Building and Using a Two-Photon Fluorescence Cross-Correlation Spectroscopy Setup Including Fluorescence Lifetime...

Area of Science:

• Biophysics
• Molecular Biochemistry
• Physical Chemistry

Background:

• Fluorescence Cross-Correlation Spectroscopy (FCCS) is a powerful technique bridging bulk and single-molecule analysis.
• Its applications span chemical kinetics, protein interactions, conformational changes, and intracellular transport.
• FCCS is adept at characterizing complex biological structures like lipid vesicles and protein complexes.

Purpose of the Study:

• To provide a detailed, step-by-step guide for setting up an FCCS instrument.
• To illustrate the versatility of FCCS, particularly when combined with fluorescence lifetime analysis.
• To enable researchers to apply FCCS for solving diverse biological problems.

Main Methods:

• Utilizing a two-photon excitation source for ease of use and maintenance.
• Integrating fluorescence lifetime analysis with FCCS.
• Detailed procedural instructions for instrument setup.

Main Results:

• Demonstrates the capability of FCCS to monitor subtle environmental changes (pH, salt concentration) using appropriate fluorophores.
• Highlights FCCS's utility in membrane docking and fusion assays.
• Provides a practical framework for implementing FCCS in biological research.

Conclusions:

• FCCS is a versatile and accessible tool for advanced biophysical studies.
• The described setup facilitates the investigation of dynamic biological processes at the molecular level.
• Combining FCCS with fluorescence lifetime analysis expands its problem-solving capacity in biochemistry.