Simulating Homo-FRET Fluorescence Decay Profiles

Polarisation resolved TCSPC data of fluorophores with a bi-exponential fluorescence decay profile undergoing homo-FRET leading to a bi-exponential anisotropy decay profile was simulated in MATLAB with a time window of 12.5 ns for a simulated pulse repetition rate of 80 MHz. The model, before accounting for incomplete decays and a simulated instrument response function takes the form where is the fractional contribution of the long component of the intensity decay and is the peak intensity. The data was simulated for two detectors polarised parallel and perpendicular to the excitation light, i.e. and . Using Equation 14, and . The model was convolved with a Gaussian IRF with a full width half maximum of 150 ps. The lifetimes of the fluorescence decay components were set to be and with a fractional contribution of the long component . The rotational correlation times were set to and , i.e. of the order expected for a fluorescent protein fusion construct undergoing homo-FRET. The pixel simulated image was split into three regions with initial anisotropy contribution of the short component set to 0.1, 0.2 and 0.3. In all three regions the total initial anisotropy . The initial intensity was set such that there were on average total integrated counts in each pixel and Poissonian noise was added to each decay using the MATLAB function poissrnd. These simulation parameters were chosen to approximate realistic values for cell imaging data, with the lifetimes selected to be similar to those of common cyan fluorescent protein variants such as ECFP and Cerulean [62] (link). In common with Experiment 5, a mean smoothing kernel was applied to each the image representing each time-bin of the data by convolution.