Evaluation of matrix effects in ion microscopic analysis of freeze‐fractured, freeze‐dried cultured cells

Abstract

SUMMARY SIMS matrix effects (mass interferences, sputter yield variations and practical ion yield variations) were evaluated in freeze‐fractured, freeze‐dried cultured cells at the ∼0.5 μm spatial resolution of the Cameca IMS‐3f ion microscope. Cell lines studied include normal rat kidney (NRK), 3T3 mouse fibroblast, L6 rat myoblast, chinese hamster ovary (CHO) and rat kangaroo kidney (PtK2) cells. High mass resolution studies indicated that the secondary ion signals of H—, C—, O—, Na+, Mg+, CN—, P—, S—, Cl—, K+ and Ca+ were free from major mass interferences. However, a large mass interference was observed for nitrogen at mass 14. No significant sputtering yield difference between the nuclear and cytoplasmic compartments of the cells studied was observed. The subcellular distributions of the major (H, C, N and O) and minor (P, S, K, Cl, Na, Mg and Ca) matrix elements were found to be largely homogeneous with the exception of Ca, which was observed mainly in the cell cytoplasm. Practical ion yield variations were compared by three different approaches: (i) by the use of cells doped with known electrolyte concentrations, (ii) by quantitative ion implantation, and (iii) by analysis of the same cell with both electron probe and ion microscope. Each approach indicated an absence of significant practical ion yield differences between the nuclear and cytoplasmic regions of these specimens. These observations indicate that secondary ion signals in this type of sample are not significantly affected by local matrix effect variations. Hence, qualitative imaging of such specimens provides a true representation of subcellular elemental distribtions. These observations should allow the development of quantitative ion imaging methodologies and enhance the applicability of ion microscopy to biomedical problems.

SIMS ion microscopy imaging of boronophenylalanine (BPA) and 13C15N-labeled phenylalanine in human glioblastoma cells: Relevance of subcellular scale observations to BPA-mediated boron neutron capture therapy of cancer

“…In the negative secondary detection mode, images of 12 C, 28 CN and 81 Br (or 79 Br) were recorded. In fractured freeze-dried cells, SIMS matrix effects (such as the practical ion yield variations and differential sputtering between the nucleus and the cytoplasm of individual cells) were not significant, and the secondary ion signals studied here did not reveal any significant mass interferences from polyatomic or hydrocarbon species [32]. Furthermore, analyses of fractured freeze-dried T98G cells not treated with BPA did not show detectable 10 B signals.…”

Quantitative imaging of free and total intracellular calcium in cultured cells.

“…The reliability of this methodology has been confirmed by successfully imaging Na+/K' transport after inhibition of the sodium pump with ouabain in several cell lines (19). The potential ion microscopic artifacts of the 39KH+ mass interference with 'Ca', differential sputtering between the cell nucleus and cytoplasm, and local matrix effect variations were found to be insignificant in 3T3 cells and several other cell lines (14). In addition, sandwichfractured freeze-dried cells examined by scanning electron microscopy revealed structural preservation far beyond the spatial resolution ofthe ion microscope (20).…”

Reconstructing accurate ToF-SIMS depth profiles for organic materials with differential sputter rates

“…Different sputter rates may also be expected in biological materials. For sample, mammalian cells have been broadly seen to sputter at the same rate across membrane, cytoplasm and nucleus 30,38,39 , while plant tissues have previously been shown to exhibit different sputter rates between cellular components 40 . Future studies investigating the interface of cells and polymeric scaffolds used for tissue engineering will certainly require consideration of multiple sputter rates.…”