SPRAY-FREEZING FREEZE-SUBSTITUTION (SFFS) OF CELL-SUSPENSIONS FOR IMPROVED PRESERVATION OF ULTRASTRUCTURE

Some unicellular organisms present challenges to chemical fixations th at lead to common, yet obvious, artifacts. These can be avoided in ent irety by adapting spray-freezing technology to ultrarapidly freeze spe cimens for freeze substitution. To freeze specimens, concentrated susp ensions of cells ranging in diameter from 0.5-30 mu m were sprayed wit h an airbrush at 140-200 kPa (1.05-1.5 torr; 20.3-29.0 psi) into a nyl on mesh transfer basket submerged in liquid propane. After freezing, t he mesh basket containing the frozen sample was lifted out of the cham ber, drained and transferred through several anhydrous acetone rinses at 188 K (-85 degrees C). Freeze substitution was conducted in 1% tann ic acid/1% anhydrous glutaraldehyde in acetone at 188 K (-85 degrees C ), followed by 1% OsO4/acetone at 277 K (4 degrees C). Freeze substitu tion was facilitated using a shaking table to provide gentle mixing of the substitution medium on dry ice. High quality freezing was observe d in 70% of spray-frozen dinoflagellate cells and in 95% of spray-froz en cyanobacterial cells. These could be infiltrated and observed direc tly; however, overall ultrastructural appearance and membrane contrast were improved when the freeze-substituted cells were rehydrated and p ost-fixed in aqueous OsO4, then dehydrated and embedded in either Spur r's; or Epon resin. Ultrastructural preservation using this ultrarapid freezing method provided specimens (C) 1997 Wiley-Liss, Inc.