Hwang Wonsang, Kim Dongeun, Kim Dugyoung
Department of Physics, Yonsei University, 50 Yonsei-ro, Seodaemun-gu, Seoul, 120-749, South Korea.
Small. 2022 Feb;18(7):e2105497. doi: 10.1002/smll.202105497. Epub 2021 Dec 4.
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.
纳米切片光学显微镜已成为与膜相关的生物医学研究中不可或缺的工具。最后，已经引入了许多纳米切片成像方案，如可变角度全内反射荧光显微镜、金属诱导能量转移（MIET）成像和超临界角荧光显微镜。然而，这些方法只能测量单层分子，测量范围低于100纳米，不足以覆盖片状伪足的厚度。本文提出了一种光学成像方案，通过在荧光寿命成像显微镜中使用MIET、轴向焦平面扫描和双指数分析，能够在扩展的测量范围内以纳米级精度识别两层分子的轴向位置。通过测量已知结构的人工样品和厚度范围为100至450纳米、精度为18.3纳米的人主动脉内皮细胞片状伪足，证明了该方法的可行性。
