Department of Physics, Yonsei University, Seoul, Republic of Korea.
Department of Microbiology, Institute for Immunology and Immunological Diseases, College of Medicine, Yonsei University, Seoul, Republic of Korea.
Commun Biol. 2021 Jan 19;4(1):91. doi: 10.1038/s42003-020-01628-3.
Total internal reflection fluorescence (TIRF) microscopy, which has about 100-nm axial excitation depth, is the method of choice for nanometer-sectioning imaging for decades. Lately, several new imaging techniques, such as variable angle TIRF microscopy, supercritical-angle fluorescence microscopy, and metal-induced energy transfer imaging, have been proposed to enhance the axial resolution of TIRF. However, all of these methods use high numerical aperture (NA) objectives, and measured images inevitably have small field-of-views (FOVs). Small-FOV can be a serious limitation when multiple cells need to be observed. We propose large-FOV nanometer-sectioning microscopy, which breaks the complementary relations between the depth of focus and axial sectioning by using MIET. Large-FOV imaging is achieved with a low-magnification objective, while nanometer-sectioning is realized utilizing metal-induced energy transfer and biexponential fluorescence lifetime analysis. The feasibility of our proposed method was demonstrated by imaging nanometer-scale distances between the basal membrane of human aortic endothelial cells and a substrate.
全内反射荧光（TIRF）显微镜的轴向激发深度约为 100nm，几十年来一直是纳米切片成像的首选方法。最近，已经提出了几种新的成像技术，例如可变角度全内反射显微镜、超临界角荧光显微镜和金属诱导能量转移成像，以提高 TIRF 的轴向分辨率。然而，所有这些方法都使用高数值孔径（NA）物镜，并且测量的图像不可避免地具有较小的视场（FOV）。当需要观察多个细胞时，小 FOV 可能是一个严重的限制。我们提出了大 FOV 纳米切片显微镜，它通过使用 MIET 打破了聚焦深度和轴向切片之间的互补关系。大 FOV 成像使用低倍率物镜实现，而纳米切片则利用金属诱导能量转移和双指数荧光寿命分析来实现。通过对人主动脉内皮细胞基底膜与基底之间的纳米级距离进行成像，验证了我们提出的方法的可行性。
