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  Quantification of membrane receptor complexes with single-molecule localization microscopy

Böger, C., Baldering, T. N., Krüger, C. L., Harwardt, M.-L.-I.-E., Mertinkus, K. R., Schröder, M. S., et al. (2019). Quantification of membrane receptor complexes with single-molecule localization microscopy. Proceedings of SPIE, 10884: 108840T. doi:10.1117/12.2522348.

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 Creators:
Böger, Carolin1, Author
Baldering, Tim N.1, Author
Krüger, Carmen L.1, Author
Harwardt, Marie-Lena I. E.1, Author
Mertinkus, Klara R. 1, Author
Schröder, Mark S.1, Author
Karathanasis, Christos1, Author
Deußner-Helfmann, Nina S.1, Author
Barth, Hans-Dieter1, Author
Malkusch, Sebastian1, Author
Dietz, Marina S.1, Author
Hummer, Gerhard2, Author                 
Wohland, Thorsten3, Author
Heilemann, Mike1, Author
Affiliations:
1Institute of Physical and Theoretical Chemistry, Goethe-Universtiy Frankfurt, Frankfurt, Germany, ou_persistent22              
2Department of Theoretical Biophysics, Max Planck Institute of Biophysics, Max Planck Society, ou_2068292              
3Departments of Biological Sciences and Chemistry, Center for Bioimaging Sciences, National University of Singapore, Singapore, ou_persistent22              

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Free keywords: Single-molecule localization microscopy; SMLM; DNA-PAINT; molecular quantification; receptor tyrosine kinases
 Abstract: Knowledge of assembly, subunit architecture and dynamics of membrane proteins in a cellular context is essential to infer their biological function. Optical super-resolution techniques provide the necessary spatial resolution to study these properties of membrane protein complexes in the context of their cellular environment. Single-molecule localization microscopy (SMLM) is particularly well suited, as next to high-resolution images, it provides quantitative information on the detection of single emitters. A challenge for current super-resolution methods is to resolve individual protein subunits within a densely packed protein cluster. For this purpose, we developed quantitative SMLM (qSMLM), which reports on molecular numbers by analyzing the kinetics of single emitter blinking. Next to theoretical models for various photophysical schemes, we demonstrate this method for a selection of fluorescent proteins and synthetic dyes and a selection of membrane proteins. We next applied this tool to toll-like receptor 4 (TLR4), and found a ligand-specific formation of monomeric or dimeric receptors. Next to fluorescent proteins, DNA-PAINT offers a novel and flexible approach for quantitative super-resolution microscopy. We demonstrate DNA-PAINT imaging of structurally defined DNA origami structures and robust quantification of target sites, as well as of membrane receptors. Molecular quantification, together with experiments following single receptor mobilities in live cells, will enlighten molecular mechanisms of receptor activation.

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Language(s): eng - English
 Dates: 2019
 Publication Status: Published in print
 Pages: 9
 Publishing info: -
 Table of Contents: -
 Rev. Type: Peer
 Identifiers: DOI: 10.1117/12.2522348
 Degree: -

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Title: Proceedings of SPIE
Source Genre: Journal
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Publ. Info: Bellingham, Washington : SPIE
Pages: - Volume / Issue: 10884 Sequence Number: 108840T Start / End Page: - Identifier: ISSN: 0277-786X
CoNE: https://pure.mpg.de/cone/journals/resource/0277-786X