Abstract
The use of viscosity-sensitive fluorescent probes in biological imaging is becoming increasingly popular, and the first measurements in various different cell types, organelles and even live animals have recently been reported. Yet there remains gaps in the understanding of ‘microviscosity’ and the various viscosity-related fluorescence parameters used to measure it.In this thesis, I moved away from a generalised notion of cellular viscosity and towards measurement of highly localised viscosity environments in biological organelles. To do this, I employed time- and polarisation-resolved fluorescence imaging and spectroscopy, using the gold-standard of viscosity probes: boron-dipyrromethene (BODIPY)-based fluorescent molecular rotors (FMRs). The objective was to achieve a high degree of local viscosity domain separation.
By combined use of chemical FMR-targeting and physical organelle extraction, I separated the two primary domains of mitochondria: matrix and membrane. This represented the first level of domain separation. Using fluorescence lifetime imaging (FLIM) and two FMRs (mitoBODIPY and BODIPY-C12), I found that both viscosity environments are responsive to small physiolog-ical or environmental changes.
The second level of domain separation was achieved through combined FMR-FLIM and time-resolved fluorescence anisotropy imaging (TR-FAIM), using BODIPY-C12. The method was applied to lipid droplet organelles and artificial analogues. Two viscosity-related parameters, FMR lifetime and rotational correlation time, were determined in the same experiment; they were non-equivalent. Furthermore, molecular dynamics simulations from a collaborator found distinct orientations of the FMR within each system which connected to empirically determined fluorophore populations, resolving the two-component signal.
Finally, I investigated a fundamental photophysical aspect of BODIPY FMRs, the transition dipole moment (TDM), which is crucial in the interpretation of TR-FAIM signals. Using the spectroscopic approach pioneered by Toptygin, I found a tilted emission TDM in the fundamental BODIPY FMR, phenyl-BODIPY. This was contextualised and explained by quantum chemical calculations carried out by a collaborator. Furthermore, I examined phenyl-BODIPY’s seemingly viscosity-dependent r0, an anisotropy parameter indicative of the angle between the absorption and emission TDM. Similar behaviour was observed in non-FMR dyes. Through a combination of higher excited state experiments and quantum chemical simulations from a col-laborator, I conclude the behaviour is due to a combination of torsional vibrations and ultra-fast librations.
| Date of Award | 1 May 2021 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Klaus Suhling (Supervisor) & Madeline Parsons (Supervisor) |