Glossary

FRET

FRET stands for (Förster) Resonance Energy Transfer and describes the mechanism of energy transfer between two light-sensitive molecules. A donor chromophore, initially in its electronic excited state, may transfer energy to an acceptor chromophore, through nonradiative dipole–dipole coupling. The fluorescence absorption and emission spectrum of the acceptor are red shifted compared to the respective spectra of the donor fluorophore. For FRET to occur, three criteria have to be fulfilled: (1) The donor fluorescence emission spectrum has to overlap with the absorption spectrum of the acceptor, (2) the fluorophores’ dipoles are sufficiently parallel and (3) the fluorophores are in close vicinity (usually < 10 nm). The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor, making FRET extremely sensitive to small changes in distance.

FRET rate constant

The FRET rate constant, \(k_{RET}\), 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.

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.

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

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

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.

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 chromophoressimultaneously [KuhnemuthS01].

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.

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.

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. The tttr capability of IMP.bff is C/C++ based to provide the capability for a broad variety of languages to interface its provided functionality.

Scatter fraction

The scatter fraction \(\gamma\) is defined by the number of photons that

Anisotropy

The steady-state anisotropy \(r_G\) in the detection channel \(G\) is formally given by the fluorescence intensity weighted integral of the time-resolved anisotropy.

\[r_G=\int F_G(t) \cdot r(t) dt \cdot \frac{1}{\int F_G(t) dt}\]

where the time-resolved anisotropy is defined by unperturbed the fluorescence intensities of an ideal detection system.

\[r_G(t)=\frac{F_{G,p}(t)-F_{G,s}(t)}{F_{G,p}(t)+2F_{G,s}(t)}\]

Through out IMP.bff two distinct anisotropies are computed: (1) background corrected anisotropies, and (2) anisotropies not accounting for the background. In single-molecule experiments the background is mainly scattered light (Raman scattering). The uncorrected anisotropy (without background correction) is computed by:

\[r = \frac{S_p - g \cdot S_s}{S_p \cdot (1 - 3 \cdot l_2) + (2 - 3 \cdot l_1) \cdot g \cdot S_s}\]

where \(S_p\) is the signal in the parallel (German: parallel=p) detection channel, \(S_s\) the signal in the perpendicular decection channel (German: senkrecht=s), \(g\) is the g-factor, \(l_1\) and \(l_2\) are factor mixing that determine the mixing of the parallel and perpendicular detection channel, respectively [KSM95] . The scatter corrected steady-state anisotropy is computed using the scatter / background corrected signals parallel \(F_p = (S_p - \gamma \cdot B_p) / (1. - \gamma)\) and perpendicular \(F_s = (S_s - \gamma \cdot B_s) / (1. - \gamma)\) fluorescence intensity. \(r = (F_p - g \cdot F_s) / (F_p \cdot (1 - 3 \cdot l_2) + (2 - 3 \cdot l_1) \cdot g \cdot F_s)\) The scatter corrected and anisotropy not corrected for scatter are computed by most fits of fit2x.

Jordi-format

In the Jordi format is a format for fluorescence decays. In the Jordi format fluorescence decays are stacked in a one dimensional array. In a typical polarization resolved Jordi file the first decay is the parallel and the subsequent decay is the perpendicular decay. In the Jordi format both decays must have the same length, i.e., the same number of micro time counting channels.

PIE Pulsed-Interleaved Excitation (PIE)

experiments excite the studied samples by multiple pulsed light sources for different dyes. The light sources excite the sample interleaved and the photons of the samples are registered by time-resolved detectors and electronics.