Development of a multibeam multiphoton multiwellplate microscope (M3M) for 2D/3D FLIM FRET assays
File(s)
Author(s)
Flanagan, William
Type
Thesis
Abstract
This thesis reports the development and application of a multibeam multiphoton multiwell plate microscope (M3M) with fluorescence lifetime imaging (FLIM) and high content analysis (HCA) capabilities based on the modular openFrame microscope stand. The instrument design, supported by numerical simulations, and its implementation and exemplar applications are described. As far as possible, all software components are open-source and modular hardware components can be easily interchanged or replaced. The factors impacting imaging speed are quantitatively analysed, including comparison with a conventional single excitation beam system. The integration of a novel polarisation based differential phase contrast (pDPC) module to provide semi-quantitative phase imaging is described with its application to multiphoton FLIM HCA. Exemplar M3M HCA applications to 2D (monolayer) and 3D (spheroid) cell cultures are presented including FLIM assays of cellular autofluorescence to investigate modulation of metabolism reported by changes in NAD(P)H fluorescence lifetime. In 2D cell cultures this FLIM assay is combined with pDPC-based quantitative phase imaging to provide complementary label-free readouts that could be directly applied to primary cells. The M3M instrument was also applied to study the interaction of rucaparib, an intrinsically fluorescent anti-cancer drug, with ovarian cancer cells, including the partitioning inside cellular compartments where FLIM reported changes in local pH and provided a quantitative visualisation of rucaparib delivery in a tumour spheroid.
Version
Open Access
Date Issued
2025-05-14
Date Awarded
01/11/2025
Advisor
French, Paul
Publisher Department
Department of Physics
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)