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Title: Imaging of metabolism in 3D culture by FLIM
Author: Chennell, George
ISNI:       0000 0004 6421 0883
Awarding Body: Imperial College London
Current Institution: Imperial College London
Date of Award: 2017
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The work presented in this thesis is aimed to develop and evaluate methodologies for noninvasive measurements of metabolism using fluorescence microscopy. The use of 3D cell cultures in biomedical research is increasing and these require appropriate tools and techniques to provide quantitative readouts for image-based studies. Fluorescence lifetime imaging microscopy (FLIM) can provide robust readouts in complex optical samples and here I have investigated its application to map changes in the response of genetically expressed biosensors utilising Förster resonance energy transfer (FRET) in spheroids. In particular, I adapted a FRET biosensor for the activity of a key metabolic enzyme, AMP activated protein kinase (AMPK), by substituting the donor fluorescent protein ECFP for mTurquoise2, in order to improve its performance in FLIM-based assays. I developed spheroid cultures expressing FRET biosensors and studied these using quantitative FRET readouts. To take account of possible influences of the microenvironment of 3D culture on the fluorescence lifetime measurements, I generated spheroids expressing simple fluorescent proteins and expressing an inactive mutation of the FRET biosensor. I evaluated the new AMPK FRET biosensor, demonstrating improved performance for fluorescence lifetime readouts, and compared dose responses for a direct activator of AMPK with the biosensor expressed in “2D” monolayer cultures and in spheroids, consistently observing a uniform response. In contrast, the dose response of an indirect activator of AMPK in spheroids presented a spatially varying AMPK activation. I further explored the application of FLIM to map the readout of a genetically expressed FRET biosensor for glucose and again observed a spatially varying response in spheroids. I then explored cell specific AMPK activities using FRET biosensors in prostate cancer cells and bone marrow stromal cells with a spheroid system of tumour stromal interactions. I also used biosensors for ATP and glucose concentration in a similar manner and undertook measurements of oxygen consumption rates using a metabolic flux analyser. I observed changes in metabolism that indicate the prostate cancer cells were metabolically benefitting from the interaction with bone marrow cells.
Supervisor: French, Paul ; Carling, David ; Sardini, Alessandro ; Dunsby, Christopher Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral