the light irradiation means. It has a detection means that senses and detects light, and the detection means is designed to continuously detect changes in the scattered light.

Further, the cell observation device according to the present invention has a container for accommodating the cells and having at least a transparent lower surface in the cell observation device according to the present invention, and the light irradiation means and the detection means are the above-mentioned containers. The incident angle, which is the angle between the optical axis of the light irradiated by the light irradiating means and the optical axis of the scattered light sensed by the detecting means, which is arranged below the lower surface, is 20 degrees or more and less than 45 degrees. It is something like that.

Further, the cell observing apparatus according to the present invention is the above-mentioned cell observing apparatus according to the present invention in which the incident angle is 20 to 40 degrees.

Further, the cell observing apparatus according to the present invention is the above-mentioned cell observing apparatus according to the present invention in which the incident angle is 30 degrees.

Further, the cell observation device according to the present invention is the above-mentioned cell observation device according to the present invention, in which the detection means is a light detector that detects the scattered light and outputs a light current, and is located after the light detector. , A current amplifier that amplifies and outputs the optical current output from the optical detector, a condenser that removes the direct current (DC) component of the current output from the current amplifier, and outputs from the condenser. It has a high-magnification amplifier that electrically amplifies a current from which a direct current (DC) component has been removed and outputs it as a voltage.

Further, the cell observation method according to the present invention is a cell observation method for observing the dynamic function of cells by irradiating the cells with light and continuously detecting changes in scattered light from the irradiated cells. It is something like that.

Further, in the cell observation method according to the present invention, in the cell observation method according to the present invention, the incident angle, which is the angle formed by the optical axis of the light irradiating the cells and the optical axis of the scattered light to be detected, is 20 degrees. It is designed to be less than 45 degrees.

Further, in the cell observation method according to the present invention, in the cell observation method according to the present invention, the incident angle is set to 20 to 40 degrees.

Further, in the cell observation method according to the present invention, the incident angle is set to 30 degrees in the cell observation method according to the present invention.

In the cell observation method according to the present invention, the cell observation method according to the present invention detects the scattered light, outputs a light current, amplifies the light current, and removes a direct current (DC) component. It is further electrically amplified and output as a voltage.

Since the present invention is configured as described above, an inexpensive and simple cell observation device capable of non-invasively observing the dynamic functions of various cells such as cultured cardiomyocytes and cultured stem cells. And it has the excellent effect of being able to provide a cell observation method.

FIG. 1 is a configuration explanatory diagram schematically showing a cell observation device according to an example of the embodiment of the present invention. FIG. 2 is an explanatory diagram showing the principle of the measurement method in the cell observation device according to the present invention. FIG. 3 is an explanatory diagram showing an incident angle in the cell observation device according to the present invention. FIG. 4 is a waveform diagram showing the experimental results using cultured cardiomyocytes in the cell observation device according to the present invention. FIG. 5 is a configuration explanatory diagram schematically showing a cell observation device according to another example of the embodiment of the present invention. 6 (a) and 6 (b) are structural explanatory views schematically showing a cell observation device according to another example of the embodiment of the present invention. FIG. 6A is a top view of the cell observation device according to another example of the embodiment of the present invention (A arrow view of FIG. 6B). FIG. 6B is a side view of the cell observation device according to another example of the embodiment of the present invention (viewing arrow B of FIG. 6A).

Hereinafter, an example of an embodiment of the cell observation apparatus and the cell observation method according to the present invention will be described in detail with reference to the accompanying drawings.

(I) First Embodiment

(1) Overall configuration

FIG. 1 shows a configuration explanatory diagram schematically showing a cell observation device according to an example of the embodiment of the present invention.

The cell observation device 10 has a sample container 12 which is a container for storing a sample Sa which is a cell to be observed by the cell observation device 10. Here, the sample Sa is various cells such as living cardiomyocytes and stem cells.

