Development and characterisation of tube-lens designs for remote-refocusing microscopy and high-speed adaptive light-sheet fluorescence microscopy with a deformable mirror
File(s)
Author(s)
Hong, Wenzhi
Type
Thesis
Abstract
Optical microscopy of bioprocesses aims at acquiring comprehensive spatiotemporal information while minimising adverse effects on the sample. Light-sheet fluorescence microscopy achieves optically sectioned imaging with relatively low photobleaching and phototoxicity. To achieve high-speed volumetric imaging without sample perturbation, employing some form of remote refocusing in the detection path is necessary.
A practical approach without using active optical elements is to use a relay-lens setup with specified magnification settings. However, this requires specific tube lens focal lengths that are not always available from stock optics. Hence, two software packages were developed to automatically design microscope tube lenses from pairs of stock achromatic doublets. Tube lens models based on matrix optics theory were also explored for remote-refocusing microscopy with interchangeable objectives, and designs for practical three-doublet configurations were developed.
To help obtain the best performance in remote-refocusing systems, a folded remote-refocusing system was developed. The mean FWHMxy of 100 nm fluorescent beads, normalised bead integrated energy, and system distortions were mapped over a remote-refocusing range and as a function of lateral image position. This allowed the estimation of the volume where diffraction-limited performance was achieved for the first time and provided insights into its variation with the system alignment.
Using the relay-lens setup still requires mechanical translation of a lens or mirror, which limits the refocusing range and/or frequency. To simultaneously achieve high-speed remote refocusing, correct system aberrations, and enhance optical throughput, a deformable mirror was employed in an adaptive LSFM system to provide higher-order defocus and aberration correction with a detection NA of 0.72-0.75. Through optimisations with continuous mirror oscillation, an estimated maximum Strehl ratio above the diffraction limit was achieved across a remote-refocusing range of 60 μm. The system capabilities were demonstrated by observing pollen grains and electrically paced cardiomyocytes at 26.3 volumes per second with 35 usable frames per volume.
A practical approach without using active optical elements is to use a relay-lens setup with specified magnification settings. However, this requires specific tube lens focal lengths that are not always available from stock optics. Hence, two software packages were developed to automatically design microscope tube lenses from pairs of stock achromatic doublets. Tube lens models based on matrix optics theory were also explored for remote-refocusing microscopy with interchangeable objectives, and designs for practical three-doublet configurations were developed.
To help obtain the best performance in remote-refocusing systems, a folded remote-refocusing system was developed. The mean FWHMxy of 100 nm fluorescent beads, normalised bead integrated energy, and system distortions were mapped over a remote-refocusing range and as a function of lateral image position. This allowed the estimation of the volume where diffraction-limited performance was achieved for the first time and provided insights into its variation with the system alignment.
Using the relay-lens setup still requires mechanical translation of a lens or mirror, which limits the refocusing range and/or frequency. To simultaneously achieve high-speed remote refocusing, correct system aberrations, and enhance optical throughput, a deformable mirror was employed in an adaptive LSFM system to provide higher-order defocus and aberration correction with a detection NA of 0.72-0.75. Through optimisations with continuous mirror oscillation, an estimated maximum Strehl ratio above the diffraction limit was achieved across a remote-refocusing range of 60 μm. The system capabilities were demonstrated by observing pollen grains and electrically paced cardiomyocytes at 26.3 volumes per second with 35 usable frames per volume.
Version
Open Access
Date Issued
2023-10
Date Awarded
2024-03
Copyright Statement
Creative Commons Attribution NonCommercial Licence
License URL
Advisor
Dunsby, Chris
Sponsor
Engineering and Physical Sciences Research Council
Publisher Department
Physics
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)