Specimen observation apparatus and specimen observation method

A specimen observation method comprising:

• an acquisition step of acquiring an electronic image of a specimen;
• and
• a subtraction step of subtracting a direct current component from a signal of the electronic image,
• wherein:
• the acquisition step is performed in a state of bright-field observation and a state in which illumination light that is transmitted through the specimen is reflected by a reflection member and is applied to the specimen again,
• the electronic image at the subtraction step is an image acquired in a predetermined state,
• in the predetermined state, a position of the specimen and a focus position of an imaging optical system are different from each other,
• the focus position is a position at which phases of zero-order diffracted light and first-order diffracted light at a spatial frequency of the specimen at a position of an image point on an optical axis are the same, and
• the following Conditional Expression (1) is satisfied:
• 10≤D×NAob2/λ≤10000 (1)
• where
• D is a distance (mm) between the position of the specimen and a reflection surface of the reflection member,
• NAob is a numerical aperture of the imaging optical system on the specimen side, and
• λ is a center wavelength (mm) of a wavelength range of the illumination light.

A specimen observation apparatus comprising:

• a light source;
• an illumination optical system;
• a stage on which a specimen is able to be disposed;
• an imaging optical system;
• and
• a reflection member disposed at a position opposed to the imaging optical system across the stage,
• wherein:
• the illumination optical system is disposed so as to apply illumination light from the light source to the specimen,
• the imaging optical system is disposed at a position at which the illumination light that is transmitted through the specimen and thereafter reflected by the reflection member to be transmitted through the specimen again enters, and is configured to form an optical image of the specimen,
• the optical image is formed in a state in which a position of the specimen and a focus position of the imaging optical system are different from each other,
• the focus position is a position at which phases of zero-order diffracted light and first-order diffracted light at a spatial frequency of the specimen at a position of an image point on an optical axis are the same, and
• the following Conditional Expression (2) is satisfied:
• 4×NAill×D+A<L (2)
• where
• NAill is a numerical aperture of the illumination optical system on the specimen side,
• D is a distance (mm) between the position of the specimen and a reflection surface of the reflection member,
• A is a diameter (mm) of an observation field of view at the position of the specimen,
• L is a diameter (mm) of an illumination range of the illumination optical system at the position of the specimen, and
• the diameter of the observation field of view and the diameter of the illumination range are diameters in a state in which the position of the specimen and the focus position of the imaging optical system are different from each other.

A specimen observation method comprising:

• an acquisition step of acquiring an electronic image of a specimen;
• and
• a subtraction step of subtracting a direct current component from a signal of the electronic image,
• wherein:
• the acquisition step is performed in a state of bright-field observation and a state in which illumination light that is transmitted through the specimen is reflected by a reflection member and is applied to the specimen again,
• the electronic image at the subtraction step is an image acquired in a predetermined state,
• in the predetermined state, a position of the specimen and a focus position of an imaging optical system are different from each other,
• the focus position is a position at which phases of zero-order diffracted light and first-order diffracted light at a spatial frequency of the specimen at a position of an image point on an optical axis are the same, and
• the following Conditional Expression (2) is satisfied:
• 4×NAill×D+A<L (2)
• where
• NAill is a numerical aperture of the illumination optical system on the specimen side,
• D is a distance (mm) between the position of the specimen and a reflection surface of the reflection member,
• A is a diameter (mm) of an observation field of view at the position of the specimen,
• L is a diameter (mm) of an illumination range of the illumination optical system at the position of the specimen, and
• the diameter of the observation field of view and the diameter of the illumination range are diameters in a state in which the position of the specimen and the focus position of the imaging optical system are different from each other.