The sample container 12 is configured as a culture container filled with various solutions So such as a culture solution and a solution that maintains physiological activity. Generally, the sample Sa is immersed in various solutions So such as a culture solution filled in the sample container 12 and a solution for maintaining physiological activity.

Further, at least the bottom surface 12a of the sample container 12 is formed of a transparent material with respect to the wavelength of the illumination light L1 emitted from the illumination light irradiation device 14 which is a light source described later.

The illumination light irradiation device 14 is arranged at a position below the bottom surface 12a of the sample container 12, and is illumination light L1 (for example, visible light or near-infrared light) having a predetermined wavelength with respect to the bottom surface 12a of the sample container 12. ) Is irradiated.

Such an illumination light irradiation device 14 can be configured as, for example, a light emitting diode (LED).

The cell observation device 10 is arranged at a position below the bottom surface 12a of the sample container 12, and includes an optical system 16 that collects the scattered light L2 generated by irradiating the bottom surface 12a of the sample container 12 with the illumination light L1. .. As the optical system 16, for example, a lens can be used.

Further, the cell observation device 10 has a photodetector 18 that detects the scattered light L2 focused by the optical system 16 and outputs a weak light current.

Further, the cell observation device 10 has a current amplifier 20 that amplifies a weak optical current output from the optical detector 18 and converts it into a current having a voltage that is easy to handle and outputs the current after the optical detector 18. The capacitor 22 that removes the direct current (DC) component of the current output from the current amplifier 20 and outputs it, and the current that removes the direct current (DC) component output from the capacitor 22 is output as a voltage V that is greatly amplified electrically. It has a high-magnification amplifier 24 and a high-magnification amplifier 24.

Therefore, the voltage V output by the high magnification amplifier 24 is a voltage output obtained by electrically and greatly amplifying a weak optical current which is a signal of the optical component of the scattered light L2 detected by the photodetector 18.

The photodetector 18 can be configured by using, for example, a photodiode.

In the above configuration, the cell observation device 10 acquires the light scattering change of the sample Sa (for example, a living and active biological cell) as a change in voltage V as a physical quantity to be measured in the present invention.

In the cell observation device 10, the illumination light L1 emitted from the illumination light irradiation device 14 is arranged so as to be obliquely incident on the bottom surface 12a of the sample container 12 to illuminate the cell. That is, the bottom surface 12a of the sample container 12 is obliquely illuminated by the illumination light L1 emitted from the illumination light irradiation device 14.

Here, since the bottom surface 12a of the sample container 12 is transparent to the illumination light L1, the illumination light L1 hits the sample Sa as it is, and scattered light scattered in all directions is generated by the light scattering of the sample Sa.

A part of the scattered light generated in this direction and scattered in all directions passes through the bottom surface 12a of the sample container 12 and becomes the scattered light L2 traveling downward of the sample container 12.

The scattered light L2 traveling downward of the sample container 12 is efficiently guided to the photodetector 18 by the optical system 16.

The photodetector 18 detects the scattered light L2 and outputs a weak optical current, and the weak photocurrent output by the photodetector 18 is converted into a voltage that is easy to handle by the current amplifier 20, and is further converted into a direct current by the condenser 22. After removing the (DC) component, it is input to the high magnification amplifier 24, and the high magnification amplifier 24 outputs the current from which the direct current (DC) component is removed, which is output from the condenser 22, as a voltage V which is greatly amplified electrically.

That is, according to the cell observation device 10, the signal of the light component of the scattered light L2, which changes from moment to moment, is greatly amplified electrically, and the change in light scattering of the sample Sa is continuously acquired as the change in voltage V. be able to. This makes it possible to dynamically and continuously observe the activity of the sample Sa, that is, the cells.

(2) Measurement principle in the cell observation device 10

As described above, the physical quantity to be measured in the present invention is the light scattering change of the sample Sa (for example, a living and active biological cell).

