Axial line-scanning stimulated emission depletion fluorescence correlation spectroscopy

2021 ◽  
Vol 46 (9) ◽  
pp. 2184
Author(s):  
Peng Gao ◽  
G. Ulrich Nienhaus
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Correlation Spectroscopy ◽  
Stimulated Emission ◽  
Axial Line ◽  
Fluorescence Correlation ◽  
Line Scanning ◽  
Stimulated Emission Depletion

scholarly journals Measuring ligand-cell surface receptor affinities with axial line-scanning fluorescence correlation spectroscopy

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Antonia Franziska Eckert ◽  
Peng Gao ◽  
Janine Wesslowski ◽  
Xianxian Wang ◽  
Jasmijn Rath ◽  
...  
Keyword(s):  
Cell Surface ◽  
Wnt Signaling ◽  
Fluorescence Correlation Spectroscopy ◽  
High Speed ◽  
Wnt Signaling Pathway ◽  
Correlation Spectroscopy ◽  
Cell Surface Receptor ◽  
Axial Line ◽  
Fluorescence Correlation ◽  
Line Scanning

Development and homeostasis of multicellular organisms is largely controlled by complex cell-cell signaling networks that rely on specific binding of secreted ligands to cell surface receptors. The Wnt signaling network, as an example, involves multiple ligands and receptors to elicit specific cellular responses. To understand the mechanisms of such a network, ligand-receptor interactions should be characterized quantitatively, ideally in live cells or tissues. Such measurements are possible using fluorescence microscopy yet challenging due to sample movement, low signal-to-background ratio and photobleaching. Here, we present a robust approach based on fluorescence correlation spectroscopy with ultra-high speed axial line scanning, yielding precise equilibrium dissociation coefficients of interactions in the Wnt signaling pathway. Using CRISPR/Cas9 editing to endogenously tag receptors with fluorescent proteins, we demonstrate that the method delivers precise results even with low, near-native amounts of receptors.

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