Factors affecting internal standard selection for quantitative elemental bio-imaging of soft tissues by LA-ICP-MS

Element response variations under different laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) operating conditions were investigated to identify important factors for selecting an internal standard (IS) for quantitative elemental bio-imaging. Analytes covering a range of atomic masses and first ionisation potentials (FIP) were selected to investigate the signal response variation with changes in laser spot diameter, mass bias and cell sampling position. In all cases, an IS improved experimental precision regardless of a close match in element mass or FIP but optimal analyte/IS combinations depended on the difference in masses of the analyte and IS. Particular attention was paid to ¹³C as this isotope is typically used as an IS in elemental bio-imaging applications. Despite its non-ideal IS characteristics (often different mass and FIP to many analytes), possibility of abundance sensitivity effects and poor signal-to-background ratio, ¹³C was a suitable IS candidate exhibiting a linear response with respect to the mass ablated, apparent independence from the high abundance of the adjacent ¹⁴N mass peak and effective analyte normalisation after background subtraction as long as the ¹³C signal from the sample was at least 6% of the gross signal.