JP2005278599A - Cultured cell observation device and method for observing cultured cell - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a cultured cell observation device capable of observing change of the cultured cells with lapse of time for a long term by keeping irradiation rate of a lamp to be proper, which rate is important in fluorometry of the cultured cells. <P>SOLUTION: The cultured cell observation device B is for observing change of cultured cells with the lapse of time, which cells exist on a carrier 6 or in a solution, wherein the device comprises a light source 70 irradiating light on the cultured cells A, a photometric part 6a measuring intensity of the light irradiated on the cultured cells A, a detecting part 40 detecting reflected light reflected from the photometric part 6a, and an analyzing part 50 analyzing the intensity of the reflected light detected by the detecting part 40 to measure the intensity of the light irradiated by the light source 70. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a cultured cell observation apparatus and a cultured cell observation method for observing changes in cultured cells over time.

  In recent years, gene analysis technology has advanced, gene sequences in many organisms including humans have been clarified, and the causal relationship between analyzed gene products (proteins) and diseases has been gradually elucidated. In the future, in order to comprehensively analyze various proteins and genes statistically, examination methods and devices using cells have begun to be considered. That is, it has been required to culture cells and observe changes over time in the cells.

Therefore, as a container for culturing cells under a microscope, as shown in FIG. 7, an observation apparatus provided with a culture container has been proposed (see Patent Document 1). In order to adjust the carbon dioxide concentration in the space S, the culture vessel forms a space S in which the temperature can be freely controlled by providing transparent heating plates 101 that can be automatically controlled to a set temperature by a temperature controller. A carbon dioxide supply port 102 and a discharge port 103 are provided in the container, and an evaporating dish 105 for keeping the humidity in the space S sealed by the seal packing 104 at a desired value is installed in the container. While observing, various cell culture conditions (temperature, carbon dioxide concentration and humidity) can be set. In the figure, reference numeral 106 denotes a microscope objective lens, 107 denotes a flow rate adjusting valve, 108 denotes water, and 109 denotes a microscope stage. In an observation apparatus provided with the culture container, a fluorescent protein is injected into a cultured cell to be observed so that it can be easily distinguished from other cultured cells, light is applied to the cultured cell, fluorescence is detected, and the behavior is observed. Therefore, the observation apparatus is provided with a lamp for irradiating the cultured cells with light. As the lamp, a xenon lamp is generally used because of the appropriateness of fluorescence detection.
JP-A-10-28576

  To observe cultured cells over a long period of time, it is important to keep the observation environment as constant as possible in order to distinguish slight differences. However, in the xenon lamp generally used for the observation apparatus, the cultured cells are irradiated with a constant light intensity for a while, but the light intensity is about 100 hours. When the irradiation time gradually decreases and the irradiation time reaches about 200 hours, the light intensity drops to about 60 to 80 percent from the beginning of use. That is, the observation can be performed in a desired environment until about 100 hours, but the observation environment is changed for observation exceeding 100 hours because the observation environment changes. Further, the light intensity of 60 to 80 percent of the xenon lamp is inappropriate for exciting weak light of cultured cells, and it is time to replace the lamp from the viewpoint of quantitativeness. Further, as an observation method, there is a method of quantifying the intensity of detected fluorescence and observing a change with time. In such a case, in order to appropriately detect the fluorescence light intensity, it is strongly required to maintain the light intensity of the irradiation light at a certain level.

  The present invention has been made in view of such circumstances, and maintains the intensity of the irradiation light of the lamp, which is important in fluorescence detection of cultured cells, to keep the changes of cultured cells over time over a long period of time. An object of the present invention is to provide a cultured cell observation apparatus that can be observed over a wide range.

The present invention provides the following cultured cell observation apparatus and cultured cell observation method as means for solving the above problems.
That is, the invention according to claim 1 is a cultured cell observation device for observing a time-dependent change of one or a plurality of cultured cells present on a carrier or in a solution, and a light source for irradiating the cultured cells with irradiation light A body, a photometric part for measuring the intensity of the irradiated light applied to the cultured cells, a detection unit for detecting reflected light reflected from the photometric part, and the reflected light detected by the detection unit And an analysis unit for measuring the intensity of the irradiation light emitted by the light source body.
In the cultured cell observation apparatus according to the present invention, the intensity of the light that the light source irradiates the cultured cell is determined by analyzing the intensity of the reflected light from the photometric part. Therefore, the observer can measure the intensity of light applied to the cultured cells of the light source body at an arbitrary timing while observing the cultured cells.

The invention according to claim 2 is the cultured cell observation apparatus according to claim 1, wherein the photometric part is provided on a carrier that bears the cultured cells.
In the cultured cell observation apparatus according to the present invention, the intensity of the light that the light source irradiates the cultured cells is obtained by analyzing the intensity of the reflected light of the carrier that bears the cultured cells to be observed. Therefore, the observer can measure the intensity of light applied to the cultured cells of the light source body at an arbitrary timing while observing the cultured cells.

