Holographic microscope and data processing method for high-resolution hologram image
The present invention can realize both a transmission type and a reflection type, and provides a holographic microscope which can exceed the resolution of the conventional optical microscope, a hologram data acquisition method for a high-resolution image, and a high-resolution hologram image reconstruction method. In-line spherical wave reference light (L) is recorded in a hologram (I LR ) using spherical wave reference light (R), and an object light (O j ) and an illumination light (Q j ) are recorded in a hologram (I j OQR ) using a spherical wave reference light (R) by illuminating the object with an illumination light (Q j , j=1, . . . , N) which is changed its incident direction. From those holograms, a hologram (J j OQL ), from which the component of the reference light (R) is removed, is generated, and from the hologram, a light wave (h j ) is generated. A light wave (c j ) of the illumination light (Q j ) is separated from the light wave (h j ), and using its phase component (ξ j =c j /|c j |), a phase adjustment reconstruction light wave is derived and added up as (H P =Σh j /ξ j ), and an object image (S P =|H P | 2 ) is reconstructed.
1. A holographic microscope, comprising:
a data acquisition unit which acquires a hologram of an object, and
an image reconstruction unit which reconstructs an image of the object from the hologram acquired by the data acquisition unit, wherein
the data acquisition unit comprises:
an optical system which generates an in-line spherical wave reference light (L), an illumination light (Q), and an off-axis spherical wave reference light (R) with a coherent light emitted by a light source, and transmits the generated lights and an object light (O) emitted from the object illuminated with the illumination light (Q);
an angle change unit which changes an incident direction of the illumination light (Q) on the object;
a photo-detector which changes a light intensity into an electric signal and outputs it; and
a storing unit which stores a reference light off-axis hologram (I LR ) of interference fringes between the in-line spherical wave reference light (L) and the off-axis spherical wave reference light (R), object light off-axis holograms (I j OR , j=1, . . . , N) of interference fringes between the off-axis spherical wave reference light (R) and object lights (O j , j=1, . . . , N) emitted from the object illuminated with the illumination lights (Q j , j=1, . . . , N) having different incident directions (θ j , j=1, . . . , N) generated from the coherent light by using the optical system and the angle change unit, and illumination light off-axis holograms (I j QR , j=1, . . . , N) of interference fringes between the illumination lights (Q j , j=1, . . . , N) and the off-axis spherical wave reference light (R), in a memory by using the photo-detector, and
the image reconstruction unit comprises:
a make-in-line modulation unit which generates an object light complex amplitude in-line hologram (J j OL ) and an illumination light complex amplitude in-line hologram (J j QL ), from the reference light off-axis hologram (I LR ), the object light off-axis hologram (I j OR ), and the illumination light off-axis hologram (I j QR ), by eliminating a component of the off-axis spherical wave reference light (R) for each of the incident directions (θ j , j=1, . . . , N);
a light wave calculation unit which derives, for each of the incident directions (θ j , j=1, . . . , N) on an arbitrary reconstruction plane (Z=Z P ), from the object light complex amplitude in-line hologram (J j OL ) and the illumination light complex amplitude in-line hologram (J j QL ), a reconstructed object light wave (h j ) of the object light (O j ), a reconstructed illumination light wave (c j ) of the illumination light (Q j ), a phase component (ξ j =c j /|c j |) contained in the reconstructed illumination light wave (c j ), and a phase adjusted reconstruction light wave (h j /ξ j ) made by removing the phase component (ξ j ) from the reconstructed object light wave (h j ); and
an image calculation unit which derives a synthetic light wave (H P =Σh j /ξ j ) on the arbitrary reconstruction plane (Z=Z P ) by adding up the phase adjusted reconstruction light waves (h j /ξ j ) for the incident directions (θ j , j=1, . . . , N), and reconstructs an object image (S P =|H P | 2 ) by using the synthetic light wave (H P ).
