Preparation of cells cultured on silicon wafers for mass spectrometry analysis

Abstract

The distribution of specific atoms and molecules within living cells is of high interest in bio-medical research. Laser secondary neutral mass spectrometry (laser-SNMS) and time-of-flight secondary ion mass spectrometry (TOF-SIMS) detect atoms with high sensitivity and spatial resolution. The application of these methods to cultured cells requires special preparation techniques preserving morphological and chemical integrity of the living cells. The cells should, therefore, be grown on a conducting material preventing charging of the sample during ion bombardment. Silicon is currently used as the preferred support material for non-biological samples in mass spectrometry. This study investigates (1) the influence of silicon surfaces on cell growth and (2) the suitability of a sandwiched, rapid freezing method to analyse transmembrane ion gradients. Human melanoma cells were grown on silicon with polished or etched surfaces. Growth kinetics were studied using the Sulforhodamine-B assay. Number, shape, and morphology of the cells were assessed by epifluorescence microscopy of calcein AM- and DAPI-stained cells. Cells were subjected to rapid freezing, freeze-fracturing, and freeze-drying prior to analysis by TOF-SIMS and laser-SNMS. While cell numbers and morphology on the rough silicon wafers were impaired, morphology and growth kinetics of cells on polished silicon were identical to control cells on cell culture tested polystyrene. TOF-SIMS and laser-SNMS resulted in high-resolution elemental images and mass spectra. Measurement of the intracellular Na+ and K+ concentrations revealed a ratio as observed in living cells. In conclusion, culturing cells on polished silicon wafers followed by sandwiched, rapid freezing is an adequate preparation method to study intracellular ion distribution with mass spectrometry.

Localization of Fatty Acids with Selective Chain Length by Imaging Time‐of‐Flight Secondary Ion Mass Spectrometry

“…Two methods have been described so far. The first one is plunge-freezing and cryostat slicing of the tissue with following freezedrying (Sjovall et al, 2004;Touboul et al, 2004aTouboul et al, , 2005b and the second one is cryofixation followed by freeze-fracturing and the freeze-drying step (Borner et al, 2006b;Roddy et al, 2002;Wittig et al, 2005). Cryostat slices, as used for this work, have a sample surface that can be assumed as plane and topographical effects in the analysis can be excluded.…”

Laser postionization secondary neutral mass spectrometry in tissue: a powerful tool for elemental and molecular imaging in the development of targeted drugs

“…This allowed cooling rates of up to 10 4 K/s on the surface of the sample. Samples were prepared on conducting support material to prevent charging of the sample during ion bombardment (22). Small pieces of tissue (<1 mm 3 ) were placed on gold specimen carriers (Baltec) and shot into the liquid propane at high velocity.…”

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

“…1 Â 1 cm 2 (7 Â 4 mm 2 for freeze-fracturing) silicon wafers 41 for 7 days while the culture medium was regularly changed every two to three days. Prior to cell seeding, the silicon wafers were cleaned with demineralized water, acetone, and ethanol.…”

“…This procedure was repeated 5-9 times until no ethanol odor could be detected at the outlet. After that, temperature was increased with 3.2 K/min to 41 C to produce supercritical CO 2 which could slowly be led out and dried the samples. The procedure was performed two times; once with osmium stained samples and once with nonosmium stained samples to avoid cocontamination.…”