Characterization of sample preparation methods of NIH/3T3 fibroblasts for ToF-SIMS analysis

The information that is obtained from single cells during time-of-flight secondary ion mass spectrometry (ToF-SIMS) analysis is influenced by the method that was used to prepare the cells. The removal of extracellular media before analysis is necessary, but the rinsing technique should not damage the plasma membrane of the cell. The presence of intracellular salts reduced the secondary ion yield an average of 2.6-fold during Bi3+/C60++ depth profiles. Chemical fixation followed by rinsing removed a majority of the intracellular salts, “recovering” the positive secondary ion yields. The formaldehyde-fixation process removed a majority of the intracellular Cl-, but other key anions were not removed in significant amounts. The data presented here is consistent the anion neutralization mechanism largely responsible for the lower ion yields. All of the organic secondary ions that were detected in the freeze-dried cells were also detected in the formaldehyde-fixed cells, suggesting that the fixation process did not remove any molecular species to an extent that is detectable by ToF-SIMS. Compared to freeze dried cells, well preserved, frozen-hydrated cells showed little increase, or a decreased yield, for most low mass ions, but an increased yield for larger mass fragments. This is consistent with a reduced damage cross section at cryogenic analysis temperatures, although proton donation from water and reduction the salt effects in the presence of water likely also play roles. Numerous ions detected from the frozen-hydrated cells were not detected from the freeze dried cells, however many of these ions were attributed to chemical combinations of water, salts and the ammonium acetate rinsing solution. Supplementary Material Additional file 1: Figure S1: (A) Na+ peak and (B) 41 K+ peak from the depth profile of FH cells. The K+/Na+ ratio is 14.7, signifying the cells were well preserved. The 41 K+ isotope peak was used due to detector saturation of the 39 K+ isotope peak. [13758_2013_15_MOESM1_ESM.pdf (42 KB)] Additional file 2: Figure S2: (A) Depth profile from cells that were freeze-dried. (B) Depth profile from cells that were chemically fixed with formaldehyde. (C) Depth profile from cells that were analyzed frozen-hydrated. The depth profiles were normalized by the Bi3 + ion dose. [13758_2013_15_MOESM2_ESM.pdf (311 KB)] Additional file 3: Table S1: All positive secondary ions detected from both the FD and FF cells. Sorted by fold difference value, smallest to largest. [13758_2013_15_MOESM3_ESM.pdf (124 KB)] Additional file 4: Table S2: Positive Ions that were detected from the frozen-hydrated cells but not the freeze-dried cells, or in new very low amounts in the FD cells. [13758_2013_15_MOESM4_ESM.pdf (29 KB)] Additional file 5: Table S3: All positive secondary ions detected from both the FH cells and the FD cells. [13758_2013_15_MOESM5_ESM.pdf (52 KB)]