Quantitative Approach for Simultaneous In Situ Profiling of Lignin, Cellulose, and Hemicellulose Using Confocal Raman...

Area of Science:

• Plant cell wall analysis
• Biomass composition
• Spectroscopic imaging

Background:

• Confocal Raman microscopy (CRM) offers high chemical specificity for in situ plant cell wall analysis.
• Simultaneous quantification of components in Gramineous species is challenging due to complex networks and spectral overlaps.
• Hemicellulose quantification is particularly difficult due to its amorphous nature and weak Raman signal.

Purpose of the Study:

• To develop a quantitative strategy combining CRM with cosine similarity (CRM-CS) for simultaneous analysis of Gramineous cell walls.
• To address the challenges of spectral overlap and weak signals in quantifying lignin, cellulose, and hemicellulose.
• To enable pixel-scale in situ quantification for applications in crop breeding and biomass utilization.

Main Methods:

• Acquisition of CRM mapping images of rice stems pretreated with acidified sodium chlorite (ASC).
• Calculation of cosine similarity (CS) values between preprocessed cell wall spectra and reference spectra (lignin, cellulose, xylan).
• Utilizing CS values as quantitative indicators for cell wall components.

Main Results:

• CS values showed significant positive correlations with lignin, cellulose, and hemicellulose content (R² > 0.90).
• The CRM-CS method accurately quantified components, outperforming conventional peak intensity approaches.
• Successfully resolved the difficulty in quantifying hemicellulose, achieving high determination coefficients.

Conclusions:

• The CRM-CS algorithm enables accurate, simultaneous in situ quantification of major components in Gramineous cell walls.
• This approach overcomes limitations of previous methods, particularly for hemicellulose analysis.
• Offers a valuable tool for plant cell wall research, crop breeding, and lignocellulosic biomass valorization.