scholarly journals Spot Variation Fluorescence Correlation Spectroscopy for Analysis of Molecular Diffusion at the Plasma Membrane of Living Cells

10.3791/61823 ◽  
2020 ◽  
Author(s):  
Sebastien Mailfert ◽  
Karolina Wojtowicz ◽  
Sophie Brustlein ◽  
Ewa Blaszczak ◽  
Nicolas Bertaux ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Fluorescence Correlation Spectroscopy ◽  
Molecular Diffusion ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Fluorescence Correlation

ANALYSIS OF MEMBRANE-BINDING PROTEIN MOBILITY IN LIVING CELLS USING TOTAL INTERNAL REFLECTION FLUORESCENCE CORRELATION SPECTROSCOPY

2006 ◽  
Vol 01 (03) ◽  
pp. 293-299 ◽  
Author(s):  
Y. OHSUGI ◽  
MASATAKA KINJO
Keyword(s):  
Signal Transduction ◽  
Plasma Membrane ◽  
Fluorescence Correlation Spectroscopy ◽  
Total Internal Reflection ◽  
Membrane Binding ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Internal Reflection ◽  
Total Internal Reflection Fluorescence ◽  
Fluorescence Correlation

Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) is an appropriate method for measuring diffusion constants and the number of fluorescent molecules very close to the coverglass surface. Recently, we have reported the application of TIR-FCS to cell biology, measuring membrane-binding farnesylated green fluorescent proteins (EGFP-F) in living cells. In this research, we measured the signal transduction molecule, protein kinase C (PKC), fused with EGFP in living HeLa cells by using TIR-FCS. We observed two different diffusional mobilities of PKCβII-EGFP, three-dimensional faster diffusion near the plasma membrane and slower lateral diffusion on the plasma membrane after adinosine tri phosphate (ATP) activation. These results indicate that it is possible to use TIR-FCS in the study of molecular dynamics and interactions of signal transduction proteins on the plasma membrane of the living cell.

scholarly journals Advanced processing and analysis of conventional confocal microscopy generated scanning FCS data

2017 ◽  
Author(s):  
Dominic Waithe ◽  
Falk Schneider ◽  
Jakub Chojnacki ◽  
Mathias Clausen ◽  
Dilip Shrestha ◽  
...  
Keyword(s):  
Confocal Microscopy ◽  
Spatial Heterogeneity ◽  
Fluorescence Correlation Spectroscopy ◽  
Large Scale ◽  
Molecular Diffusion ◽  
Correlation Spectroscopy ◽  
The Other ◽  
Statistical Accuracy ◽  
Fluorescence Correlation ◽  
Diffusion Dynamics

AbstractScanning Fluorescence Correlation Spectroscopy (scanning FCS) is a variant of conventional point FCS that allows molecular diffusion at multiple locations to be measured simultaneously. It enables disclosure of potential spatial heterogeneity in molecular diffusion dynamics and also the acquisition of a large amount of FCS data at the same time, providing large statistical accuracy. Here, we optimize the processing and analysis of these large-scale acquired sets of FCS data. On one hand we present FoCuS-scan, scanning FCS software that provides an end-to-end solution for processing and analysing scanning data acquired on commercial turnkey confocal systems. On the other hand, we provide a thorough characterisation of large-scale scanning FCS data over its intended time-scales and applications and propose a unique solution for the bias and variance observed when studying slowly diffusing species. Our manuscript enables researchers to straightforwardly utilise scanning FCS as a powerful technique for measuring diffusion across a broad range of physiologically relevant length scales without specialised hardware or expensive software.

Mobility and distribution of replication protein A in living cells using fluorescence correlation spectroscopy

2007 ◽  
Vol 82 (2) ◽  
pp. 156-162 ◽  
Author(s):  
Christophe Braet ◽  
Holger Stephan ◽  
Ian M. Dobbie ◽  
Denisio M. Togashi ◽  
Alan G. Ryder ◽  
...  
Keyword(s):  
Fluorescence Correlation Spectroscopy ◽  
Protein A ◽  
Replication Protein A ◽  
Living Cells ◽  
Correlation Spectroscopy ◽  
Replication Protein ◽  
Fluorescence Correlation
Sign in / Sign up

Export Citation Format

Share Document