Here, FIG. 2 shows an explanatory diagram showing the principle of the measurement method in the cell observation device 10. Note that FIG. 2 is an explanatory diagram showing a part of the configuration of the cell observation device 10 for explaining the principle of the measurement method, and the same configuration as that shown in FIG. 1 is indicated by the same reference numerals. Therefore, a detailed description of its configuration and operation will be omitted.

According to the cell observation apparatus 10, light scattering can be easily measured by the principle described below with reference to FIG.

That is, the illumination light L1 emitted from the illumination light irradiation device 14 irradiates the sample Sa, but most of it is transmitted light that penetrates and passes through the sample.

Further, the strongly reflected light reflected on the bottom surface 12a of the sample container 12 (not shown in FIG. 2) and the surface of the sample Sa is a line with respect to the axis symmetric with the incident illumination light L1. It will be reflected in a symmetrical position.

Here, most of the illumination light L1 that is the light emitted toward the sample Sa becomes transmitted light and reflected light, but some of the light of the illumination light L1 is the sample Sa (for example, a living cell). ) Is complicatedly reflected by the structure such as the gap (cell gap) of the sample Sa and the structure and protein, and is scattered in all directions to become scattered light.

Although it is extremely difficult to detect all of such scattered light, light scattered in a direction deviating from the optical axis of the illumination light L1, the optical axis of the transmitted light, and the optical axis of the reflected light (scattered light) should be detected. Is possible.

In the cell observation device 10, the optical system 16 and the light detector 18 are arranged in the direction directly below the center of the illumination light L1 and the reflected light, that is, below the extension direction of the axis of symmetry, and a part of the scattered light. The scattered light L2 is condensed and detected.

As can be understood from FIG. 2, the larger the aperture ratio of the optical system 16 used for light reception (when the optical system 16 is a lens, the value obtained by dividing the focal length of the lens by the radius of the aperture of the lens) is larger. , More scattered light L2 can be captured.

Here, when the sample Sa (for example, a living cell) has no structural directionality, the scattered light is scattered in all directions, and the scattered light becomes almost the same in all directions.

However, when the tissue or substance that causes the scattering of the sample Sa has a structural directionality, the scattered light is not the same depending on the scattering direction. For example, when the direction in which the tissue expands and the direction in which the tissue contracts are aligned, it is expected that there is a scattering direction depending on the alignment direction.

Therefore, the correlation between the functions of the scattered light rays scattered in a specific direction and the functions of the scattered light rays scattered in another direction is not always the same.

Therefore, in the present invention, it is possible to optimize the incident angle as described below and obtain the optimized incident angle.

(3) Optimization of incident angle

As shown in the configuration and principle described above, the cell observation device 10 can measure light scattering of a sample Sa such as a biological cell.

In the following, a point of optimizing the incident angle when the illumination light L1 is obliquely irradiated to the bottom surface 12a of the sample container 12 will be described.

Here, if the sample Sa to be observed has no structural features and is close to uniform, it is expected that the dependence on the incident angle is low.

On the other hand, if the sample Sa to be observed has structural characteristics and cannot be regarded as uniform, it is highly likely that the detected signal will differ depending on the incident angle.

FIG. 3 shows an explanatory diagram showing the incident angle in the cell observation device 10. Note that FIG. 3 is an explanatory diagram showing a part of the configuration of the cell observation device 10 for the purpose of explaining the incident angle, and the same configuration as that shown in FIG. 1 is indicated by the same reference numerals. A detailed description of its configuration and action will be omitted.

First, the definition of the incident angle in the present invention will be described with reference to FIG. As shown in FIG. 3, the light is arranged directly under the sample Sa and the sample container 12 with the axis of symmetry in the relationship between the illumination light L1 and the illumination light L1 and the reflected light having line symmetry as the main axis. The central axis of the illumination light L1 (the optical axis of the illumination light L1), which is the light emitted from the illumination light irradiation device 14 which is the light source for illumination and coincides with the optical axis of the light (scattered light L2) sensed by the detector 18. The angle formed by the main axis (the optical axis of the scattered light L2 sensed by the optical detector 18) is defined as the incident angle.

