JP2005245288A - Culturing and observing method and inverted microscope - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To enable good observation of a cell in a culture solution for a long time. <P>SOLUTION: The subject method for observing a cell in a vessel 30 while culturing the cell comprises packing a space between the top of an objective lens 8 for observing the cell in the vessel 30 and the bottom of the vessel 30 with an aqueous solution 51 of a substance having deliquescence and observing the cell with the objective lens 8 through the aqueous solution 51 of the substance having deliquescence. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a culture observation method for cell culture observation and an inverted microscope used therefor.

  Conventionally, an inverted microscope is well known as a means for observing cells. For example, as disclosed in Patent Document 1, the inverted microscope is used for observation with an objective lens from below a cover glass on which cells are placed. When using a high NA objective lens, observation is performed in a state where immersion oil is filled between the tip of the objective lens and the lower surface of the cover glass in order to enhance the resolution.

Further, when observing cells during such culture, it is common to maintain the cells in the container at a predetermined temperature. In such a case, a heater is attached to the container, and the temperature of the cells is kept constant by heat from the heater.
JP 2001-272606 A (paragraph 0023, etc.)

  However, as described in Patent Document 1, when the immersion oil is filled between the objective lens and the cover glass, if the cell to be observed is located away from the upper surface of the cover glass, the cell and the cover glass A culture solution exists between the two. Since the refractive index of this culture solution is almost equal to the refractive index of water (about 1.33), the difference between the refractive index of immersion oil (about 1.52) and the refractive index of the culture solution becomes large, and the aberration is reduced. This is inconvenient in that good observation cannot be performed.

  As a countermeasure in such a case, it is conceivable to use water instead of immersion oil. The difference in refractive index between water and the culture medium is small, and the generation of aberration can be suppressed accordingly. However, when water is used, water itself may evaporate when observed continuously for a long time. In addition, as described above, when observing while maintaining the culture solution at a constant temperature, water may evaporate more significantly due to the heat of the heater.

  The present invention has been made in view of such circumstances, and an object thereof is to provide a culture observation method and an inverted microscope capable of observing cells in a culture solution well and for a long time. It is an object.

In order to achieve the above object, the present invention provides the following means.
The present invention relates to a culture observation method for observing cells in a container while culturing cells, wherein a substance having deliquescent properties is provided between the tip of the objective lens for observing the cells in the container and the bottom surface of the container. Provided is a culture observation method in which observation is performed with the objective lens through an aqueous solution of a substance having deliquescence and filled with an aqueous solution.

In the said invention, it is preferable that the substance which has the said deliquescence is magnesium chloride, calcium chloride, sodium hydroxide, or ethyl alcohol.
Moreover, in the said invention, it is preferable that the density | concentration of the aqueous solution of the substance which has the said deliquescence is 1 mol / l or more.
Furthermore, in the said invention, it is good also as observing, humidifying the circumference | surroundings of the said objective lens.

  The present invention also provides an illumination optical system for illuminating the cells and the container in an inverted microscope for observing cells in the container while culturing them, and maintaining the humidity and temperature inside the container at predetermined values. Provided with a distal end portion arranged with a space between the cell activity maintaining device and the bottom surface of the container, and filled with an aqueous solution of a deliquescent substance between the distal end portion and the bottom surface of the container An inverted microscope having an objective lens to be held is provided.

In the said invention, it is good also as providing the receiving member which hold | maintains the aqueous solution of the substance which is arrange | positioned at the front-end | tip part of the said objective lens, and has the said deliquescent property between the bottom surfaces of the said container.
In addition, the cell activity maintaining device includes a mounting member having an opening for mounting the container and exposing at least a part of the bottom surface of the container, a heating means for heating the mounting member, Surrounded by a peripheral wall that surrounds the periphery of the mounting member, a water tank that is disposed inside the peripheral wall and stores water, a gas supply unit that supplies carbon dioxide to the water tank, and the mounting member and the peripheral wall It is good also as providing the transparent coating | coated member which covers space in the airtight state, and the heat retention means which heat-maintains this coating | coated member.

