| dc.contributor |
Corbett, Alex |
|
| dc.contributor |
Soeller, Christian |
|
| dc.creator |
Mohanan, S |
|
| dc.date |
2022-06-13T08:03:26Z |
|
| dc.date |
2022-06-13 |
|
| dc.date |
2022-06-11T07:13:30Z |
|
| dc.date |
2022-06-13T08:03:26Z |
|
| dc.date.accessioned |
2023-02-23T12:14:17Z |
|
| dc.date.available |
2023-02-23T12:14:17Z |
|
| dc.identifier |
http://hdl.handle.net/10871/129921 |
|
| dc.identifier.uri |
http://localhost:8080/xmlui/handle/CUHPOERS/258528 |
|
| dc.description |
Current developments in optical microscopy aim to visualise complex dynamic
biomolecular processes close to their native state. To capture transient phenomena,
rapid three-dimensional stacks are acquired by translating the objective or sample
stage to refocus into different depths of the specimen. Such conventional refocusing
strategies introduce vibrational artefacts when imaging specimens that are in direct
contact with the immersion media of the objective.
Remote focusing is a methodology in which agitation-free refocusing can be
performed using high numerical aperture (NA) objectives without compromising
on resolution or imaging speed. It compensates for aberrations from the imaging
objective by introducing equal and opposite aberration with a second microscope
placed in reverse to the first. As the NA of the imaging objective increases, there
are significant constraints placed on the tolerance in optical design to reach perfect
phase-matching condition.
In the first part of the thesis, the computational model developed to predict
the performance of remote focusing microscopes is presented. From the model,
the increased sensitivity of high-NA systems to magnification mismatch is inferred
where the diffraction limited volume reduces by half for a 1% error.
Informed by the sensitivity analysis, the decrease in resolution across depth for
a remote focusing microscope with a 4% magnification mismatch is demonstrated.
A protocol for magnification and resolution characterisation is presented and is
applied to a novel Spinning Disk Remote Focusing microscope. The microscope
is then applied to perform live volumetric imaging to study the normal neural
activity of Platynereis dumerilii larvae. The studies presented here paves way
for a standardised characterisation of remote focusing systems allowing for wider
implementation.
In the final part of the thesis, the spherical aberration generated by the correction
collar on an immersion objective is exploited to compensate for residual
spherical aberration in an ideal remote focusing system. The wavefront aberrations
are measured using a Shack-Hartmann sensor and sub-resolution beads are imaged
for point spread function measurements. Results from the Shack-Hartmann measurements
show a 60% increase in axial range compensated for spherical aberration.
In addition, the contribution of off-axis aberrations to the overall image quality at
defocussed positions is explored further. |
|
| dc.description |
Engineering and Physical Sciences Research Council (EPSRC) |
|
| dc.publisher |
University of Exeter |
|
| dc.publisher |
Department of Physics and Astronomy |
|
| dc.rights |
2024-06-11 |
|
| dc.rights |
Embargo required for publishing results presented in the thesis. |
|
| dc.rights |
http://www.rioxx.net/licenses/all-rights-reserved |
|
| dc.subject |
Optical microscopy |
|
| dc.subject |
Volumetric imaging |
|
| dc.subject |
Remote focusing |
|
| dc.subject |
Aberrations |
|
| dc.subject |
High numerical aperture |
|
| dc.subject |
Spinning disk microscopy |
|
| dc.subject |
Live sample imaging |
|
| dc.subject |
Correction collar |
|
| dc.title |
Remote Focusing in Optical Microscopy |
|
| dc.type |
Thesis or dissertation |
|
| dc.type |
PhD in Physics |
|
| dc.type |
Doctoral |
|
| dc.type |
Doctoral Thesis |
|