Development and application of broadly applicable microscopy methods and probes for diffraction-unlimited fluctuation imaging of dynamic biological systems.

Fluorescence imaging has seen a tremendous evolution over the last decade with the advent of methods circumventing the optical diffraction barrier, which has classically limited the fineness with which samples can be resolved to about 200 nanometers. To date, however, imaging at high spatial resolution has become reality but it comes with a cost: superresolution imaging is performed at the expense of a significantly reduced time resolution, hampering studies of e.g. dynamic biological processes; conformational dynamics, binding kinetics etc. Solving these issues requires novel fluorescence methodologies and probes. In this ambitious project, we will develop pioneering multicolor confocal fluorescence methods and concomitant probes for quantitative investigations of dynamic molecular complexes in vitro and in vivo. We will validate our novel methods by applying them to different relevant and interesting biological systems such as the bacterial secretion pathway as well as the pathology of the human immunodeficiency virus (HIV). By doing so, we can promote our novel, cutting edge methods to a large community.