  According to the present invention, there is an effect that culture observation can be favorably performed for a long time in culture observation.

(First embodiment)
Hereinafter, the culture observation method and the inverted microscope according to the first embodiment of the present invention will be described with reference to FIGS.
As shown in FIGS. 1 to 3, the inverted microscope 1 according to the present embodiment is disposed on the exit side of the xenon lamp 2 as a light source for epi-illumination and is controlled by the control device 3. An electric shutter 4, a light-reducing filter 5 disposed on the emission side of the electric shutter 4 for reducing the intensity of the light beam that has passed through the electric shutter 4, and a field stop 6 disposed on the emission side of the light-reducing filter 5 And a mirror unit 7 disposed on the exit side of the field stop 6 for deflecting the optical axis by 90 degrees, and an objective lens provided on the exit side of the mirror unit 7 and retractable from the optical axis by a revolver (not shown). 8 and.

The mirror unit 7 includes an excitation filter 9 on the incident side (xenon lamp side), a dichroic mirror 10 that reflects the light excited by the excitation filter 9 and transmits the fluorescence from the objective lens 8, and the dichroic mirror 10. And an absorption filter 11 that absorbs unnecessary light rays.
A cooling CCD camera 12 that images the light transmitted through the absorption filter 11 is disposed on the emission side of the absorption filter 11 provided in the mirror unit 7.
Furthermore, the inverted microscope 1 according to the present embodiment includes a halogen lamp 20 as a light source for transmitted illumination, an electric shutter 21 controlled by the control device 3 on the emission side of the halogen lamp 20, and an emission of the electric shutter 21. And a capacitor 22 arranged on the side.

  Furthermore, the inverted microscope 1 according to the present embodiment includes a cell activity maintaining device 31 for maintaining the activity of the cells in the container 30 in the vicinity of the objective lens 8. As shown in FIG. 2 and FIG. 3, the cell activity maintaining device 31 includes a mounting member 33 having an opening 32 at the center where the container 30 is mounted, and a peripheral wall portion 34 surrounding the mounting member 33. A donut-shaped water tank 35 held in the peripheral wall portion 34 for storing water, a supply tube 36 for supplying carbon dioxide gas from a supply device (not shown) to the water tank 35, and a mounting member A glass plate for heating the inside of the container 30 with a thermoplate 37 for warming the temperature of the container 30 and keeping the inside of the container 30 at around 37 ° C., the mounting member 33 and the peripheral wall portion 34, and equipped with a heating wire (not shown). The top heater 38 is provided. Carbon dioxide gas is supplied from the supply device with a standard of a concentration of 5% and a flow rate of 150 ml / min.

Further, the objective lens 8 is provided with a ring-shaped objective lens heater 39 that covers the outer periphery of the tip portion.
As shown in FIG. 4, the container 30 is formed with a thickness of 0.17 mm at the center of the bottom surface in order to accommodate a high NA objective lens designed with a cover glass thickness of 0.17 mm. .

  Further, an aqueous solution (hereinafter referred to as a deliquescent aqueous solution) 51 of a substance having deliquescent properties is disposed between the tip of the objective lens 8 and the bottom surface of the container. This deliquescent aqueous solution 51 is produced by dissolving any of magnesium chloride, calcium chloride, sodium hydroxide, or ethyl alcohol in water, and has a refractive index substantially equal to that of water. In addition, as a result of experiments, it has been found that the deliquescent aqueous solution 51 does not evaporate even when left at room temperature for two days or more, and is harder to evaporate than normal water.

The operation of the inverted microscope according to the present embodiment configured as described above will be described.
In the case of epi-illumination, the light emitted from the xenon lamp 2 is deflected by 90 ° by the mirror unit 7 and enters the objective lens 8. The light that has passed through the objective lens 8 passes through the deliquescent aqueous solution 51 and is irradiated to the cells in the container 30. The light reflected by the cells again passes through the deliquescent aqueous solution 51, passes through the mirror unit 7, and then enters the cooled CCD camera 12.

