Specimen observation apparatus and specimen observation method
View Patent ↗A specimen observation apparatus includes: a light source; an illumination optical system; a stage; an imaging optical system; and a reflection member disposed at a position opposed to the imaging optical system across the stage. The illumination optical system is disposed so as to apply illumination light from the light source to a 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.
1. 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 (1) is satisfied:
10≤ D ×NA ob 2 /λ≤10000 (1)
where
D is a distance (mm) between the position of the specimen and a reflection surface of the reflection member,
NA ob 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.
2. The specimen observation apparatus according to claim 1 , further comprising:
an image pickup device; and
an image processing device,
wherein:
the image pickup device is disposed at a position at which the optical image is formed, and
the image processing device generates an observation image signal from an optical image obtained in a state in which the position of the specimen and the focus position of the imaging optical system are different from each other.
3. The specimen observation apparatus according to claim 1 , wherein the following Conditional Expression (2) is satisfied:
4×NA ill ×D+A<L (2)
where
NA ill is a numerical aperture of the illumination optical system on the specimen side,
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.
4. The specimen observation apparatus according to claim 1 , wherein the reflection member is disposed at a position different from a position conjugate to a pupil of the imaging optical system.
5. The specimen observation apparatus according to claim 1 , wherein the reflection member has a positive refractive power.
6. The specimen observation apparatus according to claim 1 , wherein a reflectance of the reflection member is at least 50%.
7. The specimen observation apparatus according to claim 1 , wherein the reflection member has a reflection angle limiting structure configured to make a numerical aperture of illumination light after reflection smaller than a numerical aperture of entering illumination light.
8. The specimen observation apparatus according to claim 1 , wherein the following Conditional Expression (4) is satisfied:
0.01<NA ill /NA ob <1 (4)
where
NA ill is a numerical aperture of the illumination optical system on the specimen side.
9. The specimen observation apparatus according to claim 1 , wherein the following Conditional Expression (5) is satisfied:
0.1 μm<Δ Z ×NA ob 2 <30 μm (5)
where
ΔZ is a difference between the focus position of the imaging optical system and the position of the specimen.
10. The specimen observation apparatus according to claim 1 , wherein the following Conditional Expression (6) is satisfied:
0.05 μm<Δ Z ×NA ill <10 μm (6)
where
ΔZ is a difference between the focus position of the imaging optical system and the position of the specimen, and
NA ill is a numerical aperture of the illumination optical system on the specimen side.
11. The specimen observation apparatus according to claim 1 , wherein the illumination light is monochromatic light.
12. The specimen observation apparatus according to claim 1 , further comprising a plurality of the imaging optical systems.
13. 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 ×NA ob 2 /λ≤10000 (1)
where
D is a distance (mm) between the position of the specimen and a reflection surface of the reflection member,
NA ob 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.
14. The specimen observation method according to claim 13 , further comprising an amplification step after the subtraction step,
wherein a signal of an electronic image after the subtraction step is amplified in the amplification step.
15. The specimen observation method according to claim 13 , further comprising a step of calculating a phase amount.
16. The specimen observation method according to claim 13 , further comprising a step of acquiring an electronic image for correction,
wherein the electronic image for correction is an electronic image acquired in a state in which no specimen exists in a specimen container, and
the signal of the electronic image for correction is subtracted from the signal of the electronic image of the specimen in the subtraction step.
17. 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×NA ill ×D+A<L (2)
where
NA ill 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.
18. 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×NA ill ×D+A<L (2)
where
NA ill 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.