The invention according to claim 3 is the cultured cell observation device according to claim 1 or 2, characterized in that the photometric part is provided on a support that supports and fixes the carrier.
In the cultured cell observation apparatus according to the present invention, the intensity of the light that the light source irradiates the cultured cell is determined by analyzing the intensity of the reflected light from the support that supports and fixes the carrier that bears the cultured cell to be observed. It will be. Therefore, when the observer wants to measure the intensity of light applied to the cultured cells of the light source body, the observer can easily measure it by slightly shifting the observed part.

  According to a fourth aspect of the present invention, in the cultured cell observation apparatus according to the first to third aspects, an output unit is connected to the analysis unit, and the analysis unit has a measured intensity of the irradiation light in advance. When the intensity falls below a set intensity, an output signal is sent to the output means, and the output means outputs information indicating that the intensity of the irradiation light is below the preset intensity based on the output signal. .

  In the cultured cell observation apparatus according to the present invention, when observing the cultured cells, when the intensity of the light applied to the cultured cells by the light source body is lowered to an inappropriate level, the output means notifies the fact. Information is output and the observer can know it. Therefore, the observer can easily know that the intensity of light emitted from the light source body is at an inappropriate level, and can immediately respond to this by switching the lamp of the light source body. .

The invention according to claim 5 is the cultured cell observation apparatus according to claim 4, characterized in that the information output by the output means is an audio output by an audio output means.
In the cultured cell observation apparatus according to the present invention, the observer can confirm with the ear that the intensity of light applied to the cultured cells by the light source body has decreased to an inappropriate level. Become. Therefore, the observer can easily know that the intensity of light emitted from the light source body is at an inappropriate level, and can immediately respond to this by switching the lamp of the light source body. .

The invention according to claim 6 is the cultured cell observation device according to claim 4 or 5, wherein the information output by the output means is lighting of a lamp by a lighting means.
In the cultured cell observation apparatus according to the present invention, the observer can visually confirm that the intensity of the light emitted from the light source to the cultured cells has decreased to an inappropriate level. Become. Therefore, the observer can easily know that the intensity of light emitted from the light source body is at an inappropriate level, and can immediately respond to this by switching the lamp of the light source body. .

The invention according to claim 7 is the cultured cell observation device according to any one of claims 4 to 6, wherein the information output by the output unit is an image output by the image output unit.
In the cultured cell observation apparatus according to the present invention, the observer visually confirms that the intensity of light applied to the cultured cells by the light source body has decreased to an inappropriate level while observing the cultured cells. Will be able to do. Therefore, the observer can easily know that the intensity of light emitted from the light source body is at an inappropriate level, and can immediately respond to this by switching the lamp of the light source body. .

The invention according to claim 8 is the cultured cell observation apparatus according to claims 1 to 7, wherein the irradiation light emitted from the light source body is white light.
In the cultured cell observation apparatus according to the present invention, since white light is composed of light of various wavelengths, reflected light of a plurality of wavelengths from the cultured cells can be measured simultaneously.

The invention according to claim 9 is the cultured cell observation device according to any one of claims 1 to 7, wherein the irradiation light emitted from the light source body is fluorescence excitation light.
In the cultured cell observation apparatus according to the present invention, it is possible to limit only light having a desired wavelength and measure only a decrease in the intensity of light having that wavelength, so that measurement accuracy can be increased.

The invention according to claim 10 is the cultured cell observation apparatus according to claim 9, wherein the photometric part includes a fluorescent member that emits fluorescence having a wavelength substantially the same as the wavelength of the reflected light emitted by the cultured cell to be observed. The detection unit detects the fluorescence emitted from the fluorescent member.
In the cultured cell observation apparatus according to the present invention, it is possible to limit only light having a desired wavelength and measure only a decrease in the intensity of light having that wavelength, so that measurement accuracy can be increased.

The invention according to claim 11 is the cultured cell observation apparatus according to claims 1 to 10, wherein the output of the detection unit is corrected based on the measured intensity of the irradiation light emitted from the light source body. It is characterized by.
In the cultured cell observation apparatus according to the present invention, when the intensity of the irradiation light from the measured light source body is too strong or too weak, the output is corrected by the detection unit. Therefore, the cultured cells can be observed more favorably by the corrected output of the detection unit.

The invention according to claim 12 is the cultured cell observation device according to any one of claims 1 to 11, wherein the irradiated light is applied to the cultured cells based on the intensity of the irradiated light emitted from the measured light source body. An irradiation light adjusting unit for adjusting the intensity of the light source is provided between the light source body and the cultured cells.
In the cultured cell observation apparatus according to the present invention, when the intensity of the irradiation light from the measured light source body is too strong or too weak, the intensity of the irradiation light irradiated to the cultured cell by the irradiation light control unit Can be adjusted appropriately. Therefore, the intensity of the irradiation light applied to the cultured cells becomes appropriate and constant, and the cultured cells can be observed well.

