Correlation Techniques

Fluorescence Correlation Spectroscopy: Illuminating the Secrets of Molecular Interactions

Table of Contents:

• What does FCS/FCCS mean?
• What is FCS Used For?
• FCS and FCCS in Cells
• Comparison with FRET Techniques
• Instrumental Requirements
• Autocorrelation and Cross Correlation Functions

What does FCS/FCCS mean?

Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Cross Correlation Spectroscopy (FCCS) are used in biophysics and biochemistry to study the dynamics and interactions of fluorescently labelled molecules at the single molecule level. The techniques detect fluorescence from a small number of molecules in an extremely small observation volume. The results contain information on the diffusion speed and thus the size of the molecules, on the number and the intrinsic brightness of molecules in the observation volume,  and the viscosity of the solvent on the molecular level. By studying the fluorescence of individual molecules, researchers can explore their dynamics, conformational changes, and interactions with other molecules. This has profound implications for understanding fundamental biological processes such as enzyme kinetics, protein interactions, and protein folding.

What is FCS Used For?

The primary use is in the study of protein behaviour, protein interactions, and interactions of proteins with other molecules. FCS tells the user how large the protein is, whether it is folded or unfolded, or whether it is chemically connected to another large biomolecule. FCCS between different labelled proteins tells whether the proteins are chemically connected or not. This way, FCS enables researchers to understand the binding kinetics, stoechiometry, and affinities between different protein partners. This has profound implications for understanding fundamental biological processes such as enzyme kinetics, protein-protein interactions, DNA folding, and protein folding. An important application is drug discovery: FCS of a labelled molecule with potential therapeutic activity tells whether it has attached to a protein, FCCS of the molecule with a labelled target protein tells whether the drug molecule has attached specifically to this protein.

FCS and FCCS in Cells

FCS and FCCS can be performed in live cells. The usual procedure is that first a fluorescence or fluorescence lifetime image of the cell is recorded. Then the laser beam is placed in a spot of interest within the cell, and the FCS or FCC measurement is performed. Measurements in cells are much more difficult than measurements in solution. The concentration of labelled proteins is hard to control, and usually is much higher than optimal for FCS. Moreover, the environment in a cell is not clean in terms of background fluorescence. This interferes with the fluorescence of labelled proteins and forms an undesirable background. Finally, the excitation power in a cell is very limited, which substantially enhances to requirements to the optics and the detection system.

Comparison with FRET Techniques

It should be noted that protein interactions can also be investigated by FRET techniques. In contrast to FCS, FRET measurement can easily (and usually is) combined with imaging. FRET measurements also do not require extremely low fluorophore concentration. The photon rates are therefore much higher. A FRET image of an entire cell can be obtained within the same acquisition time as an FCS measurement in a single point. When an experiment is planned, it should therefore be decided whether it requires the single-molecule capabilities of FCS or more the imaging capabilities of FRET recording.

Instrumental Requirements

The measurement volume in FCS refers to the small three-dimensional region where the fluorescence emission is observed. It is determined by factors such as the laser focus, the optical properties of the sample, and the numerical aperture of the microscope objective. The size of the measurement volume affects the sensitivity and resolution of the FCS measurement.

The autocorrelation function is a mathematical representation of the temporal fluctuations in the fluorescence intensity. It is generated by correlating the fluorescence signal at different time intervals. The autocorrelation function provides information about the diffusion properties, concentration, and interactions of the fluorescent molecules within the measurement volume.

Autocorrelation and Cross Correlation Functions

The autocorrelation function is a mathematical representation of the temporal fluctuations in the fluorescence intensity. It is obtained by comparing the photon numbers in subsequent time intervals of the photon date stream with the photon numbers in subsequent time intervals of the same data successively shifted in time. The cross-correlation function compares the photon numbers in the time intervals of the data stream of one detection channel with the photon numbers in the data stream of a second detector. For details please see 'The bh TCSPC Handbook', chapter 'Fluorescence Correlation Spectroscopy (FCS)'.