  On the other hand, in the case of transmitted illumination, light emitted from the halogen lamp 20 passes through the capacitor 22 and is irradiated to the cells in the container 30. The light reflected by the cells passes through the deliquescent aqueous solution 51, passes through the mirror unit 7, and then enters the cooled CCD camera 12.

In the cell activity maintaining device 31, heat from the thermoplate 37 and the top heater 38 is conducted to the container 30, and the cells in the container 30 are kept at about 37 ° C. Further, the objective lens 8 is warmed by heat from the objective lens heater 39.
The water tank 35 in which water is stored is supplied with carbon dioxide gas having a concentration of 5% from the supply tube 36 at a flow rate of 150 ml / min, and the inside of the cell activity maintaining device 31 is maintained at high humidity.

The effects of the culture observation method and the inverted microscope according to the first embodiment configured as described above will be described below.
Since the culture observation method according to the first embodiment of the present invention observes cells via the deliquescent aqueous solution 51, even if the cell to be observed is located at a position away from the bottom surface of the container 30, it is an immersion. As compared with the case where oil is used, the occurrence of aberration can be suppressed, so that good observation can be performed.

Moreover, since the deliquescent aqueous solution 51 is hard to evaporate compared with water, it can observe continuously for a long time compared with water.
Furthermore, since the deliquescent aqueous solution 51 has a lower thermal conductivity than immersion oil, it is difficult to conduct heat from the thermoplate 37 or the like to the objective lens 8 side, and the cells can be efficiently kept warm.

Moreover, according to the inverted microscope which concerns on this embodiment, the inside of the cell activity maintenance apparatus 31 can be maintained at high humidity by supply of a carbon dioxide gas. For this reason, the pH of the culture solution can be maintained at a desired value.
Furthermore, according to the inverted microscope according to the present embodiment, since the container 30 and the objective lens 8 use the thermoplate 37 and the objective lens heater 39, the temperature of each part can be made constant. For this reason, there is an effect that it is possible to suppress a focus shift due to a change in the distance between a very small amount of the objective lens 8 and the cell due to a temperature change of the objective lens 8 or the like.

(Second Embodiment)
Next, an inverted microscope according to the second embodiment of the present invention will be described with reference to FIG.
The inverted microscope according to the present embodiment is obtained by improving the inverted microscope according to the first embodiment so that the deliquescent aqueous solution 51 does not flow out to the objective lens 8 side. The inverted microscope according to the first embodiment. Only the differences will be described.

  As shown in FIG. 5, the inverted microscope according to this embodiment includes a receiving member 60 at the tip of the objective lens 8. The entire receiving member 60 is made of an elastic member such as rubber, and has a cylindrical mounting portion 61 for mounting on the tip portion of the objective lens 8 and the shape of the tip portion of the objective lens 8 from the mounting portion 61. And an extending portion 62 extending to the tip side. An opening 63 is provided at the tip of the extension 62 so as not to interfere with the effective diameter of the objective lens. The opening 63 is formed with a wall 64 for receiving a deliquescent aqueous solution.

According to the inverted microscope according to the present embodiment configured as described above, the deliquescent aqueous solution 51 between the distal end portion of the objective lens 8 and the bottom surface of the container does not flow out to the objective lens 8 side by the wall portion 64. The objective lens 8 is held in a state of being interposed between the front end portion and the bottom surface.
Therefore, according to the inverted microscope according to the present embodiment, in addition to the same effects as the inverted microscope according to the first embodiment, the deliquescent aqueous solution 51 is prevented from flowing out toward the objective lens 8 side, and further for a long time. There is an effect that culture observation can be performed.
Moreover, since the receiving member 60 is attached to the objective lens by the elastic force of the elastic member, it can be easily attached and detached.

