Paper BO+AS+BI-WeA12
Effects of Different Sample Preparation Methods for Cell Imaging using TOF-SIMS

Wednesday, October 22, 2008, 5:20 pm, Room 201

Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is increasingly being used for chemical imaging of cells and tissue. A concern in these studies is that the samples need to be prepared for the vacuum environment. Several sample preparation methods exist for this purpose. In this work, effects of different preparation methods on the structure and surface chemistry of human fibroblast hTert cells were studied. Two fixation protocols, using glutaraldehyde (GA, C₅H₈O₂), and osmium tetroxide (OsO₄), respectively, were compared to a non-fixing protocol where cells were washed with ammonium formate (AF, NH₄HCOO) prior to drying. Three drying techniques were compared, namely freeze-drying (FD) after rapid plunge-freezing, critical point-drying (CPD), and alcohol ladder-drying (ALD). Imaging TOF-SIMS with Bi₃ cluster primary ions was used to compare the different preparation protocols with respect to surface chemistry, and the structure of the cells after preparation was studied using scanning electron microscopy (SEM). For the AF-washed samples, changes in cell volume was followed by interference reflection microscopy (IRM). The results show that both the fixation/washing protocols as well as the drying protocols affect the chemical information obtained in TOF-SIMS analyses. For GA-fixed samples, both CPD and ALD give rise to reduced phosphocholine (PC) signal on the cell surface by two orders of magnitude, as compared to FD, while no significant differences are seen for cholesterol and amino acid fragment ions. GA-fixed samples post-fixed using OsO₄ showed PC intensities reduced by only one order of magnitude, going from FD to CPD or ALD. The cholesterol intensity was found to be higher for AF-washed cells and cells fixed with OsO₄, than for GA fixed cells. An increase in amino acid intensity going from AF to GA to OsO₄ was also observed.