Fluorescence Photoconversion of Biarsenical-Labeled Cells for Correlated Electron Microscopy (EM)
Adapted from Live Cell Imaging, 2nd edition (ed. Goldman et al.). CSHL Press, Cold Spring Harbor, NY, USA, 2010.INTRODUCTION
Correlated light microscopy (LM)/electron microscopy (EM) analysis can be achieved by using biarsenical dyes to fluorescently label tetracysteine-tagged proteins. Once live cell imaging using LM is complete, cellular activity can be halted promptly using a glutaraldehyde-based fixative. Rapid fixation preserves cellular ultrastructure and limits diffusion of reaction products. This protocol provides details on rapid fixation of cells, followed by fluorescence photoconversion of 3,3′-diaminobenzidine tetrahydrochloride (DAB) and sample processing for EM that can be correlated with the live cell LM images.
