A novel calibration strategy for the quantitative imaging of iron in biological tissues by LA-ICP-MS using matrix-matched standards and internal standardisation

Abstract

The development of a novel and straightforward procedure for the preparation of matrix-matched calibration standards for the quantitative imaging of iron (Fe) in biological tissues by laser ablation (LA)-ICP-MS with on-tissue internal standard addition is described. This simple approach enabled on-tissue addition of Rh as internal standard to samples (with heterogeneous Fe distribution) and calibrants (with homogeneous Fe distribution). This is achieved without altering the original Fe distribution of the sample. Calibration standards were prepared by full horizontal immersion of slides with mounted homogenised sheep brain tissue section into the corresponding solution containing 0.5, 0.75, 1, 5, 10 and 20 mg kg⁻¹ Fe (each also containing 250 µg kg⁻¹ Rh as IS) in pure methanol for 30 minutes (6 immersions, each for 5 minutes). Subsequent air-drying (bench drying at room temperature) for approximately 5 minutes was undertaken in between consecutive immersions, to prevent long-term exposure of the tissue to lipid degradation. Tissue-matched standards were characterised in-house for Fe composition, homogeneity and stability (at storage temperatures of −80 °C, −20 °C, 4 °C and 25 °C for up to 2 months) in order to investigate their suitability as calibrants for quantitative LA-ICP-MS. The homogeneity data suggested that the materials are homogeneous in terms of Fe and Rh distribution with RSDs (n = 30) of 8.3% and 4.7%, respectively. The Fe measurement precision was improved by approximately a factor of 2 when normalising ⁵⁶Fe intensities to ¹⁰³Rh intensities; the RSD (n = 30) for ⁵⁶Fe/¹⁰³Rh was 3.6%. The produced calibration standards were found to be stable when stored at room temperature for approximately 50 days, suggesting that they can be reused for multiple batches. Using LA coupled to double-focusing sector field ICP-MS in medium resolution mode (m/Δm = 4000), linear calibration over a range of 107 to 1519 mg kg⁻¹ Fe (R² = 0.99) was achieved with a limit of detection of 1.84 mg kg⁻¹ Fe. Assessment of the accuracy of the method for the quantitative imaging of Fe in tissues was undertaken by comparison of the LA-ICP-MS data with that obtained by micro-XRF; the average Fe concentrations in selected tissue regions obtained by using XRF fell within the window defined by the LA-ICP-MS values and their associated standard deviations.