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.
Bibliographical noteFunding Information:
This work was supported by The National Research Foundation of Korea (NRF) through the Basic Science Program (NRF-2017R1A2B4003950 and 2020R1A2C2011942), Korea Institute for Advancement of Technology (KIAT), and the Swiss Innovation Agency (Innosuisse) through S.Korea–Switzerland Joint Innovation Project (P0011925), the Nano Material Technology Development Program (No. 2016M3A7B6908929) of the National Research Foundation (NRF) funded by the Ministry of Science and ICT.
© 2021, The Author(s).
All Science Journal Classification (ASJC) codes
- Biochemistry, Genetics and Molecular Biology(all)
- Agricultural and Biological Sciences(all)
- Medicine (miscellaneous)