The invention according to claim 13 is the cultured cell observation device according to any one of claims 1 to 12, wherein the intensity of the irradiation light emitted from the light source body is measured before the cultured cell is detected. And
In the cultured cell observation apparatus according to the present invention, the observer can confirm the intensity of the irradiated light when starting to observe the cultured cells. Therefore, the observer can observe the cultured cells better by adjusting the intensity of the light emitted from the light source body based on the intensity of the light at the beginning of observation.

According to a fourteenth aspect of the present invention, in the cultured cell observation device according to the first to thirteenth aspects, the irradiation emitted from the light source body at a predetermined time interval or a predetermined operation interval while detecting the cultured cell. It is characterized by measuring the intensity of light.
In the cultured cell observation apparatus according to the present invention, the observer can confirm the intensity of the irradiated light even while observing the cultured cells. Therefore, the observer can confirm the suitability of the intensity of the irradiation light emitted from the lamp, and can observe the cultured cells better.

The invention according to claim 15 is the cultured cell observation device according to any one of claims 1 to 14, wherein the light source body includes a plurality of light sources and a selection unit that selects the irradiation light emitted by the plurality of light sources. It is provided with.
In the cultured cell observation apparatus according to the present invention, when the intensity of the irradiation light from the light source body has dropped, the irradiation light to be irradiated can be selected from a plurality of light sources and irradiated to the cultured cells. It will be a thing. Therefore, the intensity of the irradiation light applied to the cultured cells becomes appropriate and constant, and the cultured cells can be observed well.

The invention according to claim 16 is the cultured cell observation device according to claim 15, wherein the selection means is an alternative selection means for selecting the irradiation light emitted by the plurality of light sources. .
In the cultured cell observation device according to the present invention, when the intensity of the irradiation light from the light source body has dropped, the irradiation light to be irradiated is selected from a plurality of light sources and irradiated to the cultured cells. Will be able to. Therefore, the intensity of the irradiation light applied to the cultured cells becomes appropriate and constant, and the cultured cells can be observed well.

The invention according to claim 17 is the cultured cell observation device according to claim 15 or 16, wherein the alternative selection means is configured to select the plurality of light sources based on the measured intensity of the irradiation light from the light source body. The irradiation light emitted from the light source is selectively switched.
In the cultured cell observation apparatus according to the present invention, when the intensity of the irradiation light from the light source body is likely to decrease, the irradiation light to be irradiated is selected from a plurality of light sources, and the cultured cells are irradiated with an appropriate intensity. Will be able to. Accordingly, the intensity of the irradiation light applied to the cultured cells is appropriately constant without concern for the observer, and the cultured cells can be observed favorably.

The invention according to claim 18 is a cultured cell observation method using the cultured cell observation apparatus according to claim 4, wherein the reflected light reflected from the photometric part irradiated with the irradiation light by the light source body is reflected. A procedure of detecting, a procedure of analyzing the intensity of the reflected light by the analysis unit to measure the intensity of the irradiated light irradiated by the light source body, and the intensity of the measured irradiated light being less than a preset intensity A step of outputting, by the output means, information indicating that the intensity of the irradiation light emitted from the light source body is equal to or lower than a preset intensity.
In the cultured cell observation method according to the present invention, the intensity of the irradiation light that the light source body irradiates the cultured cell is determined by analyzing the intensity of the reflected light from the photometric part, and the light source body further includes the cultured cell. When the intensity of the irradiation light applied to the light falls to an inappropriate level, information indicating that is output by the output means, and the observer can know this. Therefore, the observer can measure the intensity of the irradiation light irradiated to the cultured cells of the light source body even while observing the cultured cells, and the observer has an inappropriate intensity of the irradiation light irradiated to the light source body. It is possible to easily know that the level is low, and it is possible to immediately respond to this by switching the lamp of the light source body.

  According to the cultured cell observation apparatus and the cultured cell observation method of the present invention, the intensity of the irradiation light of the lamp, which is important in fluorescence detection of the cultured cell, is maintained in an appropriate state, and the change of the cultured cell over time is maintained. Can be observed.

Embodiments of the present invention will be described below with reference to the drawings.
A cultured cell observation apparatus 1 according to the present invention is an apparatus for continuously observing changes over time of a plurality of cultured cells A present on a carrier, as shown in FIG. That is, the cultured cell observation apparatus 1 accommodates the carrier (not shown) inside and can hold the incubator 4 capable of maintaining the cell activity of the cultured cell A on the carrier and the incubator 4. An inverted microscope 20 is provided. The incubator 4 is formed so as to accommodate a microplate 6 or a culture vessel 10 as a carrier, as will be described later.

  As shown in FIG. 1, the inverted microscope 20 images the motorized stage (movable stage) 30 that holds the incubator 4 and the cultured cells in the incubator 4 by dividing them into regions corresponding to the cells. It has a detector 40. In addition, the cultured cell observation apparatus 1 includes a computer 50 that extracts and analyzes at least one of the geometric feature amount or the optical feature amount of the cell A based on the image captured by the detector 40. . Various output devices 55 are connected to the computer 50. The detector 40 corresponds to a detection unit in the present invention, and the computer 50 corresponds to an analysis unit in the present invention. Further, the output device 55 corresponds to the output means in the present invention.

