Abstract
Abstract: Fluorescence microscopy plays an essential sensing role in biology owing to the rapid advancement of fluorescent techniques and new engineered fluorophores. While it is necessary to investigate biological processes, there is still a growing need to develop new measurement approaches that can allow in cellulo measurement of several analytes and metabolic byproducts. Among these, Reactive Oxygen Species (ROS) have been widely mentioned, along with intracellular signaling and human pathologies. ROS's different involvements in many cellular physiological roles raise the question of their intracellular roles depending on type and dose. To better quantify their spatial and temporal production, we design and synthesize organic fluorophores based on a long fluorescence lifetime probe, pyrene butyric acid. These lifetime-based sensing probes have both merits of a long fluorescence lifetime (a few hundred nanoseconds) and a mitochondrial vector, aiming at quantifying ROS in its proximity. Using fluorescence lifetime technique has the advantage in its probe concentration independence, an indispensable property when working in cells. In this thesis, we characterize the probe's photophysical behavior in solution with different ROS models (i.e., probe photostability, ROS quenching efficiency, etc.). Once their in-solution functionality is validated, we introduce the probes inside several cultured cells (adherent and not adherent) to detect cellular ROS levels. We also measure the extent of the probes' cytotoxicity to compare the probe's impact on a cell line, aiming at having a minimum cellular effect. Our goal extends to find the best fit for a Cargo-transport model featuring a lifetime-based fluorescent marker. These vectors localize in mitochondria to their positive charge and lipophilic group characteristics (i.e., mitochondrial targeting peptides (MTP), triphenylphosphonium salt (TPP+)). Several probes were synthesized using different mitochondrial vectors to improve the sensing output, assuring efficient cellular uptake and maximal mitochondrial localization.