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

Abstract

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.

Fig. 1. Schematic diagram of the top-illumination STM-TERS setup used to investigate nanoscale phase separation in supported lipid monolayers in this work.

Fig. 2. (a) STM topography image of d₆₂-DPPC : DOPC (1 : 1) monolayer supported on Au(111) measured simultaneously with the hyperspectral TERS image. Step size: 40 nm. The step edges of the Au(111) surface are highlighted with white arrows. (b) Averaged TERS spectrum and TERS images of the (c) C–D signal (2000–2200 cm⁻¹) and (d) C–H signal (2800–3200 cm⁻¹) measured in the d₆₂-DPPC : DOPC monolayer region shown in panel a. Step size: 40 nm. The averaged spectrum in panel b represents mean of the 2150 spectra measured in the TERS image. (e) Overlay of the TERS images of C–D and C–H signals shown in panels c and d, respectively. White arrows indicate the presence of nanoscale d₆₂-DPPC pockets inside DOPC-rich domains and vice versa.

Fig. 3. (a) t-SNE dimension plot of the first five PCs obtained from the hyperspectral TERS image of d₆₂-DPPC : DOPC monolayer presented in Fig. 2. (b) TERS spectra corresponding to the clusters 0 (purple), 1 (light green) and 2 (dark green) in panel a. (c) TERS intensity image of the clusters 0 (purple), 1 (light green) and 2 (dark green). (d) TERS image of the I₂₉₆₀/I₂₈₅₀ ratio.