Abstract
Plasma membrane of any cell supports a vast array of biomolecules on its surface. These are crucial in response and adaptation to the external environmental cues of the cell. The process of determining these cues is done by a plethora of plasma membrane associated proteins including receptors, channels and pores. The spatial organization including compartmentalization of these proteins is critical for cellular function. In order to generate a high spatial resolution topological map of a plasma membrane we use wide-field fluorescence super-resolution imaging technique known as Stochastic optical reconstruction microscopy (STORM). STORM works on the principle of stochastic switching of single-molecule fluorophores and precise localization of the centers of these spots. Here we labeled the plasma membrane using a small molecule lipophilic carbocyanine dye, DiD, which can photoswitch under similar conditions as previously used for Cy5 photoswitching. We stained Drosophila Schneider 2 cells with DiD and imaged them under both fixed and live conditions using 3D STORM imaging and/or single-particle tracking modes. We observed plasma membrane features including clathrin coated pit-like structures. Further experiments using multicolor STORM to image DiD together with membrane proteins will shed light on how membrane protein distribution is correlated with membrane topology.
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