Imaging green fluorescent protein-labeled neurons using light and electron microscopy

The ability to observe axons and dendrites with transmission electron microscopy (EM) after they have been previously imaged live with laser-scanning microscopy is a useful technique to study their synaptic connectivity. This protocol provides a detailed method by which neurons that were imaged in a live brain or slice culture can be reimaged using EM. First, brain tissue expressing green fluorescent protein (GFP) is chemically fixed. Then, an immunocytochemistry process is used to render the fluorescent protein electron dense so that it can first be located using light microscopy and then serial thin-sectioned for EM so that the ultrastructure of specific parts of neurites can be analyzed in three dimensions. Patterns of blood vessels observed in the live brain are used to locate the previously imaged neurons. The method described here allows for a complete three-dimensional (3D) reconstruction to be made of the imaged structures from serial electron micrographs.