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Axial super resolution topography of focal adhesion by confocal microscopy
Author(s) -
Chiu ChiLi,
Gratton Enrico
Publication year - 2013
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/jemt.22267
Subject(s) - paxillin , optics , cardinal point , focal adhesion , confocal , resolution (logic) , phasor , confocal microscopy , microscopy , diffraction , microscope , super resolution microscopy , materials science , two photon excitation microscopy , image resolution , limiting , adhesion , physics , chemistry , scanning confocal electron microscopy , computer science , fluorescence , cell adhesion , artificial intelligence , cell , engineering , biochemistry , power (physics) , electric power system , quantum mechanics , mechanical engineering , composite material
The protein organization within focal adhesions has been studied by state‐of‐the‐art super resolution methods because of its thin structure, well below diffraction limit. However, to achieve high axial resolution, most of the current approaches rely on either sophisticated optics or diligent sample preparation, limiting their application. In this report we present a phasor‐based method that can be applied to fluorescent samples to determine the precise axial position of proteins using a conventional confocal microscope. We demonstrate that with about 4,000 photon counts collected along a z‐scan, axial localization precision close to 10 nm is achievable. We show that, with within 10 nm, the axial location of paxillin, FAK, and talin is similar at focal adhesion sites, while F‐actin shows a sharp increase in height towards the cell center. We further demonstrated the live imaging capability of this method. With the advantage of simple data acquisition and no special instrument requirement, this approach could have wide dissemination and application potentials. Microsc. Res. Tech., 76:1070–1078, 2013 . © 2013 Wiley Periodicals, Inc.