title: Mirror-enhanced super-resolution microscopy.
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.
authors.affiliation: Department of Biomedical Engineering, College of Engineering, Peking University, No. 5 Yiheyuan Road, Beijing 100871, China.
authors.forename: Xusan
authors.initials: X
authors.lastname: Yang
authors.affiliation: Department of Biomedical Engineering, College of Engineering, Peking University, No. 5 Yiheyuan Road, Beijing 100871, China; Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Xie
authors.affiliation: Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
authors.forename: Eric
authors.initials: E
authors.lastname: Alonas
authors.affiliation: Department of Biomedical Engineering, College of Engineering, Peking University, No. 5 Yiheyuan Road, Beijing 100871, China; Advanced Cytometry Labs, ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, Sydney, NSW 2109, Australia.
authors.forename: Yujia
authors.initials: Y
authors.lastname: Liu
authors.affiliation: Department of Biomedical Engineering, College of Engineering, Peking University, No. 5 Yiheyuan Road, Beijing 100871, China.
authors.forename: Xuanze
authors.initials: X
authors.lastname: Chen
authors.affiliation: Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
authors.forename: Philip J
authors.initials: PJ
authors.lastname: Santangelo
authors.affiliation: Department of Biomedical Engineering, College of Engineering, Peking University, No. 5 Yiheyuan Road, Beijing 100871, China.
authors.forename: Qiushi
authors.initials: Q
authors.lastname: Ren
authors.affiliation: Department of Biomedical Engineering, College of Engineering, Peking University, No. 5 Yiheyuan Road, Beijing 100871, China; Wallace H Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA; Advanced Cytometry Labs, ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, Sydney, NSW 2109, Australia; Institute for Biomedical Materials and Devices (IBMD), Faculty of Science, University of Technology Sydney, Sydney, NSW 2007, Australia.
authors.forename: Peng
authors.initials: P
authors.lastname: Xi
authors.affiliation: Advanced Cytometry Labs, ARC Centre of Excellence for Nanoscale BioPhotonics (CNBP), Macquarie University, Sydney, NSW 2109, Australia; Institute for Biomedical Materials and Devices (IBMD), Faculty of Science, University of Technology Sydney, Sydney, NSW 2007, Australia.
authors.forename: Dayong
authors.initials: D
authors.lastname: Jin
keywords: confocal; interference; point spread function; super-resolution