Fluorescence Fluctuation Spectroscopy
Fluorescence fluctuation spectroscopy (FFS) techniques provide information at the single-molecule level with excellent time resolution. Instead of measuring the mean value of fluorescence signal, FFS analyses the fluctuations of the signal, usually coming from a few molecules in a small sample volume. Advances in FFS over the last decade has led to widespread acceptance of these methods in quantitative biology. Newer analysis methods have been developed that allows us to measure a several molecular characteristics of an analyte including translational diffusion rates, specific brightness, fluorescence anisotropy, fluorescence lifetime, spectral characteristics, and photochemical properties. All of this translates into a powerful set of applications for monitoring an enormous variety of molecular processes. The advantages of FFS techniques are:
- Small sample volumes (< 10 uL) and concentrations (picomolar)
- Single molecule sensitivity
- Measure interactions at equilibrium conditions (no washing required)
- Direct measurement (no amplification required) with stoichiometry information
- Few false negatives
- Compatible with both in vitro and live cell imaging
Experts at Cytomem have many years of experience developing advanced FFS methods using time-correlated single photon counting (TCSPC) setup in quantifying molecular interactions. We provide comprehensive consulting services in the following FFS techniques:
Fluorescence correlation spectroscopy (FCS)
is a correlation analysis of the fluorescence fluctuation in time domain. This analysis gives the average concentration and average mobility of fluorescent molecules. This technique is suited to quantify molecular interactions, such as ligand-receptor, antibody-antigen, nucleic acids (DNA and RNA) interactions.
Fluorescence cross-correlation spectroscopy (FCCS)
is a variation of FCS which uses cross-correlation of two or more fluorescence signals. This methods is more sensitive than FCS in detecting interactions where a change in mass as a result of a particular interaction is minimal. Cross-correlation of the fluorescence signal from both channels reveals information about co-movement.
Photon counting histogram (PCH) analysis
is the statistical analysis of fluorescence fluctuation in space domain. It resolves mixtures of species by differences in brightness. This method is particularly powerful in resolving small oligomerization of proteins, like formation of dimer or trimer, which is difficult for FCS to detect since the difference in the diffusion time between species is small.
Fluorescence lifetime measurement and imaging (FLIM)
Fluorescence lifetime is an intrinsic property of a fluorophore and is sensitive to a great variety of internal factors defined by the fluorophore structure and external factors that include temperature, polarity, and the presence of fluorescence quenchers. Since fluorescence lifetime does not depend on the fluorescence intensity and fluorophore concentration, it has become a separate yet complementary method to traditional fluorescence intensity measurements for detecting molecular interactions. With advances in fast and sensitive detectors, measuring fluorescence lifetime in imaging mode has been popular and can be applied with wide spatial scales, ranging from single molecules to cells.