Spectral Unmixing for Label-Free, In-Liquid Characterization of Biomass Microstructure and Biopolymer Content by...

Area of Science:

• Plant biology
• Biomass science
• Microscopy and imaging

Background:

• Lignocellulosic biomass microstructure analysis is crucial for understanding plant development and biomass recalcitrance.
• Current imaging methods lack chemical specificity, physiological compatibility, or introduce artifacts, hindering in situ studies.

Purpose of the Study:

• To develop a multi-modal, chemically specific imaging technique for label-free characterization of plant microstructure in liquid.
• To address challenges in spectral unmixing caused by sample drift in liquid environments.
• To enable detailed in situ analysis of plant tissue development and biomass properties.

Main Methods:

• Coherent anti-Stokes Raman scattering (CARS) microspectroscopy combined with simplex maximization and entropy-based spectral unmixing.
• Utilized forward-scattered CARS and epi-detected autofluorescence to mitigate spatial drift artifacts.
• Applied the technique to untreated and pretreated rapeseed straw (Brassica napus) for 3D imaging.

Main Results:

• Achieved label-free, chemically specific imaging of plant microstructure in liquid.
• Successfully mitigated artifacts from sample drift using a novel multi-modal approach.
• Demonstrated 3D imaging capabilities with chemical specificity.
• Resolved intricate details like primary/secondary cell walls, cell lumina contents, and pit structures.

Conclusions:

• The developed CARS-based technique offers a powerful tool for in situ, chemically specific characterization of lignocellulosic biomass.
• This method advances the study of plant tissue development, biomass recalcitrance, and pretreatment mechanisms.
• The technique provides unprecedented detail for understanding plant cell wall architecture and composition.