Sequential labeling and imaging in fluorescence microscopy allows the imaging of multiple structures in the same cell using a single fluorophore species.In super-resolution applications, the optimal dye suited to the method can be chosen, the optical setup can be simpler and there are no chromatic aberrations between images of different structures.We describe a method based on DNA strand Power Plug Adaptors displacement that can be used to quickly and easily perform the labeling and removal of the fluorophores during each sequence.
Site-specific tags are conjugated with unique and orthogonal single stranded DNA.Labeling for a particular structure is achieved by hybridization of antibody-bound DNA with a complimentary dye-labeled strand.After imaging, the #N/A 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.
Labeling and removal of each DNA-species requires only a few minutes.We demonstrate the concept using sequential dSTORM super-resolution for multiplex imaging of subcellular structures.