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Imaging Biological Samples with Optical Microscopy

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Optical microscopy uses optic principles to provide detailed images of samples. Antonie van Leeuwenhoek designed the first compound optical microscope in the 17th century to visualize blood cells, bacteria, and yeast cells. In 1830, Joseph Jackson Lister created an essentially modern light microscope. The 20th century saw the development of microscopes with enhanced magnification and resolution.
In optical microscopy, the specimen to be viewed is placed on a glass slide and clipped on the stage...

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Quantitative Optical Microscopy: Measurement of Cellular Biophysical Features with a Standard Optical Microscope

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Published on: April 7, 2014

Incorporation of optical profilometry volume correction in quantitative elemental bioimaging workflows.

Dayanne Mozaner Bordin¹, Thomas Lockwood¹, Mika Westerhausen¹

¹HyMaS Laboratory, School of Mathematical and Physical Sciences, University of Technology Sydney, Australia.

|October 8, 2025

Summary

Optical profilometry improves quantitative elemental bioimaging by correcting for tissue thickness variations. This volume correction enhances accuracy and interpretability, especially in complex, heterogeneous biological samples.

Keywords:

Accuracy Bioimaging LA-ICP-MS Optical profilometry Quantification

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Published on: January 22, 2017

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.