Nanoscale topographic and biomechanical studies of the human internal limiting membrane

The purpose of this article was to create a nanometer scale topographic and biomechanical profile of the human internal limiting membrane (ILM) under native conditions.
METHODS: ILMs from the posterior pole of postmortem human eyes were prepared as flat mounts and investigated by atomic force microscopy (AFM) under physiological conditions. Structural analysis was complemented by transmission electron microscopy.
RESULTS: Average thickness of the fully hydrated, native ILMs was 3488 ± 460 nm. Thickness variations from 100 nm to 4326 nm characterized the fovea, which displayed a craterlike morphology. Outside the fovea, thickness distribution was uniform. Although mean ILM thicknesses were similar, standard deviation was higher on the retinal than on the vitreal side, indicating greater roughness. Average ILM stiffness was more than fivefold higher on the retinal than on the vitreal side (227 vs. 44 kPa).
CONCLUSIONS: A detailed topographical and nanomechanical profile of native human ILM was generated using AFM. Thickness values were significantly higher than in previous studies because of the preservation of native conditions. Both thickness and stiffness showed marked variations around the fovea but were relatively uniform outside the foveal area. Interestingly, the foveal ILM displayed a craterlike morphological appearance with four distinct layers separated by comparatively steep thickness increments. ILM stiffness was considerably higher on the retinal than on the vitreal side. AFM opens new possibilities for investigating native basement membranes under physiological and pathological conditions. Transmission electron microscopy revealed higher extracellular matrix protein density on the retinal than on the vitreal side.