2. The holographic microscope according to claim 1 , wherein
the image reconstruction unit further comprises:
a pixel number increase unit which generates a pixel-increased object light complex amplitude in-line hologram (K j OL ) and a pixel-increased illumination light complex amplitude in-line hologram (K j QL ) having substantially increased number of pixels by subdividing a spatial sampling interval of the object light complex amplitude in-line hologram (J j OL ) and the illumination light complex amplitude in-line hologram (J j QL ) and performing a data interpolation to a new sampling point produced by the subdividing; and
the light wave calculation unit derives the phase adjusted reconstruction light wave (h j /ξ j ) by using the pixel-increased object light complex amplitude in-line hologram (K j QL ) and the pixel-increased illumination light complex amplitude in-line hologram (K j QL ).
3. The holographic microscope according to claim 1 , wherein
the image reconstruction unit further comprises an illumination light separation unit which separates and generates a reconstructed light wave of the illumination light (Q j ) from a hologram in which the object light (O j ) and the illumination light (Q j ) are recorded without being separated, and
the storing unit stores a hologram of interference fringes between the object light (O j ), the illumination light (Q j ), and the off-axis spherical wave reference light (R) in the memory as an object light illumination light off-axis hologram (I j OQR ) when the object light (O j ) and the illumination light (Q j ) are not separated from each other,
the make-in-line modulation unit generates an object light illumination light complex amplitude in-line hologram (J j OQL ) from the reference light off-axis hologram (I LR ) and the object light illumination light off-axis hologram (I j OQR ) by eliminating the component of the off-axis spherical wave reference light (R),
the illumination light separation unit generates, on a specific reconstruction plane (z=z S ), a reconstructed object light wave (h j ), which is a reconstructed light wave of the object light (O j ) containing the illumination light (Q j ), from the object light illumination light complex amplitude in-line hologram (J j OQL ), and separates and generates a reconstructed illumination light wave (c j ), which is a reconstructed light wave of the illumination light (Q j ), from the reconstructed object light wave (h j ),
the light wave calculation unit generates a new reconstructed object light wave (h j ) and a new reconstructed illumination light wave (c j ) by propagating the reconstructed object light wave (h j ) and the reconstructed illumination light wave (c j ) generated by the illumination light separation unit to the arbitrary reconstruction plane (z=z P ) different from the specific reconstruction plane (z=z S ), and derives the phase adjusted reconstruction light wave (h j /ξ j ) by using the new reconstructed illumination light waves.
4. The holographic microscope according to claim 3 , wherein
the image reconstruction unit further comprises a pixel number increase unit which generates a pixel-increased object light illumination light complex amplitude in-line hologram (K j OQL ) having substantially increased number of pixels by subdividing a spatial sampling interval of the object light illumination light complex amplitude in-line hologram (J j OQL ) and performing a data interpolation to a new sampling point produced by the subdividing, and
the illumination light separation unit derives the reconstructed object light wave (h j ) by using the pixel-increased object light illumination light complex amplitude in-line hologram (K j OQL ).
5. The holographic microscope according to claim 1 ,
wherein
the angle change unit comprises a condenser and a reflector, wherein the reflector makes a parallel light having a larger diameter than that of the condenser go into the condenser from an arbitrary direction leaning to an optic axis of the condenser.