  The electric stage 30 is driven by a motor (not shown) and is supported by the main mount 21 so as to be movable in the horizontal direction. An incubator fixing portion 31 for fixing the incubator 4 is provided on the electric stage 30 so that the horizontal angle can be adjusted. That is, the incubator fixing portion 31 is formed in a flat plate shape and is fixed on the electric stage 30 by surrounding mounting screws. At this time, the horizontal angle of the electric stage 30 with respect to the horizontal plane can be adjusted by adjusting the tightening amount of the surrounding mounting screws. The incubator 4 is fitted and fixed by a locking port (not shown) formed at the center of the incubator fixing portion 31. Therefore, the incubator 4 can be adjusted in the horizontal plane via the incubator fixing portion 31 and can be moved in the horizontal direction via the electric stage 30.

  Although not shown, an objective lens 41 for observing the cultured cells A on the carrier accommodated in the incubator 4 is provided below the electric stage 30, and an image is formed by the objective lens 41. The obtained image is configured to be output to a CCD camera 42 which is a part of the detector 40. The CCD camera 42 has a function of outputting the captured image to the computer 50 via an interface. Further, a light source body 70 (see FIG. 2 and subsequent figures) for irradiating the cultured cells A with light is provided below the objective lens 41 so as to be built in the main mount 21.

  Next, the main part of the present invention applied to the cultured cell observation apparatus 1 will be described with reference to FIGS. That is, the light source 70 that irradiates the cultured cell A with light, the photometric part 3 provided on the carrier, the detector 40 that detects the reflected light from the photometric part 3, and the intensity detected by the detector 40 The analysis unit 50 to be analyzed will be described in detail.

  As shown in FIG. 2, the main part B according to the first embodiment includes a xenon lamp 71 serving as a light source body 70 and an irradiation angle of irradiation light emitted from the xenon lamp 71 perpendicular to the carrier. A half mirror 72 for reflecting the reflected light from the cultured cell A and collimating the reflected light reflected from the cultured cell A by condensing the irradiated light to irradiate the cultured cell A. An objective lens 41 that converts light, a photodetector 40 that detects the reflected light, and a computer 50 that analyzes the detected numerical values are provided. The computer 50 is connected to an output device 55 including an audio output device 56, a lighting device 57, and an image output device 58, which are output means of the present invention as shown in FIG.

  In the main part B, a microplate 6 is used as a carrier for the cultured cells A, and the microplate 6 is accommodated in the incubator 4 on the electric stage 30. The microplate 6 contains cultured cells A into which fluorescent protein has been injected. The cultured cells A are irradiated with light to emit fluorescence, and the observer detects the fluorescence and determines the time of the cultured cells A over time. Observe changes.

  As the xenon lamp 71, for example, a known xenon lamp that emits white light is used, and its lamp life is about 200 hours. For example, a CCD camera 42 is used as the detector 40. The detector 40 may be a device that can amplify photoelectrons such as photomultipliers as long as it can detect light. The computer 50 has a calculation control function including a CPU, a ROM, and the like, similar to a known computer, and calculates the intensity of the detected reflected light and measures the irradiation light of the xenon lamp 71. Have Note that the computer 50 of the cultured cell observation apparatus 1 is configured to correct the output value of the detector 40 based on the measured intensity of light emitted from the xenon lamp 71.

  In addition, the computer 50 is set so that, when the intensity of the measured irradiation light is equal to or lower than a preset intensity, information notifying the observer is output to the connected output device 55. Has been. The output device 55, the sound output device (sound output means) 56 is a speaker, the lighting device (lighting device) 57 is a lamp lighting device, and the image output device (image output device) 58 is a liquid crystal monitor. On the electric stage 30, the temperature is about 37 ° C., the humidity is about 100%, and the concentration of carbon dioxide is about 5% so that the cultured cells A are preferably cultured over a long period of time. It has been preserved.

  When the cultured cell observation apparatus 1 is configured with the above-described main part B as the main part of the present invention, the irradiation light irradiated by the xenon lamp 71 is refracted at right angles by the half mirror 72 and collected by the objective lens 41. Light is applied to the bottom surface 6a of the microplate 6 as a carrier. The bottom surface 6a of the microplate 6 reflects the irradiation light, and the reflected light is converted into collimated light by the objective lens 41, passes through the half mirror 72, and includes a CCD camera 42 serving as the detection unit. Detected by the instrument 40. The computer 50 calculates and analyzes the intensity of the reflected light detected by the detector 40, and measures the intensity of the irradiation light irradiated by the xenon lamp 71.

  Further, the computer 50 corrects the output value of the detector 40 in accordance with the intensity of the irradiation light. Therefore, an image output to image output means such as a television monitor (not shown) is suitable for observing the cultured cell A.

