Fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (English fluorescence correlation spectroscopy, FCS) is a highly sensitive optical detection method that is gaining information from fluctuations in the fluorescence intensity. With FCS diffusion constants, concentrations and bonds between different diffusing species are usually measured. The method was developed in the 1970s by Douglas Magde, Elliot Elson and Watt W. Webb.

Construction

The basis for FCS usually forms a confocal microscope ( see figure). The excitation light is focused by a lens into the sample, so that the smallest possible volume of excitation is formed. Now diffuse fluorescent particles (e.g., fluorescently-labeled protein) in the excitation volume, they will be excited to fluorescence there. In this case, these particles absorb the photons of the excitation light and emit photons in turn longer wavelength, ie lower energy. The emitted photons can now use the ( impermeable to the excitation light ) beam splitter happen and are then detected by a photodetector. It is important that the readout rate of the detectors is several orders of magnitude above the typical residence time of a particle in focus. Today, avalanche photodiodes are used mainly for the FCS, can detect the single photons ( SPAD). There are also variants that are based on EMCCD cameras for example. In conjunction with cameras, are frequently used, other arrangements for the light used, such as total internal reflection fluorescence microscopy (TIR -FCS) or lens microscopy (SPIM -FCS), which also allow a spatially resolved measurement of mobility.

(Auto) correlation function

The actual measured value in the FCS, is usually referred to as a function of time time track the fluorescence intensity. The figure ( above) shows the time trace of a highly diluted sample. Each peak in the time track is a fluorescent particle that diffuses straight through the excitation volume. Each of these particles needs a certain time to pass through the focus. Therefore, the probability is high that at successive sampling times by the same particles photons are detected. It is said that the measured intensities are correlated in time. To evaluate the time traces, they are correlated with itself ( auto-correlated ). The autocorrelation function is defined as follows:

Here, the angle brackets denote an averaging over time, and.

The figure ( below) shows the autocorrelation of the fluorescence time trace, and one must note the logarithmic scale of the time axis. The decrease in the auto-correlation function of its half start value is a measurement of the diffusion time. This indicates how long the particles are, on average, takes to pass through the excitation volume. For the three-dimensional free diffusion can be shown that the autocorrelation function can be expressed as follows:

Here, the average number of particles in the excitation volume (focus ) the focal diameter and the lateral axial focus diameter. The intensity distribution of the excitation light is assumed here as a three-dimensional Gaussian function, which is a good approximation for many microscope objectives.

For leaves, the concentration of the fluorescent- active particles in the solution state, if one knows the excitation volume. The diffusion constant is given by.

For two-dimensional diffusion ( for example, cell membranes ) to the square root term is omitted.

Applications

• In molecular biophysics you can about the dependence of the diffusion coefficient from the hydrodynamic radius conclusions about the size of a protein and thus, especially in combination with the Förster resonance energy transfer ( FRET ), pull its folding state.
• In the research field of cell biology, there are increasingly working groups to carry out the FCS in living cells. Meanwhile, it is possible to carry out with suitably equipped commercial confocal microscopes fluorescence correlation measurements, FCS also extensions for older existing systems are offered.
• In the field of biotechnology, inter alia, screening robots are offered based on autocorrelation measurements.