Toward precision in nanoscale IR spectroscopy: Optimizing biological sample preparation for molecular and morphological integrity.

Biological sample preparation for nanoscale IR spectroscopy involves fixation, rinsing, and drying steps that can significantly alter biomolecular properties and cellular architecture. Chemical modifications may shift spectral band positions and intensities, compromising interpretation, while structural remodelling can distort morphology and hinder image reconstruction. In this study, we evaluated fixation protocols using paraformaldehyde, glutaraldehyde, ethanol, and osmium tetroxide, combined with different rinsing and dehydration strategies. FT-IR and AFM-IR analyses revealed that paraformaldehyde and glutaraldehyde best preserve protein content and secondary structure, whereas osmium tetroxide is more effective for stabilizing cellular lipids. Nevertheless, the post-fixation steps of rinsing and drying proved equally critical for nanoscale investigations. We found that direct rinsing with water followed by rapid nitrogen drying yields the most faithful preservation of cellular integrity, minimizing structural artefacts and ensuring reliable spectral and AFM imaging data. These findings highlight the need for optimized processing protocols tailored to nanoscale IR studies. By balancing chemical stabilization with minimal physical distortion, the proposed approach improves the accuracy of nanospectroscopic analyses of cellular components.