Using laser scanning confocal microscopy as a guide for electron microscopic study: A simple method for correlation of light and electron microscopy

Anatomic study of synaptic connections in the nervous system is laborious and difficult, especially when neurons are large of have free branches embedded among many other processes. Although electron microscopy provides a powerful tool for such study, the correlation of fight microscopic appearance and electron microscopic detail is very time-consuming. We report here a simple method combining laser scanning confocal microscopy and electron microscopy for study of the synaptic relationships of the neurons in the antennal lobe, the first central neuropil in the olfactory pathway, of the moth Manduca sexta. Neurons were labeled intracellularly with neurobiotin or biocytin, two widely used stains. The tissue was then sectioned on a vibratome and processed with both streptavidin-nanogold (for electron microscopic study) and streptavidin-Cy3 (for confocal microscopic study) and embedded in epon/araldite. Interesting areas of the labeled neuron were imaged in the epon/araldite blocks with laser scanning confocal microscopy and then thin-sectioned at the indicated depth for electron microscopic study. This method provides an easy, reliable way to correlate three- dimensional light microscopic information with electron microscopic detail, and can be very useful in studies of synaptic connections.