Further, when the intensity of the irradiation light reaches a preset intensity, the computer 50 controls the output so that the output device 55 outputs information notifying the observer of that fact. That is, the speaker 56 emits a buzzer sound, the lamp illuminator 57 is lit with a predetermined warning lamp, and the liquid crystal monitor 58 is “lamp irradiation light is reduced”. Is displayed on the monitor.
Therefore, the observer can easily know that the intensity of the irradiation light emitted from the xenon lamp 71 has been reduced by the above output, and can respond immediately to this. For example, when the intensity of the irradiation light irradiated by the xenon lamp 71 is undesirably lowered when observing the cultured cell A, the xenon lamp 71 is replaced and the observation of the cultured cell A is preferably continued. It will be possible.

  The measurement of the light intensity of the irradiation light of the xenon lamp 71 may be performed before the observation (detection) of the cultured cell A is started, or the change with time of the cultured cell A is observed ( The detection may be performed at a predetermined time interval or a predetermined operation interval. Before performing the observation (detection) of the cultured cell A, it is possible to confirm the intensity of light irradiated by the xenon lamp 71 and to ensure the observation of the cultured cell A. it can. Further, when performing at the predetermined time interval or the predetermined operation interval, the intensity of irradiation light irradiated by the xenon lamp 71 can be confirmed even during observation of the cultured cell A, and the culture is performed. Cell A can be detected quantitatively.

  The photometric part 3 in the main part B was the bottom surface 6 a of the microplate 6. Therefore, when measuring the light intensity of the irradiation light of the xenon lamp 71, the reflected light reflected from the bottom surface 6a of the microplate 6 is collected and detected, and the intensity of the reflected light is calculated and analyzed. The light intensity of the irradiation light irradiated by the xenon lamp 71 was measured. However, the photometric part 3 is not limited to this, and an electric stage 30 that supports and fixes the microplate 6 serving as the carrier may be used as the photometric part. The electric stage 30 corresponds to the support in the present invention.

Next, a main part C which is applied to the cultured cell observation apparatus 1 and is a modified example of the main part B of the present invention described above will be described. In addition, about the part comprised similarly to the principal part B which is the above-mentioned 1st Embodiment, the same code | symbol is attached | subjected and the description is abbreviate | omitted.
That is, the main part C which is this modified example is applied to the cultured cell observation device 1 similarly to the main part B, as shown in FIG. A culture vessel 10 that can cultivate a biological sample by circulating a culture solution is used. The culture vessel 10 is stored in the incubator 4. As shown in FIG. 4, the culture vessel 10 can accommodate a slide glass 11 on which cells A to be cultured are placed, and a glass member 12a for observation with an objective lens 41 is provided on the bottom surface of the culture vessel wall 10a. A glass member 12b for allowing light from the xenon lamp 71 to pass therethrough is provided in close contact with the culture vessel wall surface portion 10a. The culture vessel wall surface portion 10a is formed by molding a resin material suitable for culturing cells, and the glass member 12b on the upper surface side is detachable via a circular resin member 19 such as an O-ring, Work in the culture vessel 10 such as taking in and out of the slide glass 11 can be performed.

  As shown in FIG. 4, the culture system for supplying a culture solution to the culture vessel 10 includes a culture solution bottle 13, a culture solution pump 14, a carbon dioxide supply means 15, a culture solution pipe 16, and a control computer. 17. The culture solution bottle 13 is a container for storing a culture solution mixed with nutrients necessary for cell growth and 5% carbon dioxide. This culture solution is maintained at 37 ° C. by controlling a heater (not shown) by the culture solution bottle temperature control unit 18a, thereby preventing a decrease in cell activity due to a temperature change of the culture solution. Further, the carbon dioxide supply means 15 connected to the culture solution bottle 13 supplies the carbon dioxide to the culture bottle 13 under the control of the carbon dioxide concentration control unit 18b, so that carbon dioxide is mixed into the culture solution.

  Thus, the culture solution mixed with carbon dioxide circulates through the culture vessel 10 connected by the culture solution pipe 16 by operating the culture solution pump 14 under the control of the culture solution pump control unit 18c. Gases and nutrients are supplied to the cultured cells in the culture vessel 10. Each of the culture bottle temperature control unit 18a, the carbon dioxide concentration control unit 18b, and the culture solution pump control unit 18c receives a control signal from the computer 17.

  When the cultured cell observation apparatus 1 is configured with the above-described main part C as the main part of the present invention, the irradiation light irradiated by the xenon lamp 71 is reflected at right angles by the half mirror 72 and collected by the objective lens 41. Light and irradiate the wall surface portion 10a of the culture vessel. The wall surface portion 10a of the culture vessel reflects the irradiated light, and the reflected light is converted into collimated light by the objective lens 41, passes through the half mirror 72, and includes a CCD camera 42 serving as the detection unit. Detected by the instrument 40. The computer 50 calculates and analyzes the intensity of the reflected light detected by the detector 40, and measures the light intensity of the irradiation light irradiated by the xenon lamp 71. As described above, even in this modification, the computer 50 controls the output so that the output device 55 outputs the light when the light intensity of the irradiation light reaches a preset light intensity. .

