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.
Piehowski
Paul D
PD
Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802, USA.
Kurczy
Michael E
ME
Willingham
David
D
Parry
Shawn
S
Heien
Michael L
ML
Winograd
Nicholas
N
Ewing
Andrew G
AG
D000818:Animals; D002478:Cells, Cultured; D005613:Freeze Etching / Q000379:methods*; D051379:Mice; D018625:Microscopy, Atomic Force; D051381:Rats; D017207:Rats, Sprague-Dawley; D018629:Spectrometry, Mass, Secondary Ion / Q000379:methods*; D013499:Surface Properties; D014835:Volatilization; D014867:Water
D014867:Water