Fluorescence correlation spectroscopy: molecular complexing in solution and in living cells.

Area of Science:

• Biophysics
• Molecular Biology
• Optical Microscopy

Background:

• Classical optics have resolution limits.
• Microscopes can measure fluorescence signals from confined sample volumes (confocal volume).
• Fluorescence Correlation Spectroscopy (FCS) extends microscopic capabilities.

Purpose of the Study:

• To describe the principles and applications of FCS.
• To demonstrate FCS for studying macromolecular interactions in solution and living cells.
• To examine critical experimental parameters, instrument requirements, and data analysis for FCS.

Main Methods:

• Measuring fluorescence signals from a confocal volume using a microscope.
• Quantitating molecular dynamics and complexing via FCS.
• Analyzing diffusion times, interactions, concentrations, and reaction kinetics.

Main Results:

• FCS enables studying single molecules, surpassing classical optical resolution limits.
• FCS can measure diffusion, macromolecular interactions, absolute concentrations, and reaction kinetics.
• Practical applications include ligand-receptor binding, protein interactions, and particle aggregation studies.

Conclusions:

• FCS is a powerful technique for studying molecular stoichiometry in vitro and in vivo.
• It is applicable to soluble and membrane receptor systems.
• While in vitro FCS is straightforward, in vivo applications require careful consideration of additional factors.