Practical guidelines for dual-color fluorescence cross-correlation spectroscopy.

Area of Science:

• Biophysics
• Biochemistry
• Cell Biology

Background:

• Dual-color fluorescence cross-correlation spectroscopy (FCCS) is a powerful technique for analyzing molecular interactions.
• It enables the determination of molecular mobility, concentrations, and binding or cleavage events at very low concentrations.

Purpose of the Study:

• To provide a detailed protocol for preparing biological systems for FCCS experiments.
• To offer practical advice for performing FCCS on commercially available setups.
• To guide users in establishing successful FCCS measurements and controlling for artifacts.

Main Methods:

• Step-by-step instructions for performing FCCS experiments.
• Discussion of critical adjustments and test measurements for FCCS setup.
• Description of control experiments to identify and mitigate negative (detection volume overlap) and positive (cross-talk) artifacts.

Main Results:

• Successful application of FCCS in solutions, on membranes, and within cells.
• Analysis of dynamic colocalization during intracellular transport.
• Demonstration of FCCS's capability to accurately quantify molecular interactions.

Conclusions:

• FCCS is a valuable technique for quantitative analysis of molecular interactions and dynamics.
• The protocol addresses practical challenges and artifact control for reliable FCCS measurements.
• FCCS is expected to find broader applications in biochemical and cell biological research.