In order to obtain the optimum angle of incidence, the inventor of the present application conducted an experiment using a cell observation device 10 using cultured cardiomyocytes as sample Sa, and the change in voltage V, which is the result obtained by the experiment, is shown in FIG. It is shown. A photodiode was used as the photodetector 18.

In the waveform diagram shown in FIG. 4, the horizontal axis is the elapsed time, and the vertical axis is the minute change contained in the voltage obtained by photoelectrically converting the total amount of light incident on the photodiode 18 as the photodetector 18. It is an enlarged waveform. The scales of the vertical axis and the horizontal axis are as shown in FIG. 4, respectively.

In FIG. 4, each waveform is referred to as a waveform A, a waveform B, a waveform C, and a waveform D from the upper side to the lower side, respectively.

Here, the waveform A shows the result of measurement with the incident angle of 45 degrees, the waveform B shows the result of measurement with the incident angle of 45 degrees, the waveform C shows the result of measurement with the incident angle of 30 degrees, and the waveform D. Shows the result of measurement with the incident angle of 30 degrees.

For the cultured cardiomyocytes as sample Sa, the same cultured cardiomyocytes (cell 1) are used for waveform A and waveform C, and the same cultured cardiomyocytes different from cell 1 are used for waveform B and waveform D (cell 1). Cell 2) was used.

It was confirmed by a microscope that the cultured cardiomyocytes (cell 1 and cell 2) used in this experiment had the myocardium partially oriented, but no clearly aligned structure was observed so that the fibers could be seen. Such a structure is common and is a standard sample when cultured without intentional special manipulation.

Referring to FIG. 4, the waveforms of the waveform A and the waveform B are a little complicated, and there are two peaks in the vertical direction corresponding to the heartbeat of the myocardium, and the vertices of each can be seen.

On the other hand, the waveforms of the waveform C and the waveform D are monotonous, and the peak of the peak is one that points upward.

Further, when the waveform A and the waveform B are compared, they are not the same waveform, but when the waveform C and the waveform D are compared, they have substantially the same shape.

When the inventor of the present application tried the same experiment as the above with 20 or more cells as sample Sa, the waveform was diverse at an incident angle of 45 degrees, and about 70% had two or more vertices. Some of them had one vertex, about 20%, and some of them had small waveforms and were difficult to detect. On the other hand, when the incident angle was 30 degrees, all of the signals had almost the same shape, although the signal was large and small, and were similar to the shapes of the waveform C and the waveform D shown in FIG.

Although not shown, the same experiment as above was performed at an incident angle of 60 degrees, an incident angle of 20 degrees, and an incident angle of 10 degrees.

As a result, the incident angle of 60 degrees was similar to the result of the incident angle of 45 degrees, and the incident angle of 20 degrees was the same as the result of the incident angle of 30 degrees.

Further, when the incident angle is 10 degrees, the waveform is similar to the waveform with an incident angle of 30 degrees, but the total amount of light increases due to the increase in the directly reflected light, and the signal which is a change component is buried and the noise becomes large. The result was obtained.

From the results of the above experiments, the inventor of the present application has concluded that when cultured cardiomyocytes are used as sample Sa, an incident angle of 20 to 30 degrees is appropriate.

Although there is an optimum angle of incidence depending on the cell type and it is expected that they are not the same, according to the cell observation device 10 of the present invention, the angle of incidence is around 30 degrees, more specifically, the angle of incidence is 20 degrees or more and 45 degrees. If it is less than, more specifically, 20 to 40 degrees, it can correspond to many cell types.

(4) Explanation of the above-mentioned experiment and other experiments by the inventor of the present application.

(4-1) When cultured myocardial cells are used as the sample Sa and a general-purpose plastic container is used as the sample container 12 in the above-mentioned experiment and the experiments described in (4-2) and (4-3) described later. The amount of light incident on the light detector 18 was about 1/100 of the total amount of illumination light L1 emitted from the illumination light irradiation device 14 as a light source.

