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.