6. A data processing method for a high-resolution hologram image, comprising the steps of:
acquiring a reference light off-axis hologram (I LR ) of interference fringes between an in-line spherical wave reference light (L) and an off-axis spherical wave reference light (R);
acquiring object light off-axis holograms (I j OR , j=1, . . . , N) of interference fringes between the off-axis spherical wave reference light (R) and object lights (O j , j=1, . . . , N) emitted from an object illuminated with illumination lights (Q j , j=1, . . . , N) having different incident directions (θ j , j=1, . . . , N), and illumination light off-axis holograms (I j QR , j=1, . . . , N) of interference fringes between the illumination lights (Q 1 , j=1, . . . , N) and the off-axis spherical wave reference light (R) for each of the incident directions, wherein the off-axis holograms (I LR , I j OR , I j QR , j=1, . . . , N) are acquired under conditions that each of the object lights (O j , j=1, . . . , N) is emitted from a localized region, and the localized region and virtual-point-light-sources of the in-line spherical wave reference light (L) and the off-axis spherical wave reference light (R) are in close proximity to each other;
generating an object light complex amplitude in-line hologram (J j OL ) from the reference light off-axis hologram (I LR ) the object light hologram (I j OR ), and an illumination light complex amplitude in-line hologram (J j OL ) from the reference light off-axis hologram (I LR ) and hologram (I j QR ), respectively, by eliminating a component of the off-axis spherical wave reference light (R), for each of the incident directions (θ j , j=1, . . . , N);
deriving, for each of the incident directions (θ j , j=1, . . . , N) on an arbitrary reconstruction plane (z=z P ), a reconstructed object light wave (h j ) of the object light (O j ) from the object light complex amplitude in-line hologram (J j OL ), a reconstructed illumination light wave (c j ) of the illumination light (Q j ) from the illumination light complex amplitude in-line hologram (J j QL ), a phase component (ξ j =c j /|c j |) contained in the reconstructed illumination light wave (c j ), and a phase adjusted reconstruction light wave (h j /ξ j ) made by removing the phase component (ξ j ) from the reconstructed object light wave (h j ); and
deriving a synthetic light wave (H P =Σh j /ξ j ) on the arbitrary reconstruction plane (z=z P ) by adding up the phase adjusted reconstruction light waves (h j /ξ j ) for the incident directions (θ j , j=1, . . . , N), and then reconstructing an object image (S P =|H P | 2 ) by using the synthetic light wave (H P ).
7. The data processing method according to claim 6 , wherein
the off-axis spherical wave reference light (R) is a reflection light from a minute sphere surface.
8. The data processing method according to claim 6 , wherein
a plurality of photo-detectors are arranged so that the photo-detectors face the localized region where the object lights are emitted, and each of the holograms is acquired by using the plurality of the photo-detectors.
9. A data processing method for a high-resolution hologram image, comprising the steps of:
acquiring a reference light off-axis hologram (I LR ) of interference fringes between an in-line spherical wave reference light (L) and an off-axis spherical wave reference light (R);
acquiring object light off-axis holograms (I j OR , j=1, . . . , N) of interference fringes between the off-axis spherical wave reference light (R) and object lights (O j , j=1, . . . , N) emitted from an object illuminated with illumination lights (Q j , j=1, . . . , N) having different incident directions (θ j , j=1, . . . , N), and illumination light off-axis holograms (I j QR , j=1, . . . , N) of interference fringes between the illumination lights (Q j , j=1, . . . , N) and the off-axis spherical wave reference light (R) for each of the incident directions, wherein the off-axis holograms (I LR , I j OR , I j QR , j=1, . . . , N) are acquired under conditions that each of the object lights (Q j , j=1, . . . , N) is emitted from a localized region, and the localized region and virtual-point-light-sources of the in-line spherical wave reference light (L) and the off-axis spherical wave reference light (R) are in close proximity to each other;
deriving a synthetic light wave (H P ) by adding up light waves of the object lights (O j , j=1, . . . , N) after adjusting phases thereof on an arbitrary reconstruction plane (z=z P ); and
reconstructing an object image (S P =|H P | 2 ) by using the synthetic light wave (H P ), wherein
the adjusting of the phases is performed based on that the object light (O j ) recorded in the hologram is a superposed light of reflection lights generated by reflection of the illumination light (Q j ) at each point in the localized region of the object, and a phase of the reflection light composing the object light (O j ) at a position where the reflection light is generated and a phase of the illumination light (Q j ) generating the reflection light at the position are equal.
10. The data processing method according to claim 6 , further comprising the steps of:
generating a pixel-increased object light complex amplitude in-line hologram (K j OL ) and a pixel-increased illumination light complex amplitude in-line hologram (K j QL ) having substantially increased number of pixels by subdividing a spatial sampling interval of the object light complex amplitude in-line hologram (J j OL ) and the illumination light complex amplitude in-line hologram (J j QL ), respectively, and performing a data interpolation to a new sampling point produced by the subdividing; and
deriving the phase adjusted reconstruction light wave (h j /ξ j ) using the pixel-increased object light complex amplitude in-line hologram (K j OL ) and the pixel-increased illumination light complex amplitude in-line hologram (K j QL ).