  This modification corresponds to the photometric part 3 of the present invention is the wall surface portion 10a of the culture vessel, and reflected light is obtained by reflection of the resin surface. Therefore, the light intensity of the reflected light is stronger than the reflected light due to the reflection of the glass surface. Therefore, the measurement of the light intensity of the irradiated light becomes more accurate. Furthermore, when the culture vessel 10 is used, the culture solution is circulated suitably. Therefore, a suitable environment can be maintained when the cultured cell A is cultured for a long period of time.

Next, the main part D of the present invention, which is applied to the cultured cell observation apparatus 1 and is the second embodiment, will be described. In addition, about the part comprised similarly to the above-mentioned 1st Embodiment, the same code | symbol is attached | subjected and the description is abbreviate | omitted.
That is, the main part D of the present invention which is the second embodiment is applied to the cultured cell observation apparatus 1 as in the main part B as shown in FIG. The reference piece 60 is used instead of the microplate 6 for the portion 3. The reference piece 60 is placed on the electric stage 30 and corresponds to the fluorescent member in the present invention. Specifically, the reference piece 60 emits fluorescence having the same wavelength as the light emitted from the cultured cell A into which the fluorescent protein has been introduced, and the fluorescent plate or the fluorescent beads are the same as the cultured cell A. The electric stage 30 is provided at a height position.

  The pigment applied to the reference piece 60 has a property of emitting fluorescence when excited by irradiation with fluorescence excitation light. Therefore, in the main part D, instead of the xenon lamp 71, a fluorescent excitation lamp 73 that causes the reference piece 60 to emit fluorescence is used in the light source body 70. If the dye applied to the reference piece 60 is excited and emits fluorescence when irradiated, the fluorescence excitation lamp 73 can emit the fluorescence. An appropriate lamp such as a lamp is selected.

  Further, in the main part D, a wavelength selector 65 is provided at the position of the half mirror 72. The wavelength selector 65 is a member that transmits only an arbitrary wavelength of light. The wavelength selector 65 selects and transmits only the wavelength of light that the observer wants to detect, and transmits other wavelengths of light. It has the property which can prevent. Therefore, when detecting light, the observer can appropriately select the wavelength of desired light to be detected, such as fluorescence emitted from the cultured cell A and fluorescence emitted from the reference piece 60. The wavelength selector 72 is set so that the reference piece 60 transmits only the wavelength of fluorescence emitted when the reference piece 60 is excited by the fluorescence excitation lamp 73 and does not transmit other wavelengths of light. The half mirror 72 and the wavelength selector 65 are formed as a set that forms one filter set 66, and the filter set 66 is configured to be replaceable.

  When the cultured cell observation apparatus 1 is configured with the main part D described above as the main part of the present invention, the fluorescence excitation light irradiated by the fluorescence excitation lamp 73 is reflected by the half mirror 72 at a right angle, and the objective lens The light is condensed by 41 and irradiates the reference piece 60 provided on the electric stage 30. The irradiated reference piece 60 is excited by fluorescence excitation light to emit fluorescence, and the fluorescence is converted into collimated light by the objective lens 41 and passes through the half mirror 72. Further, the wavelength selector 65 transmits only the fluorescent collimated light emitted by the reference piece 60 among the collimated light. And it detects with the detector 40 containing CCD camera 42 used as the said detection part. The computer 50 calculates and analyzes the intensity of the fluorescence detected by the detector 40 and measures the intensity of the fluorescence excitation light emitted by the fluorescence excitation lamp 73.

  As in the first embodiment described above, even in the second embodiment, when the intensity of the fluorescence excitation light reaches a preset intensity, the computer 50 causes the output device 55 to The output is controlled so that it is output. That is, the sound output device 56 emits a warning sound, the lighting device 57 turns on a predetermined warning lamp, and the image output device 58 indicates that “the fluorescence excitation light has decreased. Etc. "is displayed. Therefore, the observer can easily know that the light intensity of the fluorescence excitation light emitted from the fluorescence excitation lamp 73 has been lowered by the above output, and can respond immediately to this. It will be a thing. For example, when the intensity of light irradiated by the fluorescent excitation lamp 73 is undesirably reduced in observing the cultured cell A, the fluorescent exciting lamp 73 is replaced to cope with the observation of the cultured cell A. It will be able to continue preferably.

  Further, the observer can know the decrease in the light intensity by using the reference piece by limiting to the wavelength used for observation. Therefore, in white light or the like, not all wavelengths are reduced uniformly, so it is possible to accurately know the intensity reduction for light of a desired wavelength, and more suitable for observation of cultured cells A. It will be something that can be. The wavelength selector 65 is included in a replaceable filter set 66. Therefore, when fluorescent proteins having different wavelengths are introduced into the cultured cells, if the filter set 66 is replaced, it becomes suitable for observation of the cultured cells.