Hereinafter, experimental examples for testing the performance of the deliquescent aqueous solution used in the culture observation method according to each of the above embodiments will be shown.
The experiment was performed by dissolving magnesium chloride (manufactured by Wako Pure Chemicals, model number 135-00165) in ultrapure water obtained by filtering tap water with an ion exchange water device (manufactured by Organo) and MILLIQ (manufactured by Milliboa). .
For example, in order to produce a magnesium chloride aqueous solution having a concentration of 1 mol / liter (1M (mora)), first, the weight of magnesium chloride is measured at 203.3 g, and ultrapure water is added thereto to finally make 1 liter. did. Similarly, magnesium chloride solutions with concentrations of 10 mM, 33.3 mM, 100 mM and 3M were prepared.

The magnesium chloride aqueous solution thus produced was put in an Eppendorf tube, accommodated in a low-temperature aluminum block high-temperature bath (MG-1000 manufactured by Tokyo Rika Co., Ltd.), and heated at 37 ° C. at a constant temperature.
In addition, the lid of the Eppendorf tube was opened slightly so that the internal vapor pressure was not increased.
FIG. 6 shows temporal changes in the weight of magnesium chloride aqueous solution and ultrapure water when heated to 37 ° C. in a high-temperature bath.
According to this figure, the magnesium chloride aqueous solution having a concentration of 1M and 3M decreases in weight until the number of heating days is about 5 days, but there is almost no change in weight thereafter, and it is difficult to evaporate. ing. Further, in the case of the concentration of 3M, the change in weight until the fifth day is sufficiently smaller than that in the case of 1M, so that it is further difficult to evaporate.

It is a mimetic diagram explaining the whole inverted microscope concerning a 1st embodiment of the present invention. It is a longitudinal cross-sectional view which shows the cell activity maintenance apparatus of the inverted microscope of FIG. It is a top view of the cell activity maintenance apparatus of FIG. It is an enlarged view which shows the state between the objective lens and container of the inverted microscope of FIG. It is an enlarged view which shows the state between the objective lens and container of the inverted microscope which concerns on the 2nd Embodiment of this invention. It is a graph which shows the mode of a mass change with time of the magnesium chloride aqueous solution as an example of the deliquescent aqueous solution used for the culture observation method which concerns on this invention.

Explanation of symbols

8 Objective Lens 30 Container 31 Cell Activity Maintenance Device 33 Placement Member 34 Peripheral Wall 35 Water Tank 36 Supply Tube 37 Thermoplate 38 Top Heater 51 Deliquescent Aqueous Solution 60 Receiving Member 64 Wall

Claims (7)

  In the culture observation method for observing cells in the container while culturing, the space between the tip of the objective lens for observing the cells in the container and the bottom surface of the container is filled with an aqueous solution of a substance having deliquescence, A culture observation method characterized by observing with the objective lens through an aqueous solution of a substance having deliquescence.   The culture observation method according to claim 1, wherein the deliquescent substance is magnesium chloride, calcium chloride, sodium hydroxide, or ethyl alcohol.   The culture observation method according to claim 2, wherein the concentration of the aqueous solution of the substance having deliquescence is 1 mol / l or more.   The culture observation method according to claim 1, wherein the periphery of the objective lens is observed while being humidified.   In an inverted microscope for observing cells in a container while culturing, an illumination optical system that illuminates the cells, a cell activity maintaining device that stores the containers and maintains the humidity and temperature therein at predetermined values, An objective lens that is provided with a distal end portion that is spaced from the bottom surface of the container, and that is held in a state filled with an aqueous solution of a deliquescent substance between the distal end portion and the bottom surface of the container; An inverted microscope characterized by comprising.   The inverted body according to claim 5, further comprising a receiving member that is disposed at a distal end portion of the objective lens and holds an aqueous solution of the deliquescent material between the bottom surface of the container. microscope.   The cell activity maintaining device includes a mounting member having an opening for mounting the container and exposing at least a part of a bottom surface of the container, a heating means for heating the mounting member, A space surrounded by a peripheral wall part surrounding the member, a water tank disposed inside the peripheral wall part for storing water, a gas supply means for supplying carbon dioxide to the water tank, and the mounting member and the peripheral wall part. The inverted microscope according to claim 5 or 6, further comprising: a transparent covering member that covers the covering member in a sealed state; and a heat retaining unit that retains the temperature of the covering member.

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