Abstract
In any complex biological system, a mosaic of molecular states and reaction pathways exist in parallel. Single molecule observation allows dissecting this complexity, providing a direct look at the distribution of molecular behaviors. Facilitated by innovations in optimizing photon collection efficiency of modern microscopes and spectroscopes, observing fluorescence from a single dye has become a standard capability in many laboratories. Still, creating a meaningful reporter system of molecular plasticity seems out of reach for most biological systems. I will present several novel approaches how to make complex biological systems accessible to high resolution single molecule and superresolution technologies in vitro and in vivo. I will show how quantitative microscopy techniques permit deciphering the molecular architecture of extremely large cellular structures like the nuclear pore complex. Furthermore, I will demonstrate how biocompatible ultrafast inverse-electron-demand Diels-Alder can be encoded into proteins site-specifically and used for the purpose of multicolor live cell labelling and super-resolution microscopy with very small and photostable fluorescent dyes. I will discuss the limitations and future prospects of these technologies and also introduce a freeware analysis platform that permits to estimate the specific advantages of using small sized labels for high end microscopy.