Glossary

FRET rate constant

The FRET rate constant, $latex k_{RET}$, quantifies the FRET process by the number of quanta transferred from the donor’s excited state to the acceptor per time. It depends on the mutual dipole orientation of the donor and the acceptor fluorophore, the distance between the donor and acceptor, \(R_{DA}\), the Förster radius, \(R_0\) of the dye pair, and the corresponding fluorescence lifetime of the donor in the absence of FRET, \(\tau_{0}\). The orientation factor \(\kappa^2\) captures The mutual dipole orientation.

\[k_{RET} = \frac{1}{\tau_0} \kappa^2 \left( \frac{R_0}{R_{DA}} \right)^{6}\]

Note, for the calculation of the FRET rate constant the fluorescence lifetime has to match the Förster radius. Meaning the fluorescence lifetime of the corresponding donor fluorescence quantum yield, \(\Phi_{F}^{D0}\) should be used.

CLSM confocal laser scanning microscopy

PDA Photon Distribution Analysis

MFD (Multiparameter Fluorescence Detection)

A MFD experiments is a time-resolved fluorescence experiment which probes the absorption and fluorescence, the fluorescence quantum yield, the fluorescence lifetime, and the anisotropy of the studied chromophores simultaneously (see [KS01])

FCS (Fluorescence correlation spectroscopy)

FCS is a method which relies on fluctuations on the recorded signals to characterize molecular interaction such as binding and unbinding, chemical reaction kinetics, diffusion of fluorescent molecules (see [EM74] and [MEW72])

FRET efficiency

The FRET efficiency is the yield of a FRET process. A FRET process transfers energy from the excited state of a donor fluorophore to an acceptor fluorophore. The number of donor molecules in an excited state which transfers energy to an acceptor defines the yield of this energy transfer.

\[E = \frac{transfered}{excited}\]

Practically, mostly the donor and acceptor fluorescence intensities are used to obtain an experimental estimate for this yield.

FRET induced donor decay

The FRET-induced donor decay is a time-resolved intensity independent measure of FRET similar to the time-resolved anisotropy defined by the ratio of the donor fluorescence decay in the presence and the absence of FRET (see: [POS17]).

Instrument response function (IRF)

IRF stands for instrument response function. In time-resolved fluorescence measurements the IRF is the temporal response of the fluorescence spectrometer to a delta-pulse. Suppose a initially sharp pulse defines the time of excitation / triggers the laser, then recorded response of the fluorescence spectrometer is broadened due to: (1) the temporal response of the exciting light source, (2) the temporal dispersion due to the optics of the instrument, (3) the delay of the light within the sample, and (4) the response of the detector. As the most intuitive contribution to the IRF is the excitation profile, the IRF is sometimes called ‘lamp function’. The IRF is typically recorded by minimising the contribution of (3), e.g., by measuring the response of the instrument using a scattering sample, or a short lived dye.

Time-tagged time resolved (TTTR)

TTTR stands for time tagged time-resolved data or experiments. In TTTR-datasets the events, e.g., the detection of a photon, are tagged by a detection channel number. Moreover, the recording clock usually registers the events with a high time resolution of a few picoseconds. For long recording times of the detected events, a coarse and a fine clock are combined. The fine clock measures the time of the events relative to the coarse clock with a high time resolution. The time of the coarse and the fine clock is usually called macro and micro time, respectively.

FRET positioning system (FPS)

FRET positioning system, FPS, is an approach to determine structural models based on a set of FRET measurements. FPS explicitly considers the spatial distribution of the dyes. This way experimental observables, i.e., FRET efficiencies may be predicted precisely. The FPS-toolkit is available from the web page of the Seidel group of the Heinrich Heine University. An implementation of accessible volume simulations (AV) used in FPS are available as open source.

Time correlated single photon counting (TCSPC)

Time correlated single photon counting (TCPSC) is a technique to measure light intensities with picosecond resolution. Its main application is the detection of fluorescent light. A pulsed light source excites a fluorescent sample. A single photon detector records the emitted fluorescence photons. Thus, per excitation cycle, only a single photon is detected. Fast detection electronics records the time between the excitation pulse and the detection of the fluorescence photon. A histogram accumulates multiple detected photons to yield a time-resolved fluorescence intensity decay.

SWIG

SWIG is a software development tool that connects programs written in C and C++ with a variety of high-level programming languages. SWIG can be used with different types of target languages including common scripting languages such as Javascript, Perl, PHP, Python, Tcl and Ruby and non-scripting languages such as C#, D, Go language, Java, Octave, and R. SWIG is free software and the code that SWIG generates is compatible with both commercial and non-commercial projects. tttrlib is C/C++ based to provide the capability for a broad variety of languages to interface its provided functionality.