Mirror-enhanced super-resolution microscopy

• Type of Material: Article
• Authors: Xusan Yang, Peking University; Hao Xie, Peking University; Eric Alonas, Georgia Institute of Technology; Yujia Liu, Peking University; Xuanze Chen, Peking University; Philip Santangelo, Emory University; Qiushi Ren, Peking University; Peng Xi, Peking University; Dayong Jin, Macquarie University
• Language: English
• Date: 2016-06-17
• Publisher: Nature Publishing Group: Open Access Journals - Option C
• Publication Version: Final Published Version
• Copyright Statement: © 2016, Rights Managed by Nature Publishing Group
• License: Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.1038/lsa.2016.134
• Title of Journal or Parent Work: Light: Science and Applications
• ISSN: 2095-5545
• Volume: 5
• Start Page: 16134
• End Page: 16134
• Grant/Funding Information: Support for E Alonas was provided by the National Institute of Health (GM094198 to PJS).; This research is supported by the National Instrument Development Special Program (2013YQ03065102), the ‘973’ Major State Basic Research Development Program of China (2011CB809101), the Natural Science Foundation of China (31327901, 61475010, 61428501) and the Australian Research Council Centre of Excellence for Nanoscale BioPhotonics (CE140100003).
• Keywords: (IM)-M-5; NUCLEAR-PORE COMPLEX; Science & Technology; ARCHITECTURE; Optics; NM AXIAL RESOLUTION; super-resolution; LIGHT; Physical Sciences; STED NANOSCOPY; CELLS; FLUORESCENCE MICROSCOPY; SCALE RESOLUTION; confocal; OPTICAL NANOSCOPY; interference; point spread function
• Research Categories: Engineering, Biomedical; Health Sciences, General

Abstract

Axial excitation confinement beyond the diffraction limit is crucial to the development of next-generation, super-resolution microscopy. STimulated Emission Depletion (STED) nanoscopy offers lateral super-resolution using a donut-beam depletion, but its axial resolution is still over 500 nm. Total internal reflection fluorescence microscopy is widely used for single-molecule localization, but its ability to detect molecules is limited to within the evanescent field of ~ 100 nm from the cell attachment surface. We find here that the axial thickness of the point spread function (PSF) during confocal excitation can be easily improved to 110 nm by replacing the microscopy slide with a mirror. The interference of the local electromagnetic field confined the confocal PSF to a 110-nm spot axially, which enables axial super-resolution with all laser-scanning microscopes. Axial sectioning can be obtained with wavelength modulation or by controlling the spacer between the mirror and the specimen. With no additional complexity, the mirror-assisted excitation confinement enhanced the axial resolution six-fold and the lateral resolution two-fold for STED, which together achieved 19-nm resolution to resolve the inner rim of a nuclear pore complex and to discriminate the contents of 120 nm viral filaments. The ability to increase the lateral resolution and decrease the thickness of an axial section using mirror-enhanced STED without increasing the laser power is of great importance for imaging biological specimens, which cannot tolerate high laser power.