Improving workflows of quantitative elemental bioimaging using optical profilometry

D.M. Bordin^1,2; T. Lockwood²; M. Westerhausen²; P. Doble¹

1Atomic Medicine Initiative (AMI) & 2HyMaS Laboratory, School of Mathematical and Physical Sciences, University of Technology Sydney

Dayanne.bordin@uts.edu.au

Introduction: Modern laser ablation–inductively coupled plasma-mass spectrometry (LA-ICP-MS) instruments have vastly improved acquisition times for quantitative two-dimensional elemental bioimaging of thin biological specimens. It is a mature technology with an ever-increasing application potential in medical, biological, clinical, forensic, and environmental sciences. However, precise and accurate quantification is a persistent challenge due to instrumental drift, matrix effects, and sample thickness variations of disparate cell populations and densities, or irregular and inconsistent cutting procedures. Typical analytical workflows remove all tissue from the sample substrate, usually glass microscope slides. Effective methods to overcome instrumental drift and matrix effects are well developed, however, specimen thickness variations and cutting artefacts are usually ignored or presumed non-significant contributors to method statistical uncertainties [1-3].

Method: We propose a novel approach to minimise contributions of statistical method uncertainties associated with calibration standards and specimen thickness variations and anomalies by introducing volume normalisation to the analytical workflow using optical profilometry. Ablated volumes per voxel were measured to normalise ablated mass differences by calculating the volume of standard removed during calibration post-ablation, and pre-ablation acquisition of detailed maps of sample thickness and surface topography. This approach was cross-validated by non-contact mode atomic force microscopy (NC-AFM) measurements [2-3].

Results: Our data demonstrated that the integration of ablation volume normalization improved the precision, accuracy, and reliability of quantification, particularly for heterogeneous tissue structures. Profilometry data highlighted discrepancies between the assumed tissue thickness (30 µm) and the actual average tissue thickness (15 µm), which is crucial for accurate quantification procedures. Despite variations in concentration, the elemental distribution in areas of interest remained consistent. However, normalisation proved critical in addressing concentration variations in thicker regions and improving the correlation between elemental concentrations and tissue characteristics. Additionally, while C12 and phosphorus were considered as potential internal standards or tissue thickness indicators, they showed weak or no meaningful correlation with tissue thickness or profiles. This integrated approach represents the first report to overcome the final challenge of absolute quantification workflows, advancing our understanding of elemental distributions in biological samples. Ultimately, this facilitates discoveries of hidden biochemical pathways where elements play a significant role, such as in cancer pathogenesis and regenerative medicine.

Figure 1. Elemental distribution profiles of a meningioma tissue sample analysed using both methods—with and without volume normalisation

References

[1] P.A. Doble, R. Gonzalez de Vega, D.P. Bishop, D.J. Hare, D. Clases; Chem. Rev., 2021, 121, 11769–11822.

[2] T.E. Lockwood, M.T. Westerhausen, P.A. Doble; Anal. Chem., 2021, 93, 10418–10423.

[3] M.T. Westerhausen, T.E. Lockwood, R. Gonzalez De Vega, A. Röhnelt, D.P. Bishop, N. Cole, P.A. Doble, D. Clases; Analyst, 2019, 144, 6881.