Nanoscale Characteristics of Ocular Lipid Thin Films Using Kelvin Probe Force Microscopy

Abstract

Purpose: To describe the use of Kelvin probe force microscopy (KPFM) to investigate the electrical surface potential of human meibum and to demonstrate successful use of this instrument on both human meibum and a meibum model system (six-lipid stock [6LS]) to elucidate nanoscale surface chemistry and self-assembly characteristics.

Materials and methods: 6LS and meibum were analyzed in this study. Mica-supported thin films were created using the Langmuir-Blodgett trough. Topography and electrical surface potential were quantified using simultaneous atomic force microscopy/KPFM imaging.

Results: Both lipid mixtures formed thin film patches on the surface of the mica substrate, with large aggregates resting atop. The 6LS had aggregate heights ranging from 41 to 153 nm. The range in surface potential was 33.0 to 125.9 mV. The meibum thin films at P = 5 mN/m had aggregates of 170 to 459 nm in height and surface potential ranging from 15.9 to 76.1 mV, while thin films at P = 10 mN/m showed an aggregate size range of 147 to 407 nm and a surface potential range of 11.5 to 255.1 mV.

Conclusions: This study showed imaging of the differences in electrical surface potential of meibum via KPFM and showed similarities in nanoscale topography. 6LS was also successfully analyzed, showing the capabilities of this method for use in both in vitro and ex vivo ocular research.

Translational relevance: This study describes the use of KPFM for the study of ocular surface lipids for the first time and outlines possibilities for future studies to be carried out using this concept.

Keywords: Kelvin probe force microscopy; atomic force microscopy; meibum/meibomian gland secretion; tear film.

Figure 1.

Illustrated depiction of sample preparation and analysis. Samples were prepared using the Langmuir-Blodgett trough (A) prior to analysis using AFM (B) and KPFM (C). AFM uses a physical probe to scan across the surface of a sample, giving topographical imaging with nanoscale resolution. KPFM is based on an AFM setup and uses a conducting AFM probe with a voltage applied to it, allowing for the mapping of local electrical surface potential in air. B and C altered from Drolle et al. This figure is intended as a simplified guide and is not meant as an exact representation of the complex lipid makeup, structures, or organizations.

Figure 2.

Representative AFM topography images of the 6LS at P = 10 mN/m, illustrating the lipid distribution in thin films deposited atop an atomically flat substrate. (A) Topography image at dimensions of 50 × 50 µm with cross section (B) marked across one surface feature. The area outlined is magnified to 30 × 30 µm (C) and further magnified to 12 × 12 µm (E). Cross sections of the highest magnification are shown in F. The most likely identity of the patches seen in the topography images is an amphiphilic lipid multilayer atop a continuous monolayer, with nonpolar lipid aggregates interacting with the nonpolar tail groups of the multilayer (D, intended as a simplified guide and is not meant as an exact representation of the complex lipid makeup, structures, or organizations).

Figure 3.

Simultaneous imaging of topography (gold) and distribution of differences in surface potential (blue) for 6LS thin films at P = 10 mN/m. Topographical features seen in A and C show corresponding differences in surface potential (B and D, respectively). All images taken were 30 × 30 µm.

Figure 4.

Representative AFM topography images of meibum at P = 5 mN/m, illustrating the lipid distribution in thin films deposited atop an atomically flat substrate. (A) Topography image at dimensions of 50 × 50 µm with cross section (B) marked across one aggregate on the surface. The area outlined is magnified to 25 × 25 µm (C) with a cross section of multilayers surrounding the aggregates illustrated via the cross section in (D), which is largely hidden due to the large nature of the aggregates.

Figure 5.

Simultaneous imaging of topography (gold) and distribution of differences in surface potential (blue) for meibum thin films at P = 5 mN/m. Topographical features seen in A and C show corresponding differences in surface potential (B and D, respectively). All images taken were 30 × 30 µm.

Figure 6.

Simultaneous imaging of topography (gold) and distribution of differences in surface potential (blue) for meibum thin films at P = 10 mN/m. Topographical features seen in A and C show corresponding differences in surface potential (B and D, respectively).