Measurement of neuronal surface area using high-voltage electron microscope tomography.

High-voltage electron microscopy (HVEM) and axial tomography were used to reconstruct the three-dimensional structure of dendrites of intracellularly stained neostriatal spiny neurons. Neurons were stained using iontophoretic injection of horseradish peroxidase from an intracellular micropipet. Thick sections were cut on a vibratome, reacted with diaminobenzidine, and embedded in plastic. High-voltage electron microscopy was used to obtain high-resolution images of neuronal processes in 2- to 3-micron plastic sections cut from those blocks. Series of high-voltage electron micrographs were taken at 2 degrees increments of specimen tilt over a range of at least +/- 60 degrees, and three-dimensional reconstructions were generated from the series using an R-weighted backprojection method. An interactive procedure was used to draw the dendritic profiles from slices through the reconstructed volume and to measure volumes and surface areas of the dendrites. Values obtained in this manner matched previous findings using reconstruction from serial thin sections. The HVEM-tomography method offers an alternative to the serial-thin-section method for the quantitative analysis of neuronal shape.