Dual-color fluorescence cross-correlation spectroscopy with continuous laser excitation in a confocal setup
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Beitragende
Abstract
Fluorescence correlation spectroscopy evaluates local signal fluctuations arising from stochastic movements of fluorescent particles in solution. The measured fluctuating signal is correlated in time and analyzed with appropriate model functions containing the parameters that describe the underlying molecular behavior. The dual-color extension, fluorescence cross-correlation spectroscopy (FCCS) allows for a comparison between spectrally well-separated channels to extract codiffusion events that reflect interactions between differently labeled molecules. In addition to solution measurements, FCCS can be applied with subcellular resolution and is therefore a very promising approach for a quantitative biochemical assessment of molecular networks in living cells. To derive thermodynamic and kinetic reaction parameters, the influence of a number of other factors like background noise, illumination intensity profiles, photophysical processes, and cross talk between the channels have to be treated. Here, we provide a roadmap to derive binding reaction data with dual-color FCCS using continuous wave laser excitation, as it is now accessible with many state-of-the-art confocal microscopes.
Details
Originalsprache | Englisch |
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Titel | Fluorescence Fluctuation Spectroscopy (FFS), Part A |
Herausgeber (Verlag) | Elsevier Academic Press Inc |
Seiten | 43-70 |
Seitenumfang | 28 |
ISBN (Print) | 9780123884220 |
Publikationsstatus | Veröffentlicht - 2013 |
Peer-Review-Status | Ja |
Publikationsreihe
Reihe | Methods in Enzymology |
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Band | 518 |
ISSN | 0076-6879 |
Schlagworte
ASJC Scopus Sachgebiete
Schlagwörter
- Binding models, Confocal microscopy, Diffusion, Fluorescence correlation spectroscopy, Fluorescence cross-correlation spectroscopy, Fluorescence fluctuation analysis, Fluorescent dye, Fluorescent protein, Ligand-receptor interactions, Living cells, Protein-protein interactions, Stoichiometry