Fluorescence microscopy and spectroscopy are in-vivo compatible techniques that are currently standard tools to address questions related to the organization and the dynamics of biological systems. The development of super-resolution microscopy over the last 30 years has enabled scientists to investigate such systems below the diffraction limit of light with a near-molecular spatial resolution. Although fluorescence is by nature an environment-sensitive process, common fluorophores for super-resolution microscopy typically lack chemical sensitivity to their environment and are thus merely used as labels of the position of the molecule to which they are attached or bind. Based on this observation, the work presented in this thesis addresses novel possibilities to introduce both chemical and physical sensitivity in fluorescence spectroscopy and imaging studies relying on fluorescent probes compatible with both biological environments and single-molecule based super-resolution microscopy.