Of the amount of light incident on the photodetector 18, the signal derived from the change in light scattering accompanying the activity of cultured cardiomyocytes was about 1/1000 to 1/3000 of the amount of light incident on the photodetector 18.

(4-2) Using cultured cardiomyocytes as sample Sa, the wavelength dependence of the illumination light L1 irradiated from the illumination light irradiation device 14 was experimentally confirmed.

In the experiment, as the illumination light irradiation device 14, a light emitting diode that emits infrared light (wavelength 830 nm), a light emitting diode that emits red (660 nm), a light emitting diode that emits yellow (600 nm), and a green (530 nm) ), A light emitting diode that emits green-blue (505 nm), and a light emitting diode that emits blue (465 nm), respectively.

As a result, even if the wavelength of the illumination light L1 was changed, no difference was observed in the shape of the acquired voltage V waveform.

However, regarding the amplitude of the waveform, green, blue-green, and blue were about 20% larger than infrared light and red. The reason seems to depend on the absorption / reflection characteristics of the cultured cardiomyocytes, which are the sample Sa, but it can be concluded that there is almost no wavelength dependence of the illumination light L1 qualitatively.

(4-3) As shown in FIGS. 1 to 3, in the above experiment, a single (one) light emitting diode is used as the illumination light irradiation device 14.

The inventor of the present application arranges two light emitting diodes symmetrically with respect to the main axis below the bottom surface 12a of the sample container 12 as an illumination light irradiation device 14, and irradiates the bottom surface 12a with illumination light from both sides with respect to the main axis. The experiment was conducted.

Further, the inventor of the present application arranges four light emitting diodes evenly around the main axis below the bottom surface 12a of the sample container 12 as the illumination light irradiation device 14, and irradiates the bottom surface 12a with illumination light from all directions. An experiment was conducted.

As a result of the experiment, it was confirmed that there was almost no difference in the shape of the obtained voltage V waveform.

The sample container 12 and the sample Sa are placed on the base above the optical system 16, and noise is generated due to the influence of vibration or the like. Therefore, rather than irradiating the bottom surface 12a of the sample container 12 with the illumination light L1 from one side of the main shaft using a single (one) light emitting diode as the illumination light irradiation device 14, the main shaft uses two light emitting diodes. When the bottom surface 12a of the sample container 12 was irradiated with the illumination light from both sides, the sensitivity of the noise caused by vibration and the like was lowered, and a good signal could be observed.

Here, from the viewpoint of eliminating the noise caused by the vibration described above, when comparing the case where two light emitting diodes are used and the case where four light emitting diodes are used, four light emitting diodes are used. The case was slightly better, but no significant improvement was seen.

(II) Second embodiment

FIG. 5 shows a configuration explanatory diagram schematically showing a cell observation device according to another example of the embodiment of the present invention.

In FIG. 5, the same or equivalent configurations as those shown in FIG. 1 are designated by the same reference numerals, and detailed description of the configurations and actions thereof will be omitted.

From the viewpoint of optimizing the incident angle described above, the cell observation device 100 has an incident angle of the illumination light L1 emitted from the illumination light irradiation device 14 of 30 degrees, and the sample container 12 described above has the incident angle of 30 degrees. From the viewpoint of reducing noise caused by vibration, two illumination light irradiation devices 14 are arranged symmetrically with respect to the main axis.

As each of the two illumination light irradiation devices 14, a blue-green light emitting diode was used, and a bullet-shaped general-purpose product having a high brightness and a narrow irradiation angle and a standard of 5 mm was used.

As the optical system 16, a plastic lens having a diameter of 6 mm and a focal length of 6 mm was used.

Further, the distance α from the lens of the optical system 16 to the bottom surface 12a of the sample container 12 and the distance β from the lens of the optical system 16 to the surface of the photodiode used as the photodetector 18 are set to about 12 mm, respectively. It was adjusted so that it was in focus at a magnification of 1: 1.

