LAUSR

Nanometer-sectioning optical microscopy has become an indispensable tool in membrane-related biomedical studies. Finally, many nanometer-sectioning imaging schemes, such as variable-angle total internal reflection fluorescence microscopy, metal-induced energy transfer (MIET) imaging, and supercritical-angle fluorescence microscopy have been introduced. However, these methods can measure a single layer of molecules, and the measurement ranges are below 100 nm, which is not large enough to cover the thickness of lamellipodium. This paper proposes an optical imaging scheme that can identify the axial locations of two layers of molecules with an extended measurement range and a nanometer-scale precision by using MIET, axial focal plane scanning, and biexponential analysis in fluorescence lifetime imaging microscopy. The feasibility of the proposed method is demonstrated by measuring an artificial sample of a known structure and the lamellipodium of a human aortic endothelial cell whose thickness ranges from 100 to 450 nm with 18.3 nm precision.