Next, the main part E of the present invention which is applied to the cultured cell observation apparatus 1 and is the third embodiment will be described. In addition, about the part comprised similarly to the above-mentioned 2nd Embodiment, the same code | symbol is attached | subjected and the description is abbreviate | omitted.
That is, the main part E of the present invention, which is the third embodiment, is applied to the cultured cell observation device 1 in the same manner as the main part D, as shown in FIG. In addition to the configuration of the main part D, an irradiation light adjusting unit 80 for adjusting the light intensity of the irradiation light applied to the cultured cell A is provided between the light source body 70 and the cultured cell A. The irradiation light adjusting unit 80 adjusts the intensity of irradiation light emitted from the light source body 70 based on the measured intensity of irradiation light emitted from the light source body 70 and causes the cultured cells A to emit irradiation light. As will be described in detail later, the light source body 70 in the main part E is configured to include a plurality of light sources and a selection unit that selects light emitted from the plurality of light sources. Xenon lamps are used for the.

  Specifically, a filter wheel 81 is used for the irradiation light adjusting unit 80. As shown in FIG. 6B, the filter wheel 81 has a wheel body 82 having a circular cross section, and the wheel body 82 is provided with a plurality of holes 83 like a revolver. In this hole 83, transmission filters having different light transmittances are set. As described above, the filter wheel 81 rotates the wheel body based on the measured intensity of light emitted from the light source body 70 (70A in this embodiment), and the transmission filters have different light transmittances. Is appropriately selected to adjust the light intensity of the irradiation light emitted by the light source body 70A.

  Further, the light source body 70A of the main part E is configured to be provided with xenon lamps as two light sources so as to irradiate the filter wheel 81 with light. Specifically, the light source body 70A is provided so that the half mirror 75 maintains an angle of 45 degrees diagonally downward to the right with respect to the filter wheel 81, and the half mirror is in the same direction as the irradiation direction of the light source body 70A. A xenon lamp 71a provided with a shutter 74a is provided between them and a xenon lamp 71b provided with a shutter 74b below the half mirror 75. As with the half mirror 72, the half mirror 75 has the property of reflecting light at half intensity and transmitting light at half intensity. The shutters 74a and 74b have a property of shielding light. Further, the main part E is provided with two types of reference pieces 61 and 62 that emit fluorescence having the same wavelength as the two types of fluorescent proteins.

  When the cultured cell observation apparatus 1 is configured with the above-described main part E as the main part of the present invention, for example, when the xenon lamp 71b is turned off, the shutter 74a is opened and the xenon lamp 71a is turned on, the xenon lamp 71a is turned on. The intensity of half the irradiation light emitted from the light source is emitted as the light source body 70A. When the intensity of the irradiation light emitted from the xenon lamp 71a decreases with time and the intensity of the irradiation light becomes unsuitable for detecting fluorescence of the cell, the xenon lamp 71b is turned on. When the shutter 74a is closed, half the intensity of the irradiation light emitted from the xenon lamp 71b is emitted as the light source body 70A. When the intensity of the irradiation light emitted from the xenon lamp 71b decreases and the intensity of the irradiation light becomes unsuitable for detecting fluorescence of cells, the xenon lamp 71a is opened when the shutter 74a is opened. The light emitted from the xenon lamp 71b and the light emitted from the xenon lamp 71b are emitted as the light source 70A. Therefore, the cultured cells A can be irradiated with a preferable light intensity over a long period of time, compared to the case where the light source body is constituted by one xenon lamp. When the shutter 74a is closed and the xenon lamp 71b is turned on, the observer replaces the xenon lamp 71a with a new one, and when the intensity of irradiation light emitted from the xenon lamp 71b decreases, the xenon lamp 71a again. May be turned on and the shutter 74b may be closed.

Further, by rotating a wheel body provided in the filter wheel 81 and appropriately selecting a transmission filter having a different light transmittance, it is possible to further finely adjust the intensity of irradiation light emitted by the light source body 70A. The observation environment for observing the cultured cell A becomes better. Further, as described above, when the transmission filter is appropriately selected based on the intensity of light emitted from the light source 70A measured by the computer 50, the selection of the transmission filter is more precise. Thus, the cultured cell A can be observed in a stable observation environment over a long period of time. Accordingly, the complexity of lamp replacement and the like is eliminated, leading to cost reduction.
Furthermore, the two types of reference pieces 61 and 62 emit fluorescence having the same wavelength as the two types of fluorescent proteins. Therefore, even when a plurality of types are observed separately, the light intensity of the fluorescence excitation light of each wavelength can be examined, and the range of observation of the cultured cells is widened.

In addition, this invention is not limited to said embodiment, In the range which does not deviate from the meaning, an appropriate selection is possible. For example, the mirror that reflects the irradiation light or the fluorescence excitation light is half-mirrors 72 and 75 as an example in the configuration. However, the present invention is not limited to this, and a mirror that totally reflects is provided at an appropriate position. It may be configured.
The irradiation light adjusting unit 80 does not set a transmission filter in each of the holes 83 provided in the filter wheel main body 82 of the filter wheel 81, but the filter wheel main body 82 itself is configured with a filter whose transmittance gradually changes. It may be. Further, the present invention is not limited to the filter wheel 81, and a configuration may be adopted in which transmission filters having different light transmittances can be selectively changed so that irradiation light emitted from the light source body 70A is appropriately adjusted.
Moreover, the said reference piece can provide multiple types suitably the same as the wavelength which the fluorescent protein inject | poured into a cultured cell emits. Furthermore, when the light source body is composed of a plurality of light sources, the light source composed of two xenon lamps can be an appropriate number of light sources.
Further, the irradiation light may be white light composed of light of various wavelengths, and the intensities of the light of a plurality of types of wavelengths reflected from the cultured cells may be measured simultaneously.

