Quantification of membrane receptor complexes with single-molecule localization microscopy

Carolin Böger; Tim N. Baldering; Carmen L. Krüger; Marie-Lena I. E. Harwardt; Klara R. Mertinkus; Mark S. Schröder; Christos Karathanasis; Nina S. Deussner-Helfmann; Hans-Dieter Barth; Sebastian Malkusch; Marina S. Dietz; Gerhard Hummer; Thorsten Wohland; Mike Heilemann

doi:10.1117/12.2522348

22 February 2019 Quantification of membrane receptor complexes with single-molecule localization microscopy

Carolin Böger, Tim N. Baldering, Carmen L. Krüger, Marie-Lena I. E. Harwardt, Klara R. Mertinkus, Mark S. Schröder, Christos Karathanasis, Nina S. Deussner-Helfmann, Hans-Dieter Barth, Sebastian Malkusch, Marina S. Dietz, Gerhard Hummer, Thorsten Wohland, Mike Heilemann

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Proceedings Volume 10884, Single Molecule Spectroscopy and Superresolution Imaging XII; 108840T (2019) https://doi.org/10.1117/12.2522348
Event: SPIE BiOS, 2019, San Francisco, California, United States

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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Carolin Böger, Tim N. Baldering, Carmen L. Krüger, Marie-Lena I. E. Harwardt, Klara R. Mertinkus, Mark S. Schröder, Christos Karathanasis, Nina S. Deussner-Helfmann, Hans-Dieter Barth, Sebastian Malkusch, Marina S. Dietz, Gerhard Hummer, Thorsten Wohland, and Mike Heilemann "Quantification of membrane receptor complexes with single-molecule localization microscopy", Proc. SPIE 10884, Single Molecule Spectroscopy and Superresolution Imaging XII, 108840T (22 February 2019); https://doi.org/10.1117/12.2522348

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KEYWORDS

Receptors

Proteins

Fluorescent proteins

Microscopy

Imaging systems

Calibration

Luminescence

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