Development of widefield time-resolved luminescence anisotropy imaging on short and long time scales using novel time resolved cameras.

The time-resolved fluorescence anisotropy of a probe can reveal information about the rotational dynamics, or structure of a probe's local environment. To study this phenomenon, researchers are required to measure the changing dipole orientation of a fluorescent probe during its tumbling in-between absorption and emission events under polarised excitation. Fluorescence anisotropy studies of biological samples are commonly used to observe molecular interactions using single point measurements in cuvettes without any spatial information of the sample. Yet, 2-dimensional (2D) time resolved fluorescence anisotropy imaging (TR-FAIM) of biological samples has been reported only a handful of times. Despite the lack of progression of TR-FAIM methods, 2D images of the rotational diffusion of rigid dyes using TR-FAIM has great potential for viscosity studies of biological systems. Partly because the investigator can choose a probe whose fluorescence lifetime is sensitive to another cellular environment parameter, and simultaneously capture fluorescence lifetime imaging (FLIM) and TR-FAIM images of the biological sample. This is possible because TR-FAIM data inherently contains all the necessary information to construct a FLIM image. Additionally, the investigator can introduce more control in their experiments by choosing a probe with an affinity for a specific structure in the biological sample. One reason for the lack of progression of TR-FAIM could be due to the lack of available photo-detectors practical for measuring the tens of thousands of photons required in each pixel to accurately resolve mono-exponential anisotropy decays. Due to this restriction, laser scanning techniques using single point detectors typically require long acquisition times (hours) to gain the required photon counts in each pixel. Yet, the recently developed single-photon avalanche detector (SPAD) array and Timepix3 emerge as excellent candidates for such measurements.