Definition of fluorescence correlation spectroscopy

Excelitas Technologies Corp. - X-Cite Vitae LB 11/24

Photonics Dictionary

fluorescence correlation spectroscopy: Fluorescence correlation spectroscopy (FCS) is a powerful analytical technique used to study the dynamics and interactions of fluorescently labeled molecules in solution at the single-molecule level. It provides information about molecular diffusion, concentration, binding kinetics, and molecular interactions with high sensitivity and temporal resolution.

Principle: FCS is based on the analysis of fluctuations in the fluorescence intensity emitted by fluorescently labeled molecules as they move through a small detection volume illuminated by a laser beam. These fluctuations arise from changes in the number of fluorescent molecules present in the detection volume over time due to diffusion, binding, and photophysical processes.

Experimental setup: A focused laser beam is used to excite the fluorescent molecules in solution, typically with a confocal microscope setup to ensure precise control of the excitation volume. Fluorescent emission from the illuminated volume is collected by a sensitive photodetector, such as a photomultiplier tube (PMT) or avalanche photodiode (APD). The emitted fluorescence signal is recorded as a time-series of intensity fluctuations.

Analysis:

Autocorrelation analysis: The recorded fluorescence signal is analyzed using autocorrelation analysis, which calculates the correlation between the fluorescence intensity at different time intervals.

Autocorrelation function: The autocorrelation function provides information about the timescale of fluctuations in the fluorescence intensity, which is related to the diffusion time and kinetics of molecular interactions.

Fitting models: The autocorrelation function can be fitted to mathematical models to extract quantitative parameters such as diffusion coefficients, molecular concentrations, and binding kinetics.

Applications:

Biomolecular dynamics: FCS is widely used to study the dynamics of biomolecules such as proteins, nucleic acids, lipids, and small molecules in solution. It provides insights into processes such as protein-protein interactions, enzyme kinetics, DNA-protein binding, and membrane dynamics.

Cellular imaging: FCS can be applied to study molecular dynamics and interactions in living cells. It allows researchers to investigate processes such as receptor-ligand binding, intracellular transport, and diffusion of biomolecules within cellular compartments.

Drug discovery: FCS is used in drug discovery and development to study ligand-receptor interactions, protein-ligand binding kinetics, and drug-target interactions. It provides valuable information for drug screening, lead optimization, and pharmacokinetic studies.

Material science: FCS can be used to study the dynamics of fluorescently labeled nanoparticles, polymers, colloids, and other materials in solution. It is valuable for characterizing nanomaterials, understanding self-assembly processes, and studying surface interactions.

Advantages:

High sensitivity: FCS can detect and analyze fluorescence from single molecules, providing high sensitivity and resolution.

Label-free detection: FCS can be performed with fluorescently labeled molecules or without labels, allowing for label-free detection of intrinsic fluorescence or autofluorescence.

Real-time measurements: FCS provides real-time measurements of molecular dynamics and interactions, enabling kinetic studies with high temporal resolution.

Challenges and considerations:

Photobleaching: Continuous illumination by the laser beam can lead to photobleaching of fluorescent molecules, limiting the duration of measurements.

Background noise: Background fluorescence and scattering can introduce noise in the fluorescence signal, affecting the accuracy of measurements.

Sample requirements: FCS requires samples with low background fluorescence, stable conditions, and appropriate concentrations of fluorescent molecules for reliable measurements.