Here, the photodiode used as the photodetector 18 is a 2.65 mm square silicon PIN type photodiode, and a general-purpose product having a sensitivity of about 950 nm to 400 nm was used.

Further, a general-purpose low-noise operational amplifier was used to configure the current amplifier 20 and the high-magnification amplifier 24 having an amplification factor of 50 times.

As the feedback resistor 102 that experimentally determines the current amplification factor, 1 MΩ to 10 MΩ was appropriate.

Further, between the output of the current amplifier 20 and the input of the high magnification amplifier 24, there is a DC cut circuit composed of a constant shown in FIG. 5, that is, a capacitor 22 having a capacitance of 1 μF and a resistor 104 having a resistance of 2 MΩ. It has been inserted. The time constant composed of the capacitance and the resistance depends on the sample Sa to be measured.

Although not shown, a reset circuit or a sample circuit may be added as needed.

The voltage V output from the high magnification amplifier 24 is used for analysis by connecting to an AD converter having a 16-bit resolution and a conversion period of 100 KHz, and continuously storing digital data in a memory and transferring it to a personal computer. You can do it.

(III) Third Embodiment

6 (a) and 6 (b) show a configuration explanatory diagram schematically showing a cell observation device according to another example of the embodiment of the present invention.

In FIGS. 6 (a) and 6 (b), the same or equivalent configurations as those shown in FIGS. 1 and 5 are designated by the same reference numerals, and detailed description of the configurations and actions thereof will be omitted. .. Further, in FIG. 6, in order to facilitate the understanding of the present invention, a part of the configuration of the cell observation apparatus 100 shown in FIG. 5 is shown in a simplified manner without being shown.

The cell observation device 200 shown in FIGS. 6 (a) and 6 (b) has a 96-well pitch (9 mm) generally used in culture research, and eight cell observation devices 100 shown in FIG. 5 are arranged side by side at the same time. It is possible to observe and record the activity of cells from a culture vessel which is 12 sample containers.

Further, as described above, the cell observation apparatus 200 in which eight cell observation apparatus 100s were arranged were arranged in three rows, and at the same time, the cell activity was recorded from the culture vessels of 24 sample containers 12 and the experiment was conducted.

As a result, it was confirmed that the activity of cultured cardiomyocytes derived from iPS cells (induced pluripotent stem cells) can be accurately measured by using the cell observation device 200.

(IV) Description of Other Embodiments and Modifications

It should be noted that the above-described embodiment is merely an example, and the present invention can be carried out in various other embodiments. That is, the present invention is not limited to the above-described embodiment, and various omissions, replacements, and changes can be made without departing from the gist of the present invention.

For example, the above-described embodiment may be modified as shown in (1) to (5) below.

(1) In the above-described embodiment, a case where a light emitting diode is used as the illumination light irradiation device 14 as a light source has been described. However, it goes without saying that the illumination light irradiation device 14 as a light source is not limited to the light emitting diode. As the illumination light irradiation device 14 as a light source, for example, a laser may be used as a light source instead of the light emitting diode. As a feature required for the illumination light irradiation device 14 as a light source, it is desirable that the spread (viewing angle) of the irradiation light is 5 degrees or less, and it is desirable that the emission intensity is extremely stable. Therefore, the light emitting diode and the laser are examples of a desirable light source as the illumination light irradiation device 14.

(2) In the experiment in the above-described embodiment, a light emitting diode having a blue-green emission color is used as the illumination light irradiation device 14 as a light source, but the illumination light irradiation device 14 as a light source is not limited to this. Of course. That is, as described above, in the present invention, the wavelength dependence of the illumination light L1 emitted from the illumination light irradiation device 14 as the light source is almost nonexistent, and the emission wavelength of the illumination light irradiation device 14 as the light source May select an appropriate wavelength suitable for the absorption / reflection characteristics of the sample Sa.

