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

Abstract

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 μm 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°C). Freeze substitution was conducted in 1% tannic acid/1% anhydrous glutaraldehyde in acetone at 188 K (−85°C), followed by 1% OsO4/acetone at 277 K (4°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. Microsc. Res. Tech. 38:315–328, 1997. © 1997 Wiley‐Liss, Inc.

Use of luminometry and flow cytometry for evaluating the effects of cryoprotectants in the gorgonian coral endosymbiont Symbiodinium

“…Toxicity of Gly was species‐dependent as it was more harmful than DMSO for cryopreservation of Euglena gracilis but could be used at low concentration for the cryopreservation of Nannochloropsis oculata (Poncet and Veron ). Fields et al () found that Gly could cause the loss of membrane integrity in unicellular organisms. MeOH has shown variable success for cryopreservation of marine algae.…”

Phycobiliprotein genes of the marine photosynthetic prokaryote Prochlorococcus: evidence for rapid evolution of genetic heterogeneity

“…In SS120, the PE genes are part of a larger cluster, within which other phycobiliprotein-related genes (cpeZ, cpeY, mpeX, Phycobiliprotein genes of Prochlorococcus ppeC) can be found . But there is no evidence that PE forms part of a cyanobacterial-like phycobilisome structure in Prochlorococcus (Chisholm et al, 1988 ;Fields et al, 1997 ;C. S. Ting and others, unpublished).…”

Silica nanoparticles aid in structural leaf coloration in the Malaysian tropical rainforest understorey herb Mapania caudata

“…For more precise measurements of cell wall structures we used a high-pressure freezing technique with leaf discs (2 mm in diameter) punched from blue -green iridescent leaves. Frozen leaf discs were then incubated with or without 1 % OsO 4 in distilled acetone for 20 days at -90 8C and processed according to techniques described in Fields et al (1997). Samples were slowly warmed to -25 8C, transferred to 4 8C and then to room temperature, rinsed in distilled acetone and rehydrated to 100 % water in a graded series.…”