Probing cell chemistry with time-of-flight secondary ion mass spectrometry: development and exploitation of instrumentation for studies of frozen-hydrated biological material.
Imaging static secondary ion mass spectrometry (SIMS) offers a powerful method of obtaining molecular information from biological systems with good spatial resolution. However, the technique needs further development to make it suitable for routine analysis of cells. We report here the development of a new freeze-facture device to facilitate the manipulation and analysis of biological cell material, with the cell chemistry preserved intact by rapid freezing. We illustrate performance characteristics with high-contrast images of freeze-fractured, frozen-hydrated liposomes with the drug clofazamine constrained within the lipid bilayer providing a marker to determine the fracture plane across the liposome structure. By monitoring and imaging clofazamine on the surface of yeast cells in the frozen-hydrated state, and demonstrating its absence within molecular information from a cell fractured to reveal the cell ultrastructure, we demonstrate that the molecule does not penetrate the cell wall.
D041221:Candida glabrata / Q000166:cytology*; D002473:Cell Wall / Q000378:metabolism; D002991:Clofazimine / Q000032:analysis; D005614:Freeze Fracturing; D005615:Freezing; D008081:Liposomes / Q000737:chemistry*; D018629:Spectrometry, Mass, Secondary Ion / Q000295:instrumentation* / Q000379:methods*
D008081:Liposomes; D002991:Clofazimine