Surface-dependent reorganization of Chlorella vulgaris cell wall components revealed by AFM and SRS microscopy

Characterization of microalgae cell wall properties using Atomic Force Microscopy (AFM) depends critically on the immobilization strategy employed. In this study, we investigated how various immobilization surfaces influence the biophysical properties of the <i>Chlorella vulgaris</i> cell wall as probed by AFM. Cells were immobilized on three distinct surfaces, each leveraging different types of interactions with cells: polyoctyl-(PO)-Chitosan-coated glass slides promoting hydrophobic interactions, Superfrost™ positively charged glass slides that interact hydrophobically and electrostatically with cells, and PDMS chambers that immobilize cells through mechanical confinement. AFM imaging, nanoindentation, and force spectroscopy revealed that the biophysical properties of the <i>Chlorella vulgaris</i> cell wall strongly depend on the immobilization strategy. At the same time, all other experimental conditions were kept identical. Cells attached to PO-Chitosan through hydrophobic interactions displayed high surface roughness, higher rigidity, hydrophobic behavior, and positive surface charges. In contrast, cells immobilized on positively charged but less hydrophobic Superfrost™ slides exhibited smoother surfaces, lower rigidity, reduced hydrophobicity, and no detectable positive charges. Cells confined in PDMS chambers exhibited the smoothest and softest cell walls, characterized by hydrophilic and uncharged surfaces. Complementary Stimulated Raman Scattering (SRS) microscopy experiments confirmed distinct lipid rearrangements associated with each immobilization method. Together, these results support the hypothesis formulated in this study that immobilization surfaces can cause the reorganization of cell wall components-polysaccharides, proteins, and lipids-thereby reshaping the physico-chemical and mechanical surface properties. This study thus demonstrates the great significance of immobilization strategies to ensure the reliability of nanoscale biophysical measurements in microalgae research.