Investigation of homo-FRET and rotational mobility via time-resolved fluorescence microscopy to probe the fluorophore’s environment

Abstract

The aim of this thesis is the development of a multi-modal fluorescence microscopy setup, with the main objective of maximising the amount of information from a single limited photon budget. Fluorescence in an image can be characterised by position, intensity, lifetime, wavelength and polarisation. The more of these parameters are measured, the more information about the probe’s environment is gained. Here, I show how position, intensity, lifetime and polarisation can be measured in a single experiment. The time-resolved polarisation in particular can give information about rotational diffusion, and homo-FRET. Several fluorescence microscopy techniques were combined in a based Time-Correlated Single Photon Counting (TCSPC) framework: Fluorescence Lifetime IMaging (FLIM), time-resolved Fluorescence Anisotropy IMaging (tr-FAIM) and Fluorescence Recovery After Photo-bleaching (FRAP). The set-up was built in-house and calibrated with rhodamine 6G (R6G) solutions in water/glycerol mixtures and further tested in single lipid bilayers (SLBs) with the environmentally-sensitive dye di-4-ANEPPDHQ. With this multi-modal fluorescence microscopy setup the hydrodynamic radius of the fluorescence probe can be calculated without any a priori viscosity knowledge when the solution is isotropic and homogeneously distributed. Protein dimerisation in cells triggers many biological processes such as cell signalling, which is crucial for the right functioning of the cell itself. For this reason, protein dimerisation was also investigated by firstly studying two enhanced green fluorescence protein (EGFP) constructs - monomer & dimer - in buffer/glycerol solution mixtures. Time-resolved fluorescence anisotropy measurements were taken for both EGFP constructs and the F¨orster Resonance Energy Transfer (FRET) from the EGFP dimer was extracted by fitting the time-resolved fluorescence anisotropy data with a stretched exponential model, whose FRET energy values were encoun-tered within the range provided by the molecular dynamic (MD) simulation re-sults. As a real example, the protein homo-dimerisation of Coxsackievirus and Adenovirus Receptor (CAR) tagged with GFP within the cell membrane of Human Bronchial Epithelial (HBE) cells was investigated, where CAR arranges itself as a dimer. Steady-state anisotropy information revealed the distinction of different cell stages from the disruption of the CAR-GFP dimer configuration via the infection of cells with adenovirus. Lastly, the amount of photons needed in time-resolved fluorescence anisotropy measurements is investigated. An expression for the uncertainty associated with the rotational correlation time θ , based on the Perrin equation, is derived. The validation of this expression is undertaken by comparison with experimental data. Some simulations provide information in regards to the optimal boundary conditions that must be set in the experiment to achieve low rotational correlation time uncertainties ∆θ . As part of imaging, the distribution of the rotational correlation time θ across an isotropic and homogeneous solution is investigated, based on the fluorescence lifetime τ, steady-state anisotropy r distributions and the application of the Perrin equation.

Date of Award	1 Jun 2020
Original language	English
Awarding Institution	King's College London
Supervisor	Klaus Suhling (Supervisor) & Amelle Zair (Supervisor)