A new method for scanning and transmission electron microscopy of synaptic vesicles isolated from the cerebral cortex.

Abstract

Spheroid and flattened synaptic vesicles were isolated from the brain homogenate of guinea pigs by a modified purification method. For scanning and transmission electron microscopy, a simple dipping method of preparation was developed and used. The purest and richest fraction of synaptic vesicles was obtained from a 0.1 M sucrose fraction of density gradients. The pellets of synaptic vesicles were easily resuspended without aggregate after ultracentrifugation at 40,000 rpm for 5 min. The isolated synaptic vesicles were dispersed as a monolayer on the surface of a copper grid covered with Formvar membrane. Adequate contrast was obtained by metal impregnation of specimens and gold coating at magnifications as high as 100,000 times using an acceleration voltage of 25 to 40 kV. The specimens were fixed in 0.75% glutaraldehyde (0.1 M phosphate buffer, pH 7.3) and then postfixed in 1% osmium tetroxide. After dipping for 1 to 2 min each in tannic acid, phosphotungstic acid, lead citrate and uranyl acetate, they were dehydrated with graded ethanol and coated with gold by ion sputtering at 400 to 560 volts for 4 min. The preparation method is reported on and technical problems are discussed.