Work

Incorporation of optical profilometry volume correction in quantitative elemental bioimaging workflows

Year: 2025

Type: article

Abstract: Quantitative elemental bioimaging workflows are well established and rely upon the preparation of matched matrix standards, or other substrates such as gelatin, to provide comparable and repeatable instrument calibrations. However, variations of specimen tissue thicknesses, microtome cutting artefacts, biological cell density distributions and other factors are assumed by necessity to be of negligible significance to method uncertainties. Various approaches are routinely employed to mitigate these factors, including endogenous signal normalization with 12C or 31P, complete ablation of thin sections, or analyses of thicker specimens with stable laser fluences that ablate approximately the same amount of tissue per acquisition. Although these methods of measurement error mitigation may be effective for homogeneous specimens that consist of similar cell populations under constrained criteria, analyses of tissues with disparate anatomical structures, different cell populations and densities, or sourced from various suppliers with limited oversight of sectioning accuracy, introduce uncontrolled and unknown measurement anomalies. These anomalies may produce misleading results and/or erroneous interpretation of elemental changes in biological pathways, particularly for heterogeneous tissues where cell populations and elemental distributions respond to disease or external insult. Accordingly, we report the introduction of optical profilometry into quantitative elemental bioimaging workflows to map surface topographies of tissue sections for correction of specimen thickness variations. Topographic maps were constructed for gelatin standards and representative tissue sections with unform and variable cell populations, different thicknesses and roughness. These topographical measurements were registered against quantified elemental images and volume normalized. In all cases there were significant changes of absolute elemental concentrations upon volume correction, with similar relative trends of distributions in homogenous sections, and considerable deviations within heterogeneous tissues. We also assessed tissue thickness normalization with 12C, and cell density correlations with 31P.

Source: Talanta

DOI: 10.1016/j.talanta.2025.128923