Correlative light and electron microscopy of GFP.

The correlation of light and electron microscopy (EM) is a powerful tool as it combines the investigation of dynamic processes in vivo with the resolution power of the electron microscope. The green fluorescent proteins (GFPs) and its derivatives revolutionized live-cell light microscopy. Hence, this review outlines correlative microscopy of GFP through photo-oxidation, a method that allows for the direct ultrastructural visualization of fluorophores upon illumination. Oxygen radicals generated during the GFP bleaching process photo-oxidize diaminobenzidine (DAB) into an electron dense precipitate that can be visualized both by routine EM of thin sections and by electron tomography for 3D analysis. There are different levels of correlative microscopy, i.e. the correlation of certain areas, cells, or organelles from light to EM, where photo-oxidation of DAB through GFP allows the highest possible degree--the correlation of specific molecules.