A Strategy for Quantitative Imaging of Lanthanide Tags in A549 Cells Using the Ratio of Internal Standard Elements
Billimoria, K., et. al.
Analytical Chemistry
DOI: https://doi.org/10.1021/acs.analchem.4c02763
This recent publication in Analytical Chemistry, led by researchers from the UK National Measurement Laboratory at LGC and the Bundesanstalt für Materialforschung und -prüfung (BAM) utilized the icpTOF 2R coupled to a Bioimage 266 nm laser ablation system from Elemental Scientific Lasers to introduce a novel strategy for intracellular imaging using lanthanide-tagged cells (Eu and Ho) with laser ablation inductively coupled time-of-flight mass spectrometry (LA-ICP-TOFMS). A major challenge in spatially resolved elemental quantification of biological samples is the lack of a suitable internal standard (IS). The authors utilized a layer containing Ga and In as IS elements. Normalizing the lanthanide signal to the IS ratio significantly improved calibration accuracy and reduced bias in quantifying Eu and Ho, improving consistency across standards and samples.
In vitro testing of 2D and 3D cell models is important for screening disease pathways and treatment efficacy before animal or human trials. However, bulk cell population measurements lack detailed insights into individual cell responses. Elemental mapping techniques like LA-ICP-MS are gaining traction for single-cell analysis, allowing for better understanding of intracellular drug uptake and treatment outcomes. Recent advancements in ICP-TOFMS have improved data resolution and speed, making it possible to map individual cell elements. However, LA-ICP-TOFMS imaging still faces challenges such as lack of standardization, calibration materials, and effective signal normalization. This paper introduces a novel strategy using lanthanide mapping and a gelatin layer with internal standards (Ga and In) for signal normalization in LA-ICP-TOFMS. This method addresses the variability in sample preparation and aims to improve the accuracy and reliability of intracellular element mapping. The approach is validated using a quality control sample containing europium (Eu) and holmium (Ho).
By adding a gelatin layer with known amounts of Ga and In as IS elements onto lanthanide-tagged cell samples (Eu and Ho) and gelatin calibration standards, signal normalization on a pixel-to-pixel basis is achieved. This enables accurate quantification of Eu and Ho at the intracellular level using LA-ICP-TOFMS. Normalizing the lanthanide signal to the ratio of internal standard IS elements, Ga and In, significantly improved calibration accuracy. Correlation coefficients for europium (Eu) and holmium (Ho) increased from 0.9885 to 0.9971 and from 0.9805 to 0.9980, respectively. Without IS normalization, the concentrations of Eu and Ho were overestimated by approximately 20% compared to expected values. This overestimation was also observed in the lanthanide concentration distribution of cell samples, highlighting the importance of IS normalization for accurate intracellular quantification. Normalizing the lanthanide signal to the IS ratio also improved recovery rates for Eu and Ho from 123 ± 5.30% to 93.7 ± 4.90% and from 125 ± 2.90% to 95.0 ± 4.02%, respectively.
The strategy outlined in this study offers a simple method for incorporating an IS for quantitative intracellular imaging of biological samples in combination with our cutting-edge icpTOF imaging technology. This method significantly enhances accuracy and reduces bias in multitagging techniques used for intracellular analysis, benefiting disease diagnosis and treatment of chronic illnesses such as cancer.
