Mechanical Quantification in Lipid Membrane and Live Cells using Fluorescence-based Molecular Sensors

Abstract

Fluorescence is a well-studied physical phenomenon and has been the basis for many research tools. Yet the recent advancements in ultrafast lasers and fluorescent molecules have opened the door to new ways to study the dynamical forces acting inside living cells. Fluorescence lifetime imaging microscopy, or FLIM, can move the fluorescence microscopy beyond being a tool of simple tracking and identification of cellular features and enables live monitoring of cellular activities and dynamics. New fluorophores and their arrangements have helped to take advantage of FRET, fluorescence resonance energy transfer process which enables measurement of qualities previously difficult to measure. The thesis focuses on demonstration of these newly developed tools and their applications as molecular sensors in biomedical research. Flipper-TR, a recently arrived molecular probe, shows how it can report via FLIM on lipid packing orders in different lipid membranes and how they change with the varying cholesterol contents. The measurements from FLIM-Flipper-TR are compared to those by deuterium NMR, a well-established method to study the structures and phase transitions within lipid membranes, to test an implicit hypothetical relationship between the intermolecular spacing reported by the fluorescence probe and order parameters derived from NMR spectra. The result yields a strong linear correlation between the two techniques and measurements obtained from them; additional experiment reveals a still incomplete picture but a definite potential of interchangeability for the two methods. Additionally, the work lists demonstrations of diverse molecular sensors based on FRET and how their utilization can aid in characterization of tumor cells and development of novel cancer treatments, not to mention study of general cellular dynamics. In summary, the thesis work offers a glimpse into the world of new fluorescence-based molecular sensors and their promising applications in biomedical research.