Photobleaching is a general hurdle of fluorescence-based techniques especially in high-resolution microscopy that relies on prolonged and complex illumination. Strategies to reduce photobleaching require chemical modifications of the cell medium, which often compromise physiological cellular conditions. Here, we outline an all-optical strategy to minimize photobleaching in reversibly switching fluorescent proteins (RSFPs), a class of probes used in super-resolution and protein-multiplexing imaging techniques. By identifying the photobleaching pathways, we develop imaging schemes to increase the number of on-off photoswitching cycles, either modulating the on-switching light or co-irradiating the RSFPs with light at longer wavelengths with respect to fluorescence excitation. We apply the optimized imaging scheme to achieve imaging multiplexing at high-spatiotemporal resolutions and to record longer time-lapse imaging of sub-cellular structures with both confocal microscopy and parallelized RESOLFT nanoscopy.

The formation of macromolecular complexes can be measured by detection of changes in rotational mobility using time-resolved fluorescence anisotropy. However, this method is limited to relatively small molecules (~0.1–30 kDa), excluding the majority of the human proteome and its complexes. We describe selective time-resolved anisotropy with reversibly switchable states (STARSS), which overcomes this limitation and extends the observable mass range by more than three orders of magnitude. STARSS is based on long-lived reversible molecular transitions of switchable fluorescent proteins to resolve the relatively slow rotational diffusivity of large complexes. We used STARSS to probe the rotational mobility of several molecular complexes in cells, including chromatin, the retroviral Gag lattice and activity-regulated cytoskeleton-associated protein oligomers. Because STARSS can probe arbitrarily large structures, it is generally applicable to the entire human proteome.

Arc (or Arg3.1), Activity Regulated-Cytoskeleton associated-protein is pivotal to mediate plastic responses in neuronal cells. In vitro and in vivo studies suggest its ability to form high- and low-order oligomers which are potentially involved in neuronal trafficking. Despite its important function, no direct observation of Arc oligomers in cells has been presented due to its highly regulated spatiotemporal expression, the small size of the structures, the lack of appropriate labelling strategies and the background associated to free diffusing cytosolic proteins. Here, we take advantage of several complementary advanced fluorescence microscopy and spectroscopy techniques to observe and quantify Arc oligomeric states in cellular environment especially in the synapses. In cells, we uncovered Arc-Arc intermolecular interactions, Arc tendency to form liquid condensates and to interact with lipid bilayers. High-order oligomers are found to localize at the excitatory synaptic compartment and to directly affects AMPA receptor surface levels. Together, our observations support the model by which Arc oligomerization mediates plasma- membrane negative inward curvature favoring AMPA receptors endocytosis.