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Sequential labeling and imaging in super-resolution fluorescence microscopy allows the imaging of multiple structures in the same cell using a single fluorophore species. In this work, we describe a method based on DNA strand displacement that can be used to quickly and easily perform the labeling and removal of the fluorophores during each sequence. Labeling for a particular structure is achieved by hybridization of site-specific antibody-bound ssDNA with a complimentary dye-labeled strand. After imaging, the dye is removed using toehold-mediated strand displacement, in which an invader strand competes off the dye-labeled strand than can be subsequently washed away. We demonstrate the concept using sequential dSTORM super-resolution for multiplex imaging of subcellular structures.


Fig2: Data validating rapid kinetics of strand displacement and low residual cross-talk fluorescence after invader treatment.

Fig3: Data containing image stacks from six total rounds of sequential imaging of sub-cellular structures in HeLa cell.

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