Quantification in bioimaging by LA-ICPMS - Evaluation of isotope dilution and standard addition enabled by micro-droplets
Andreas Schweikert, Sarah Theiner, Martin Šala, Petra Vician, Walter Berger, Bernhard K. Keppler, Gunda Koellensperger
Abstract
This study explores quantitative bioimaging as enabled by laser ablation-inductively coupled plasma time-of-flight mass spectrometry (LA-ICP-TOFMS), designing standardization methods based on robotic micro-droplet dispensing. The potential of producing controlled and highly precise pL-volume droplets was exploited to establish on-tissue isotope dilution and standard addition. Both strategies eliminate matrix effects and offer high metrological order traceable to SI units. The absolute quantity was obtained for μm-sized regions of interest in tissue samples, as defined by the extension of the deposited pL-volume droplet. While the gold standard isotope dilution (ID) was restricted to the accurate quantification of a single element, i.d. platinum in different tissue samples (mouse liver, spleen and tumor tissue), multiplexed matrix-matched calibration was obtained by on-tissue standard addition by depositing a dilution series of certified multi-element standards. Here, the working range was determined by the heterogeneity of the tissue samples and the background levels of elements intrinsically present and/or artificially introduced during sample preparation. Both methods, ID and standard addition served as reference methods for validation of external calibration using gelatin-based micro-droplet standards. Given full ablation, these external standards revealed a high dynamic range together with an excellent repeatability. Where applicable, the cross-validation revealed consistent quantitative results for the three quantification approaches. The comparable sensitivity obtained for standard addition and external standardization, respectively expressed as slope of the calibration function, provided proof that gelatin-based micro-droplets could serve as matrix-matched calibrations. Therefore, gelatin micro-droplets offer a valid tool for multiplexed matrix-mimicking standardization at high-throughput.