It is a block diagram of the cultured cell observation apparatus which concerns on this invention. It is a block diagram about the principal part B of the cultured cell observation apparatus shown in FIG. 1 used as 1st Embodiment. It is a block diagram about the principal part C used as the modification of the principal part B of the cultured cell observation apparatus shown in FIG. It is a schematic block diagram of a culture system. It is a block diagram about the principal part D of the cultured cell observation apparatus shown in FIG. 1 used as 1st Embodiment. It is a block diagram about the principal part E of the cultured cell observation apparatus shown in FIG. 1 used as 1st Embodiment. It is the conventional observation apparatus provided with the culture container.

Explanation of symbols

A Cultured cells B, C, D, E Main parts of cultured cell observation device 1 Cultured cell observation device 6 Microplate (carrier)
6a Bottom of microplate (photometric part)
40 Detector (Detector)
50 Computer (Analysis Department)
70 Xenon lamp (light source)

Claims (18)

A cultured cell observation device for observing a change with time of one or more cultured cells present on a carrier or in a solution,
A light source for irradiating the cultured cells with irradiation light;
A photometric part for measuring the intensity of the irradiation light irradiated to the cultured cells;
A detection unit for detecting reflected light reflected from the photometric part;
A cultured cell observation apparatus comprising: an analysis unit that analyzes the intensity of the reflected light detected by the detection unit and measures the intensity of the irradiation light emitted from the light source body.   The cultured cell observation apparatus according to claim 1, wherein the photometric part is provided on a carrier that bears the cultured cells.   The cultured cell observation apparatus according to claim 1, wherein the photometric part is provided on a support that supports and fixes the carrier. Output means is connected to the analysis unit,
The analysis unit sends an output signal to the output unit when the measured intensity of the irradiation light is equal to or lower than a preset intensity, and the output unit determines the intensity of the irradiation light based on the output signal. 4. The cultured cell observation apparatus according to claim 1, wherein information that is equal to or less than a set intensity is output.   The cultured cell observation apparatus according to claim 4, wherein the information output by the output unit is an audio output by an audio output unit.   The cultured cell observation apparatus according to claim 4 or 5, wherein the information output by the output means is lighting of a lamp by a lighting means.   The cultured cell observation apparatus according to claim 4, wherein the information output by the output unit is an image output by an image output unit.   The cultured cell observation apparatus according to claim 1, wherein the irradiation light emitted from the light source body is white light.   The cultured cell observation apparatus according to claim 1, wherein the irradiation light emitted from the light source body is fluorescence excitation light. The photometric part comprises a fluorescent member that emits fluorescence having a wavelength substantially the same as the wavelength of the reflected light emitted by the cultured cells to be observed,
The cultured cell observation apparatus according to claim 9, wherein the detection unit detects the fluorescence emitted from the fluorescent member.   The cultured cell observation apparatus according to claim 1, wherein the output of the detection unit is corrected based on the measured intensity of the irradiation light emitted from the light source body.   An irradiation light adjusting unit that adjusts the intensity of the irradiation light applied to the cultured cells based on the measured intensity of the irradiation light emitted from the light source body is provided between the light source body and the cultured cells. The cultured cell observation apparatus according to claim 1, wherein:   The cultured cell observation apparatus according to claim 1, wherein the intensity of the irradiation light emitted from the light source body is measured before the cultured cell is detected.   The cultured cell observation according to any one of claims 1 to 13, wherein the intensity of the irradiation light emitted from the light source body is measured at a predetermined time interval or a predetermined operation interval while detecting the cultured cell. apparatus.   The cultured cell observation apparatus according to claim 1, wherein the light source body includes a plurality of light sources and a selection unit that selects the irradiation light emitted by the plurality of light sources.   The cultured cell observation apparatus according to claim 15, wherein the selection unit is an selection unit that selects the irradiation light emitted from the plurality of light sources.   16. The alternative selection unit is configured to selectively switch the irradiation light emitted from the plurality of light sources based on the measured intensity of the irradiation light from the light source body. 16. The cultured cell observation apparatus according to 16. A cultured cell observation method using the cultured cell observation device according to claim 4,
The procedure of detecting the reflected light reflected from the photometric part irradiated with the irradiation light by the light source body, and analyzing the intensity of the reflected light by the analysis unit and the irradiation light irradiated by the light source body The procedure for measuring the intensity, and when the measured intensity of the irradiated light is less than or equal to a preset intensity, information that the intensity of the irradiated light irradiated by the light source body is less than or equal to a preset intensity, A method of observing cultured cells, comprising a procedure of outputting by an output means.

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