(3) In the above-described embodiment, the case where one, two, or four light source illumination light irradiation devices 14 are used has been described, but the number of light source illumination light irradiation devices 14 to be arranged is limited to this. Of course, it is not something that can be done. That is, three or five or more illumination light irradiation devices 14 as light sources may be arranged in a ring shape around the main axis. Even when three or five or more illumination light irradiation devices 14 are arranged, the incident angle of each is about 30 degrees, more specifically, the incident angle is 20 degrees, as in the above-described embodiment. It is set to less than 45 degrees, more specifically, 20 to 40 degrees.

(4) In the experiment in the above-described embodiment, the case where the photodiode is used as the photodetector 18 has been described, but it goes without saying that the photodetector 18 is not limited to the photodiode. Here, the characteristics required for the photodetector 18 are sensitivity and low noise, and the effective noise is sensitivity and low noise that sufficiently captures a change of 1/1000 or less with respect to the total amount of incident light. It is desirable to have, and at the same time, a sufficient response speed is required to capture the signal. If such a condition can be satisfied, for example, a multi-element photodiode or an image sensor can be used as the photodetector 18. According to the experiment by the inventor of the present application, a low noise and high speed image sensor could be used.

(5) Of course, the above-described embodiment and the various embodiments and modifications shown in the above-mentioned (1) to (4) may be appropriately combined.

The present invention can be used for research and industry using organisms such as cultured cells. In particular, according to the present invention, the activities of cultured cardiomyocytes and nerve cells can be easily observed. Therefore, the present invention is mainly used for research on the effects and side effects of drugs or quality control of cultured cells. Can be done.

10 Cell observation device 12 Sample container (container)
12a Bottom surface 14 Illumination light irradiation device (light irradiation means)
16 Optical system 18 Photodetector (detection means)
20 Current amplifier 22 Capacitor 24 High magnification amplifier 100 Cell observation device 102 Feedback resistance 104 Resistance 200 Cell observation device

Claims (6)

In a cell observation device that observes the dynamic function of cells
A first light irradiator and a second light irradiator that irradiate cells with light,
It is a cell observation device having a detection means for detecting a change in the scattered light by sensing the scattered light from the cells irradiated with light from the first light irradiation device and the second light irradiation device . hand,
It has a container that houses the cells and has a transparent bottom surface at least.
The first light irradiation device, the second light irradiation device, and the detection means are arranged below the lower surface of the container.
The incident angle, which is the angle between the optical axis of the light irradiated by the first light irradiation device and the second light irradiation device and the optical axis of the scattered light sensed by the detection means, is 20 degrees or more and 45 degrees. Is less than
The first light irradiation device and the second light irradiation device are arranged symmetrically with respect to the optical axis of the scattered light.
A cell observation device characterized by this. In the cell observation apparatus according to claim 1,
The angle of incidence is 20 to 40 degrees.
A cell observation device characterized by this. In the cell observation apparatus according to claim 1 ,
The incident angle is 20 to 30 degrees.
A cell observation device characterized by this. In the cell observation apparatus according to claim 1 ,
A cell observation device characterized in that the incident angle is 30 degrees. In the cell observation apparatus according to any one of claims 1, 2, 3 or 4.
The detection means is a photodetector that detects the scattered light and outputs a photocurrent.
After the photodetector,
A current amplifier that amplifies and outputs the photocurrent output from the photodetector, and
A capacitor that removes the direct current (DC) component of the current output from the current amplifier and outputs it.
A cell observation apparatus characterized by having a high-magnification amplifier that electrically amplifies a current from which a direct current (DC) component output from the capacitor has been removed and outputs it as a voltage. In a cell observation method for observing the dynamic function of cells,
The cell observation apparatus according to any one of claims 1, 2, 3, 4 or 5 is used.
The cells are irradiated with light from the first light irradiation device and the second light irradiation device, and the cells are irradiated with light.
The detection means continuously detects changes in the scattered light from the cells.
A cell observation method characterized by this.

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