Nanoscale Probing of Cholesterol-Rich Domains in Single Bilayer Dimyristoyl-Phosphocholine Membranes Using Near-Field Spectroscopic Imaging

Abstract

Cholesterol is believed to induce the formation of membrane domains, “rafts”, which are implicated in a range of natural and pathologic membrane processes. Therefore, it is important to understand the role that cholesterol plays in the formation of these structures. Here, we use label-free spectroscopic imaging to investigate cholesterol fractioning in supported bilayer membranes at nanoscale. Scattering-type scanning near-field optical microscopy (s-SNOM) was used to visualize the formation of cholesterol-induced domains in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes. Our results revealed the coexistence of phase separated domains in DMPC lipids with 10 mol % cholesterol content, whereas a mostly homogeneous bilayer was found at low (5 mol %) and high (15 mol %) cholesterol content. Near-field nano-FTIR spectroscopy was used to identify the cholesterol-rich domains based on their qualitative chemical compositions. It was determined that cholesterol binds to phosphodiester and alkyl glycerol ester moieties, likely via hydrogen bonding of the alcohol to either of the ester oxygens. The results also confirm the existence of an ideal cholesterol-lipid mixture ratio (∼15:85) with a geometrically defined packing. At lower cholesterol content there is phase separation between liquid ordered and almost neat DMPC domains. Thus, the liquid ordered phase exists at an energy minimum at a given lipid–cholesterol ratio.

Nanoscale Probing of the Supramolecular Assembly in a Two‐Component Gel by Near‐Field Infrared Spectroscopy

“…The interferometric recording of the scattered light provides infrared spectra at a spatial resolution approximately equivalent to that of the tip (20 nm) [21–28] . sSNOM has been used for the analysis of polymeric thin film surfaces, [24,29–35] imaging of cells, [36–40] viruses [41,42] and neurons [43] and the structural elucidation of protein fibrils [44–46] …”

“…The interferometric recording of the scattered light provides infrared spectra at a spatial resolution approximately equivalent to that of the tip (20 nm). [21][22][23][24][25][26][27][28] sSNOM has been used for the analysis of polymeric thin film surfaces, [24,[29][30][31][32][33][34][35] imaging of cells, [36][37][38][39][40] viruses [41,42] and neurons [43] and the structural elucidation of protein fibrils. [44][45][46] Although the secondary structure of protein fibrils has been successfully studied by sSNOM, in supramolecular gels the selfassembly of the gelator molecules (e.g.…”

Multi-feature round silicon membrane filters enable fractionation and analysis of small micro- and nanoplastics with Raman spectroscopy and nano-FTIR

“…Examples are the characterization of thin lms and blends, 22,23 single type and core-shell polymer nanoparticles, 21,24 multilayer structures, 25 phospholipid membranes and the distribution of cholesterol within them as well as viruses. 21,[26][27][28] Nano-FTIR spectra comply well with references from macroscopic IRtechniques and are thus suited for library-based identication. 20,23,27,29 Methodological and theoretical details of scattering-type scanning near eld optical microscopy (s-SNOM) and nano-FTIR are explained in ref.…”

Super-resolution mid-infrared spectro-microscopy of biological applications through tapping mode and peak force tapping mode atomic force microscope

“…By depositing the sample on the epitaxial graphene on a SiC substrate, the authors found that the near-field graphene plasmon map is a good indicator with enhanced image contrast at domain boundaries. In parallel, near-field spectroscopic visualization of cholesterol-rich domains at the bilayer of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) was conducted by s-SNOM [100]. Three nano domains with different cholesterol concentrations -phaseseparated cholesterol-rich domains, cholesterol-poor DMPC bilayer, and cholesterol-rich DMPC bilayer, were discovered, confirming the optimal mixture ratio of cholesterol/DMPC to be 15/85.…”