"Fluorescence lifetime imaging and fluorescence polarization for elucidation of molecular level events: peptide self-assembly, NP dissassembly in cells, and phage binding."

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Fluorescence based tools are a cost effective means (compared to pulsed nMR which is the only technique that can provide similar information) of elucidating molecular level events. This is primarily due to the wealth of information that can be obtained from fluorescence emission, which can be quantified in obvious ways like intensity and position, as well as less obvious ways like lifetime, polarization and wavelength. Fluorescence lifetime imaging (FLIM), for example, can provide detailed information regarding photophysical events otherwise impossible to detect using fluorescence intensity imaging techniques. FLIM technique is relatively insensitive to probe concentration, but susceptible to environmental changes like pH, viscosity, ion or oxygen concentration, refractive index, polarity and quenching via Forster resonance energy transfer (FRET). Fluorescence polarization (anisotropy) may yield information that cannot be obtained from intensity, lifetime or emission spectrum data, including: molecular aggregation, orientation, rotational mobility and the migration of energy between identical fluorophores (homo-FRET). Moreover, fluorescent polarization has been shown to be proportional to molecular size and solution viscosity, whilst being inversely proportional to fluorescence lifetime and solution temperature. Specifically, the Stokes-Einstein equation can be used to relate the rotational diffusion time to molecular size (ie. hydrodynamic volume), viscosity, and solution temperature. This methodology has been used to allow the determination of molecular size and viscosity gradients within lipid membranes or live cells. This equipment will allow for significant advances in the molecular level understanding of how peptide chemistry affects peptide self-assembly, nanofiber formation, and molecular binding. Work considered here is interdisciplinary by nature, facilitating the training of HQP across biomedical, biophysics, pharmacy, medicine, and biochemical engineering.