Freeze-etching and vapor matrix deposition for ToF-SIMS imaging of single cells.

Freeze-etching, the practice of removing excess surface water from a sample through sublimation into the vacuum of the analysis environment, has been extensively used in conjunction with electron microscopy. Here, we apply this technique to time-of-flight secondary-ion mass spectrometry (ToF-SIMS) imaging of cryogenically preserved single cells. By removing the excess water which condenses onto the sample in vacuo, a uniform surface is produced that is ideal for imaging by static SIMS. We demonstrate that the conditions employed to remove deposited water do not adversely affect cell morphology and do not redistribute molecules in the topmost surface layers. In addition, we found water can be controllably redeposited onto the sample at temperatures below -100 degrees C in vacuum. The redeposited water increases the ionization of characteristic fragments of biologically interesting molecules 2-fold without loss of spatial resolution. The utilization of freeze-etch methodology will increase the reliability of cryogenic sample preparations for SIMS analysis by providing greater control of the surface environment. Using these procedures, we have obtained high quality spectra with both atomic bombardment as well as C 60 (+) cluster ion bombardment.

High-resolution TOF-SIMS imaging of eukaryotic cells preserved in a trehalose matrix.

A novel, trehalose-glycerol matrix was utilized to generate high-resolution, TOF-SIMS images of macrophages and glial cells. Viable cells incubated in 50 mM trehalose, then lyophilized in a 50 mM trehalose, 10-15% (w/w) glycerol rinse, are preserved and chemically profiled. These experiments demonstrate the utility of the disaccharide matrix as an efficient, cost-effective alternative to cryogenics for SIMS and other ultrahigh-vacuum (UHV) analyses of biological species. Cellular processes on oligodendrocytes and astrocytes, 1-3 mum in width, were well resolved for cells in the trehalose-glycerol matrix. The viscous cell matrixes were fractured and analyzed at room temperature and maintained their three-dimensional integrity under UHV. Images have been generated with a Au primary ion source near the static limit of 10(12) ions/cm2. Though these nucleated cells do not remain viable after desiccation, TOF-SIMS imaging and subsequent rehydration reveals structural and morphological preservation. Eliminating the inherent obstacles associated with cryogenic analysis opens the door to greater utility of SIMS as a bioanalytical tool, such as lipid mapping of single cells in the nervous system.