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  4. Combining PALM and SOFI for quantitative imaging of focal adhesions in living cells
 
conference paper

Combining PALM and SOFI for quantitative imaging of focal adhesions in living cells

Deschout, Hendrik  
•
Lukes, Tomas  
•
Sharipov, Azat  
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2017
Proc. of SPIE
SPIE Photonics West

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.

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Type
conference paper
DOI
10.1117/12.2252865
Web of Science ID

WOS:000406422600002

Author(s)
Deschout, Hendrik  
Lukes, Tomas  
Sharipov, Azat  
Feletti, Lely  
Lasser, Theo  
Radenovic, Aleksandra  
Date Issued

2017

Publisher

Spie-Int Soc Optical Engineering

Publisher place

Bellingham

Published in
Proc. of SPIE
ISBN of the book

978-1-5106-0583-1

978-1-5106-0584-8

Total of pages

7

Series title/Series vol.

Single Molecule Spectroscopy and Superresolution Imaging

Volume

100710E-1

Subjects

PALM

•

SOFI

•

focal adhesion

•

quantitative imaging

•

live cell imaging

•

super-resolution imaging

Editorial or Peer reviewed

REVIEWED

Written at

EPFL

EPFL units
LBEN  
LOB  
Event nameEvent placeEvent date
SPIE Photonics West

San Francisco

27 January-2 February 2017

Available on Infoscience
July 7, 2017
Use this identifier to reference this record
https://infoscience.epfl.ch/handle/20.500.14299/138831
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