Inverted microscope and sample observation method

1. An inverted microscope comprising:

a stage configured to support directly or indirectly a bottom member and capable of retaining a sample above the bottom member, the bottom member being optically transparent;

an immersion objective disposed facing vertically upward such that a distal end thereof faces a bottom face of the bottom member supported by the stage;

an alignment unit configured to move at least one of the stage and the immersion objective in an optical-axis direction extending along an optical axis of the immersion objective;

a liquid injecting unit configured to inject liquid between the bottom face of the bottom member and the distal end of the immersion objective;

a liquid discharging unit that discharges the liquid from between the distal end of the immersion objective and the bottom face of the bottom member; and

a controller configured to control the liquid injecting unit, in a state where the bottom face of the bottom member and the distal end of the immersion objective are disposed with a gap therebetween in the optical-axis direction greater than a height of a droplet that can be formed on the distal end, so as to inject the liquid onto the distal end to form the droplet, and to control the alignment unit so as to relatively bring the bottom member and the immersion objective closer to each other in the optical-axis direction to bring the droplet on the distal end into contact with the bottom face of the bottom member, thereby forming a liquid column constituted of the liquid being in contact with the distal end and the bottom face,

wherein the controller is configured to perform control to move the stage and the immersion objective relative to each other in the optical-axis direction while maintaining the liquid column, and

wherein the controller controls the liquid discharging unit so as to reduce an amount of the liquid on the distal end in a case where the stage and the immersion objective are moved relative to each other in the optical-axis direction by the alignment unit so as to bring the bottom member and the immersion objective closer to each other while maintaining the liquid column, and the controller controls the liquid injecting unit so as to replenish the liquid onto the distal end in a case where the stage and the immersion objective are moved relative to each other in the optical-axis direction by the alignment unit so as to move the bottom member and the immersion objective away from each other while maintaining the liquid column.

2. An inverted microscope according to claim 1 , wherein the liquid injecting unit and the liquid discharging unit are driven by a mutually common driving source.

3. An inverted microscope according to claim 2 , wherein the driving source is a peristaltic pump.

4. An inverted microscope according to claim 1 , wherein the stage is provided so as to be movable in an intersecting direction intersecting the optical-axis direction, and

wherein the controller controls the liquid injecting unit so as to replenish the liquid onto the distal end in a case where the stage is moved in the intersecting direction to shift the bottom member and the immersion objective relative to each other in the intersecting direction in the state where the liquid column has been formed.

5. An inverted microscope according to claim 1 , wherein a distance from the distal end of the immersion objective to a focal position thereof is longer than the height of the droplet that can be formed on the distal end.

6. An inverted microscope according to claim 1 , wherein a distance from the distal end of the immersion objective to a focal position thereof is not less than 1.5 mm.

7. A sample observation method comprising:

a droplet forming step of, in a state where a bottom member that retains a sample thereabove and that is optically transparent and an immersion objective that is disposed facing vertically upward with a distal end thereof facing a bottom face of the bottom member are disposed with a gap therebetween greater than a height of a droplet that can be formed on the distal end of the immersion objective, injecting liquid onto the distal end to form the droplet;

a liquid-column forming step of relatively bringing the bottom member and the immersion objective closer to each other in an optical-axis direction extending along an optical axis of the immersion objective to bring the droplet formed on the distal end into contact with the bottom face of the bottom member, thereby forming a liquid column constituted of the liquid being in contact with the distal end and the bottom face; and

a focal-position adjusting step of moving the bottom member and the immersion objective relative to each other in the optical-axis direction, while maintaining the liquid column, to adjust a focal position of the immersion objective on the sample retained above the bottom member,

wherein an amount of the liquid on the distal end is reduced in a case where the bottom member and the immersion objective are moved relatively in a direction closer to each other while maintaining the liquid column, and the liquid is replenished onto the distal end in a case where the bottom member and the immersion objective are moved relatively in a direction away from each other while maintaining the liquid column.

8. A sample observation method according to claim 7 , wherein the liquid is replenished onto the distal end of the immersion objective in a case where the bottom member and the immersion objective are shifted relative to each other in an intersecting direction intersecting the optical-axis direction in the state where the liquid column has been formed.