Quantifying multiple stain distributions in bioimaging by hyperspectral X-ray tomography.

Area of Science:

• Biomedical Imaging
• Materials Science
• Analytical Chemistry

Background:

• Chemical staining enhances soft tissue contrast but lacks precise elemental identification, especially for similar densities.
• Distinguishing multiple stains and their distribution is challenging with conventional imaging methods.

Purpose of the Study:

• To develop and validate a hyperspectral imaging routine for non-destructive identification and mapping of elemental composition in biological specimens.
• To enable direct measurement of chemical stain concentrations and analyze stain overlap and uptake.
• To advance understanding of multi-staining procedures and heavy metal uptake in various fields.

Main Methods:

• Utilized hyperspectral X-ray computed tomography (XCT) with high-resolution energy channels for simultaneous multi-element detection.
• Employed elemental calibration phantoms for direct stain concentration measurement using spectral absorption edge markers.
• Applied the routine to double- and triple-stained biological specimens to analyze stain interactions.

Main Results:

• Successfully distinguished multiple chemical agents based on their unique elemental signatures, irrespective of contrast similarity.
• Quantified stain concentrations and mapped their distribution within biological tissues.
• Analyzed the extent of stain overlap and specific uptake regions for common contrast agents.

Conclusions:

• Hyperspectral XCT provides a non-destructive method for unambiguous identification, segmentation, and elemental mapping of multiple stains in biological samples.
• The developed routine enables accurate measurement of stain concentrations and analysis of stain interactions.
• This technique offers significant potential for optimizing multi-staining protocols and investigating heavy metal uptake in diverse applications.