Improving STED-Based Super-resolution-Imaging with Fluorescence Lifetimes

The neuronal cytoskeleton and synaptic organization play crucial roles in neuronal information processing. The cytoskeleton is composed of individual filaments that differ in their posttranslational modifications, which often reflect differences in stability or dynamics. Similarly, chemical synapses consist of multiple functionally specialized compartments within which proteins are organized into nanodomains. Understanding the spatial organization of these components requires imaging techniques with sufficient resolution to visualize multiple marker proteins simultaneously and in z-stacks that can be used to display the three-dimensional organization. Super-resolution methods such as Stimulated Emission Depletion (STED) microscopy are essential to achieve this purpose. STED overcomes the diffraction-limited resolution of conventional fluorescence microscopy by narrowing down the area of detected fluorescence. As an extension of that, tauSTED uses the information of fluorescence lifetime to further enhance lateral, axial, and temporal resolution, thus offering several advantages for biological imaging. These advanced microscopy methods, however, require sample preparation with additional considerations compared to regular fluorescent microscopy. Thorough planning is critical to successfully acquire high-quality super-resolution images using STED microscopy. In this chapter, we place a particular focus on tauSTED nanoscopy. We address key aspects, including sample preparation, understanding the underlying theoretical principles and finally the selection of suitable imaging parameters.