Spray-freezing freeze substitution (SFFS) of cell suspensions for improved preservation of ultrastructure

Some unicellular organisms present challenges to chemical fixations that lead to common, yet obvious, artifacts. These can be avoided in entirety by adapting spray-freezing technology to ultrarapidly freeze specimens for freeze substitution. To freeze specimens, concentrated suspensions of cells ranging in diameter from 0.5-30 pm were sprayed with an airbrush at 140-200 kPa (1.05-1.5 torr; 20.3-29.0 psi) into a nylon mesh transfer basket submerged in liquid propane. After freezing, the mesh basket containing the frozen sample was lifted out of the chamber, drained and transferred through several anhydrous acetone rinses at 188 K (-85 degrees C). Freeze substitution was conducted in 1% tannic acid/1% anhydrous glutaraldehyde in acetone at 188 K (-85 degrees C), followed by 1% OsO4/acetone at 277 K (4 degrees C). Freeze substitution was facilitated using a shaking table to provide gentle mixing of the substitution medium on dry ice. High quality freezing was observed in 70% of spray-frozen dinoflagellate cells and in 95% of spray-frozen cyanobacterial cells. These could be infiltrated and observed directly; however, overall ultrastructural appearance and membrane contrast were improved when the freeze-substituted cells were rehydrated and post-fixed in aqueous OSO4, then dehydrated and embedded in either Spurr's or Epon resin. Ultrastructural preservation using this ultrarapid freezing method provided specimens that were consistently superior to those obtainable in even the best comparable chemical fixations.