A RAPID METHOD FOR COMBINED LASER-SCANNING CONFOCAL MICROSCOPIC AND ELECTRON-MICROSCOPIC VISUALIZATION OF BIOCYTIN OR NEUROBIOTIN-LABELED NEURONS

Intracellular recording and dye filling are widely used to correlate t he morphology of a neuron with its physiology. With laser scanning con focal microscopy, the complex shapes of labeled neurons in three dimen sions can be reconstructed rapidly, but this requires fluorescent dyes . These dyes are neither permanent nor electron dense and therefore do not allow investigation by electron microscopy. Here we report a tech nique that quickly and easily converts a fluorescent label into a more stable and electron-dense stain. With this technique, a neuron is fil led with Neurobiotin or biocytin, reacted with fluorophore-conjugated avidin, and imaged by confocal microscopy. To permit long-term storage or EM study, the fluorescent label is then converted to a stable elec tron-dense material by a single-step conversion using a commercially a vailable ABC kit. We find that the method, which apparently relies on recognition of avidin's excess biotin binding sites by the biotin-pero xidase conjugate, is both faster and less labor intensive than photo-o xidation procedures in common use. The technique is readily adaptable to immunocytochemistry with biotinylated probes, as we demonstrate usi ng anti-serotonin as an example.