Novel fluorescent probes and analysis methods for single-molecule and single-cell microscopy

By directly visualizing biological processes at the level of individual cells down to single proteins, many fundamental biological questions can be answered. Fluorescence microscopy has emerged as the most widely used tool for this purpose as it has unique advantages. The technique is minimally perturbative, and state of the art fluorescent labels are used which yield high specificity. Single fluorescent emitters can be imaged with high signal-to-noise ratios. By circumventing ensemble averaging, underlying heterogeneity in molecules can be resolved and dynamic events are observed without the need of synchronization. The aim of this thesis is to address some of the current limitations in fluorescence microscopy. Specifically, the focus is on the development of new fluorescent probes used to acquire images, and the development software for its analysis. New fluorescent derivatives were developed which exhibited enhanced resistance against irreversible destruction by the high illumination intensities applied in single-molecule fluorescence microscopy. This allows users to observe samples for extended periods of time. The probes were tested in optical super-resolution techniques and were found to have superior performance, thereby further pushing the spatial resolution that can be achieved. Next, the thesis describes the development of an open-source software package for the analysis of fluorescence microscopy data of single cells, aimed to stimulate open and reproducible. Finally, a novel imaging probe is described, and we characterize its ability to selective image gram-negative bacteria and its potential for combating antimicrobial resistance through photodynamic therapy.