Advanced Fluorescence Microscopy for Pharmaceutical Studies

The biomedical field is constantly looking for noninvasive, more sensitive techniques to study pharmaceuticals and their interactions with cells. In this thesis, two advanced fluorescence imaging methods, fluorescence lifetime imaging microscopy (FLIM) and video tracking, were used to study solid-state drug formulations and extracellular vesicle (EV)–mediated drug delivery. FLIM creates a spatial map of the fluorescence decay rates in a microscopic sample, providing a unique environmental sensitivity which is not possible to reach by mapping only the fluorescence intensity. The video tracking is typically based on the intensity detection but adds another dimension to the conventional fluorescent microscopy: the nanoparticle trajectories recorded in high spatio-temporal resolution allow the detailed analysis of the nanoparticle movements. The lifetime detection provided sensitivity to the early stages of indomethacin crystallization on the sample surface, offering an important addition to the traditional methods used mainly for following the bulk crystallization. Furthermore, changes in the fluorescence lifetime revealed alternative cellular distributions of a fluorescent taxol conjugate when loaded into different EV subtypes, suggesting different internalization and drug release pathways of the EV populations. Motivated by this observation, the intracellular EV movements were directly studied by video tracking with a particular focus on the improvement of the analysis for the complex nanoparticle trajectories. The intracellular trajectory patterns and the related dynamics were close to similar for both studied EV types and resembled those reported for intravesicular trafficking along the cytoskeleton. The EVs required fluorescent labelling for being visualized by the fluorescent methods. The removal of the excess dye from the labelled EVs was shown to be challenging with the typical methods used for the EV purification and depend on the properties of the dye. Therefore, a fluorescence-based method to evaluate the purification outcome was proposed. The imaging methods used in this thesis were shown to provide sensitivity and information not available via other methods. The presented results increase the current understanding of the crystallization mechanisms of the amorphous solids, the adequate EV sample preparation for fluorescent imaging, and the EV-mediated drug delivery on a single-cell level.