11. The data processing method according to claim 9 , wherein
the off-axis spherical wave reference light (R) is a reflection light from a minute sphere surface.
12. The data processing method according to claim 9 , wherein
a plurality of photo-detectors are arranged so that the photo-detectors face the localized region where the object lights are emitted, and each of the holograms is acquired by using the plurality of the photo-detectors.
13. A data processing method for a high-resolution hologram image, comprising the steps of:
acquiring a reference light off-axis hologram (I LR ) of interference fringes between an in-line spherical wave reference light (L) and an off-axis spherical wave reference light (R),
acquiring object light illumination light off-axis holograms (I j OQR , j=1, . . . , N) of interference fringes between the off-axis spherical wave reference light (R), object lights (O j , j=1, . . . , N) emitted from an object illuminated with illumination lights (Q j , j=1, . . . , N) having different incident directions (θ j , j=1, . . . , N), and the illumination lights (Q j , j=1, . . . , N) for each of the incident directions when the object light (O j ) and the illumination light (Q j ) are not separated from each other, wherein the off-axis holograms (I LR , I j OQR , j=1, . . . , N) are acquired under conditions that each of the object lights (O j , j=1, . . . , N) is emitted from a localized region, and the localized region and the virtual-point-light-sources of the in-line spherical wave reference light (L) and the off-axis spherical wave reference light (R) are in close proximity to each other;
generating object light illumination light complex amplitude in-line holograms (J j OQL ) from the reference light off-axis hologram (I LR ) and the object light illumination light off-axis holograms (I j OQR ), by eliminating a component of the off-axis spherical wave reference light (R) for each of the incident directions (θ j , j=1, . . . , N);
generating, on a specific reconstruction plane (z=z S ), a reconstructed object light wave (h j ), which is a reconstructed light wave of the object light (O j ) containing the illumination light (O j ), from the object light illumination light complex amplitude in-line hologram (J j OQL ), and separating a reconstructed illumination light wave (c j ), which is a reconstructed light wave of the illumination light (Q j ), from the reconstructed object light wave (h j ) for each of the incident directions (θ j , j=1, . . . , N);
generating a new reconstructed object light wave (h j ) and a new reconstructed illumination light wave (c j ) by propagating the reconstructed object light wave (h j ) and the reconstructed illumination light wave (c j ) to an arbitrary reconstruction plane (z=z P ) different from the specific reconstruction plane (z=z S ), and deriving, on the arbitrary reconstruction plane (z=z P ), a phase component (ξ j =c j /|c j |) contained in the new reconstructed illumination light wave (c j ) and a phase adjusted reconstruction light wave (h j /ξ j ) by eliminating the phase component (ξ j ) from the new reconstructed object light wave (h j ); and
deriving a synthetic light wave (H P =Σh j /ξ j ) on the arbitrary reconstruction plane (z=z P ) by adding up the phase adjusted reconstruction light waves (h j /ξ j ) for the incident directions (θ j , j=1, . . . , N), and then reconstructing an object image (S P =|H P | 2 ) using the synthetic light wave (H P ).
14. The data processing method according to claim 13 , further comprising the steps of:
generating a pixel-increased object light illumination light complex amplitude in-line hologram (K j OQL ) having substantially increased number of pixels by subdividing a spatial sampling interval of the object light illumination light complex amplitude in-line hologram (J j OQL ) and performing a data interpolation to a new sampling point produced by the subdividing; and
deriving the reconstructed object light wave (h j ) using the pixel-increased object light illumination light complex amplitude in-line hologram (K j OQL ).
15. The data processing method according to claim 13 , wherein
the off-axis spherical wave reference light (R) is a reflection light from a minute sphere surface.
16. The data processing method according to claim 13 , wherein
a plurality of photo-detectors are arranged so that the photo-detectors face the localized region where the object lights are emitted, and each of the holograms is acquired by using the plurality of the photo-detectors.
17. The data processing method according to claim 13 , wherein
the illumination lights (Q j , j=1, . . . , N) have a condensing point and recorded in the object light illumination light off-axis holograms (I j OQR ) in a spread state after passing the condensing point.