Cerebellar mossy, parallel, basket and Golgi axons were used to study the variables contributing to vesicle shape.

It has been found that, when aldehydes are used as a primary fixative, synaptic vesicles can be either small round vesicles (less than 400 Å in diameter), large round vesicles (400–500 Å in diameter) or flat, with a ratio of large to small diameters of 2 or greater (500–600 Å × 250 Å). The proportion of round vesicles as well as the presence and the number of flat vesicles in each axon depend basically upon the osmotic pressure of the buffer. Concentration of aldehydes, duration of the fixation and dehydration, temperature of the fixative, etc. do not affect the synaptic vesicle shape. With buffers (phosphate and sodium cacodylate) of low osmolarity, mossy and parallel axons exhibit a round synaptic vesicle population and no flat vesicles are seen at all. With the same low osmolarity buffers, basket and Golgi axons have a synaptic vesicle population that is mainly round with a low proportion of flat vesicles. As the osmolarity of the buffers is increased, the number of flat vesicles in basket and Golgi axons increases. In mossy and parallel axons the flat vesicles are present with highest osmolarities but always in a lower proportion than in basket and Golgi axons.

Quantitative observations indicate that each axon type, under given conditions of fixation, exhibits a mixed vesicle population with a characteristic vesicle ratio. Although this vesicle ratio is modified significantly with changes in the osmotic pressure of the buffer, each axon type exhibits a different and characteristic modification of its vesicle ratio. These observations show that the vesicle ratio and its changes in different conditions of fixation are useful parameters for classifying axon.