A versatile light-sheet microscope, platform for advanced 3D fluorescence imaging and research

Garbellotto, Chiara (2021) A versatile light-sheet microscope, platform for advanced 3D fluorescence imaging and research. PhD thesis, University of Glasgow.

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Abstract

Light-sheet microscopy has become one of the leading techniques for 3D in vivo imaging, thanks to its optical sectioning capability, its imaging speed and its reduced photo-damaging effects on the specimen. This work presents the SLM-SPIM, a Selective Plane Illumination Microscope (SPIM, a cylindrical-lens-based light-sheet microscope) with a phase-only Spatial Light Modulator (SLM) integrated into its illumination arm. The SLM is placed in a Fourier plane, and it can be used to move and modulate the light-sheet to perform a variety of existing imaging techniques for improving image quality, such as structured illumination, tiling, pivoting, autofocusing and pencil beam scanning. The SLM-SPIM configuration presented in this work has been designed to allow all of these techniques to be employed on an easily reconfigurable optical set-up, compatible with the OpenSPIM design. This work presents results obtained by applying the above mentioned imaging techniques on samples of fluorescent beads, zebrafish embryos, and optically cleared whole mouse brain samples, thereby demonstrating the flexibility and performance of the system.

This work also presents two innovative light-sheet modulations evaluated using the SLM-SPIM, which have been specifically designed to help reduce the effect of photobleaching and, more in general, to improve the system’s light-efficiency.

The first light-sheet modulation proposed is specifically designed to be used while performing the imaging technique known as tiling, which makes it possible to obtain a more uniform illumination by moving the highest-resolution region of the light-sheet across the imaging field of view. The proposed light-sheet aims at better confining the illumination light, thereby helping reduce the undesired extra photobleaching generated by the sample over-exposure typical of the tiling implementation. The simulated and experimental results obtained so far suggest that the advantages of using the modified light-sheet over a traditional Gaussian light-sheet would only be minimal (minimal reduction of photo-bleaching of the sample for the same number of images acquired). Nevertheless, the results obtained trying this imaging technique on the SLM-SPIM also demonstrate how such a versatile, flexible system can be a very useful tool when wanting to develop and try new ideas and imaging techniques.

The second innovative imaging technique proposed involves the generation of a depth-modulated light-sheet array which can be used to perform more light-efficient (i.e. faster and less photodamaging) 3D imaging, following the principles of compressive sensing. This thesis presents simulated and experimental results obtained using this compressed imaging scheme to reconstruct the 3D image of sparse solutions of fluorescent beads suspended in agarose. The results obtained so far seem to suggest that, for samples which are sparse enough in the spatial domain, the proposed compressed scheme allows for a successful reconstruction of a 3D image of the sample using half (or less) of the light-sheet images normally required by the traditional plane-by-plane 3D imaging scheme.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Supported by funding from the Engineering and Physical Sciences Research Council (EPSRC).
Subjects: Q Science > QC Physics
Colleges/Schools: College of Science and Engineering > School of Physics and Astronomy
Supervisor's Name: Taylor, Dr. Jonathan M.
Date of Award: 2021
Depositing User: Theses Team
Unique ID: glathesis:2021-82429
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 08 Sep 2021 14:16
Last Modified: 08 Sep 2021 14:16
Thesis DOI: 10.5525/gla.thesis.82429
URI: http://theses.gla.ac.uk/id/eprint/82429
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