LAUSR

The communication and exchange of material between subcellular organelles is critical to many cellular processes. Areas of close apposition between organelles, termed membrane contact sites (MCS), have recently become a focus of study for intracellular communication. Through enrichment of specific proteins and protein complexes, these sites are implicated in the exchange of lipids and metabolites such as calcium. As MCS characteristically display inter-membrane distances of 10-30 nm, electron microscopy is often used to visualize these phenomena. However, the molecular specificity of the proteins involved in MCS is of great interest. In order to address these questions more closely, we have developed a hyperspectral localization microscopy technique to spectrally distinguish far-red fluorophores using a Versachrome thin film tunable filter (Semrock) integrated into the emission path of an Olympus IX-83 dual deck inverted TIRF microscope. Using this hyperspectral 3D Direct Stochastic Optical Reconstruction Microscopy (dSTORM) imaging approach, we were able to spectrally and spatially distinguish between Alexa 647 and Alexa 680 labels in our studies of the nanoscale spatial distribution of ER and peroxisome membranes under WT and MCS knockdown conditions and the clustering of MCS proteins at key sites of membrane apposition.