Large field-of-view nanometer-sectioning microscopy by using metal-induced energy transfer and biexponential lifetime analysis

Author:

Hwang Wonsang,Seo Jinwon,Kim DongEun,Lee Chang Jun,Choi In-HongORCID,Yoo Kyung-Hwa,Kim Dug YoungORCID

Abstract

AbstractTotal 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.

Funder

National Research Foundation of Korea

Korea Institute for Advancement of Technology

Publisher

Springer Science and Business Media LLC

Subject

General Agricultural and Biological Sciences,General Biochemistry, Genetics and Molecular Biology,Medicine (miscellaneous)

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