Share Email Print
cover

Proceedings Paper

Combining PALM and SOFI for quantitative imaging of focal adhesions in living cells
Author(s): Hendrik Deschout; Tomas Lukes; Azat Sharipov; Lely Feletti; Theo Lasser; Aleksandra Radenovic
Format Member Price Non-Member Price
PDF $14.40 $18.00
cover GOOD NEWS! Your organization subscribes to the SPIE Digital Library. You may be able to download this paper for free. Check Access

Paper Abstract

Focal adhesions are complicated assemblies of hundreds of proteins that allow cells to sense their extracellular matrix and adhere to it. Although most focal adhesion proteins have been identified, their spatial organization in living cells remains challenging to observe. Photo-activated localization microscopy (PALM) is an interesting technique for this purpose, especially since it allows estimation of molecular parameters such as the number of fluorophores. However, focal adhesions are dynamic entities, requiring a temporal resolution below one minute, which is difficult to achieve with PALM. In order to address this problem, we merged PALM with super-resolution optical fluctuation imaging (SOFI) by applying both techniques to the same data. Since SOFI tolerates an overlap of single molecule images, it can improve the temporal resolution compared to PALM. Moreover, an adaptation called balanced SOFI (bSOFI) allows estimation of molecular parameters, such as the fluorophore density. We therefore performed simulations in order to assess PALM and SOFI for quantitative imaging of dynamic structures. We demonstrated the potential of our PALM–SOFI concept as a quantitative imaging framework by investigating moving focal adhesions in living cells.

Paper Details

Date Published: 21 February 2017
PDF: 7 pages
Proc. SPIE 10071, Single Molecule Spectroscopy and Superresolution Imaging X, 100710E (21 February 2017); doi: 10.1117/12.2252865
Show Author Affiliations
Hendrik Deschout, Ecole Polytechnique Fédérale de Lausanne (Switzerland)
Tomas Lukes, Ecole Polytechnique Fédérale de Lausanne (Switzerland)
Czech Technical Univ. in Prague (Czech Republic)
Azat Sharipov, Ecole Polytechnique Fédérale de Lausanne (Switzerland)
Lely Feletti, Ecole Polytechnique Fédérale de Lausanne (Switzerland)
Theo Lasser, Ecole Polytechnique Fédérale de Lausanne (Switzerland)
Aleksandra Radenovic, Ecole Polytechnique Fédérale de Lausanne (Switzerland)


Published in SPIE Proceedings Vol. 10071:
Single Molecule Spectroscopy and Superresolution Imaging X
Jörg Enderlein; Ingo Gregor; Zygmunt Karol Gryczynski; Rainer Erdmann; Felix Koberling, Editor(s)

© SPIE. Terms of Use
Back to Top