Preparation of Photoswitchable Labeled Antibodies for STORM Imaging

Abstract

Stochastic optical reconstruction microscopy (STORM) is a method for superresolution fluorescence imaging based on the high accuracy localization of individual fluorophores. It uses optically switchable fluorophores: molecules that can be switched between a nonfluorescent and a fluorescent state by exposure to light. Many synthetic fluorescent dye molecules show photoswitchable fluorescence emission. In particular, photoswitchable cyanine fluorophores such as Cy5, Alexa 647, and Cy7, may be paired with a second fluorophore, which serves as an activator, determining the wavelength of light that re-activates the fluorescence of the photoswitchable molecule. This protocol describes the preparation of antibodies labeled with one such pairing scheme of synthetic fluorophores, Alexa 405 and Alexa 647. It may easily be adapted for labeling with other fluorophores or for labeling other substrate molecules.