Incorporation of optical profilometry volume correction in quantitative elemental bioimaging workflows

Area of Science:

• Biomedical imaging
• Analytical chemistry
• Materials science

Background:

• Quantitative elemental bioimaging relies on matrix-matched standards for calibration.
• Variations in tissue thickness and surface topography are often assumed to have negligible effects on accuracy.
• Existing methods like endogenous signal normalization may fail in heterogeneous tissues, leading to misinterpretation.

Purpose of the Study:

• To incorporate optical profilometry into quantitative bioimaging workflows.
• To directly measure tissue surface topographies and correct for thickness variations.
• To improve the accuracy, reproducibility, and interpretability of elemental quantification in heterogeneous tissues.

Main Methods:

• Optical profilometry was used to acquire topographic maps of standards and diverse tissues (murine kidney, multi-organ arrays, human meningioma, emphysematous lung).
• Topographic data were registered with LA-ICP-MS elemental images for volume normalization.
• Elemental quantification was reassessed after volume correction.

Main Results:

• Significant deviations from nominal thicknesses and heterogeneous surface roughness were observed in all analyzed samples.
• Volume correction substantially altered elemental quantification, with increases of 4-10 fold for Cu, Fe, and Zn in kidney and meningioma.
• Correlations between endogenous signals (¹²C, ³¹P) and tissue thickness were tissue-dependent and often weak, highlighting limitations of normalization strategies.

Conclusions:

• Profilometry-based volume correction is crucial for accurate elemental quantification in bioimaging.
• This approach overcomes limitations of traditional methods in heterogeneous samples.
• The findings underscore the importance of accounting for 3D sample geometry in quantitative elemental bioimaging.