Understanding complex biological systems is essential for developing new approaches for the treatment of diseases. Super-resolution (SR) fluorescence microscopy serves as an important tool to assist in gathering this key knowledge, as it enables highly specific and detailed visualization of fluorescently labeled molecules. Technological development over the last decades has continuously improved both resolution and labeling approaches. Especially, the development of the technique expansion microscopy (ExM) offered new possibilities for a wide range of researchers as it permits three- dimensional high-resolution imaging of multiple targets using conventional fluorescence microscopes. By embedding biological samples in a hydrogel and physically expanding them, ExM provides several advantages, particularly when employing a post-expansion labeling variant of the method. Among the benefits of post-labeling are improved epitope accessibility and elimination of steric hindrance leading to a higher labeling density. Additionally, non-specific staining is reduced and the linkage error between the fluorescent dye and the target structure is minimized. However, several challenges concerning the isotropy and expansion quality of different structures still remain. This thesis aims to optimize and apply post-labeling ExM in combination with or in addition to other SR methods to investigate the organization of sphingolipids and proteins in cells. The first part of the thesis focuses on the enzyme neutral sphingomyelinase 2 (NSM2), which is activated upon T-cell antigenic stimulation. NSM2 is an important component of the sphingolipid pathway catalyzing the production of ceramide at the cytosolic leaflet of the plasma membrane. We analyzed the plasma membrane topology of NSM2 with or without T-cell stimulation using single-molecule localization microscopy (SMLM), confocal and live-cell imaging. Although no oligomerization of NSM2 was observed, the enzyme was strongly accumulated at the artificial immunological synapse, suggesting a potential role in its formation and possible influence on membrane properties through increased ceramide production. Functionalized ceramides offer a smart strategy for visualizing sphingolipids by ExM, since most lipids lack functional groups required for anchoring into the hydrogel. To enable simultaneous visualization of ceramide and NSM2 distribution an already established functionalized C6-ceramide was tested for post-labeling ExM and analyzed via mass spectrometry concerning its conversion by cellular enzymes. This yielded important insights for the interpretation of future ExM experiments. Utilizing a newly developed functionalized C16-ceramide, optimal results were obtained when combined with post-expansion click-staining, possibly due to better accessibility to the functional group for fluorescent click-staining. In subsequent chapters of the thesis, efforts were concentrated on methodological refinement of a post-labeling ExM approach for integration with other super-resolution techniques to achieve molecular resolution. We could demonstrate an improved epitope accessibility and reduced linkage error thereby enabling detailed multicolor 3D-visualization of components of the neuronal synapse. However, several cases showed deficiencies in expansion quality when using the mild homogenization method essential for post-labeling. To overcome this limitation we developed an enhanced iterative post-labeling approach with double homogenization (dTREx), which allows efficient expansion of protein complexes at the nanoscale. Following the improvement of cyanine dye survival during polymerization, the dTREx approach was integrated with re-embedding in a non-shrinking gel for direct stochastic optical reconstruction microscopy (dSTORM) and validated using the reference structures clathrin-coated pits (CCPs) and microtubules. Finally, this approach was used to investigate components of the active zone at the neuronal presynapse. This enabled us to elucidate the molecular organization of Munc13-1 and RIM, two essential proteins of the presynaptic exocytosis apparatus. In conclusion, the refined post-labeling ExM approach represents a promising strategy for investigating cellular protein arrangements at the molecular level through efficient homogenization while retaining benefits of post-expansion-labeling.
Janna Eilts (Thu,) studied this question.