Nanoscale visualization of phase separation in binary supported lipid monolayer using tip-enhanced Raman spectroscopy

Supported lipid membranes are an important model system to study the phase separation behavior at the nanoscale. However, the conventional nanoanalytical tools often fail to provide reliable chemical characterization of the phase separated domains in a non-destructive and label-free manner. This study demonstrates the application of scanning tunneling microscopy-based tip-enhanced Raman spectroscopy (TERS) to study the nanoscale phase separation in supported d₆₂-DPPC : DOPC lipid monolayers. Hyperspectral TERS imaging successfully revealed a clear segregation of the d₆₂-DPPC-rich and DOPC-rich domains. Interestingly, nanoscale deposits of d₆₂-DPPC were observed inside the DOPC-rich domains and vice versa. High-resolution TERS imaging also revealed the presence of a 40-120 nm wide interfacial region between the d₆₂-DPPC-rich and DOPC-rich domains signifying a smooth transition rather than a sharp boundary between them. The novel insights obtained in this study demonstrate the effectiveness of TERS in studying binary lipid monolayers at the nanoscale.