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

Abstract

The information that is obtained from single cells during 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.

Assessment of different sample preparation routes for mass spectrometric monitoring and imaging of lipids in bone cells via ToF-SIMS

“…Robinson and Castner compared different preparations and had also a focus on depth profiles. 18 In comparison to freeze-drying, it was suggested that formaldehyde fixation does not remove any molecular species to an extent detectable via ToF-SIMS. Analysis of frozenhydrated samples showed increased yields for larger mass fragments.…”

“…17,18,26,31,33 Usually, only some lipid fragments are considered 17,26,33 or the investigation was carried out only in the positive ion mode, 26,33 while important lipid fragments such as fatty acids can only be seen in the negative ion mode. 18 Published comparisons are not comprehensive, e.g., Robinson and Castner 18 only analyzed formalin fixed cells; fixation via glutaraldehyde and secondary fixation via osmium tetroxide was not considered, and parameters of the freeze-drying procedure remain unclear. In particular, fixation by osmium tetroxide should be tested, as it is a known procedure to stain and fix tissues for electron microscopy based on the fixation of unsaturated lipids 34,35 and it might prevent lipid extraction during sample preparation.…”

“…The sample was then submerged into liquid propane which was produced by leaking propane into a liquid nitrogen cooled Polytetrafluoroethylene beaker like described in Ref. 18 with a setup similar to that shown in Ref. 42.…”

“…14,15 Sample preparation is thus a crucial factor to maintain sample integrity, and it has to be well adapted to the analytes of interest. 14,16 A suitable preparation routine must preserve the native state concerning morphology, 17 chemical composition and distribution of the analytes, 14,17 remove excess salts from culture media 17,18 and be reproducible. 19 Systematic cell studies require that the preparation routine is applicable to large numbers of samples with high reproducibility in order to obtain reliable statistics.…”

ToF-SIMS of tissues: “Lessons learned” from mice and women

“…[12][13][14][15][16] Studies have been carried out in a variety of areas including the analysis of cancer cells, [17][18][19] cancer tissues, 20,21 muscular dystrophy, 22,23 and drug permeation into skin. 24 Several groups have discussed sample preparation methods for cells [25][26][27][28] and tissues. 14,29 However, the use of ToF-SIMS is still currently in early stages of development for answering biologically related questions.…”