Förster resonance energy transfer (FRET)
With the FRET method, the distance between two fluorescent probes, the donor and acceptor, can be measured. FRET is used throughout the natural sciences as it allows probing many molecular properties, such as interactions, activity (kinase,…), forces or structure, but also analytes (pH, calcium, chloride…) can be quantified via FRET biosensors.
Illustration of FRET.
We use FRET at the ensemble (cuvette or droplet) level, but over the years, we’ve also specialized in single molecule (sm) implementations of FRET. SmFRET is particularly popular in structural biology studies, as it is one of the few methods that can simultaneously quantify molecular conformation and conformational dynamics. In our research we characterized different FRET dye pairs, continuously refine our FRET analysis methods and try to increase the accuracy/precision of structure measurements, and push the time scales of conformational dynamics that we can probe. We also investigate different biological systems with smFRET.
Illustration of smFRET. Adapted from Schrimpf et al., 2018.
Fluorescence fluctuation spectroscopy (FFS)
A family of methods that allow quantifying molecular properties such as concentrations, mobility, stoichiometry, interaction affinities of diffusing molecules at equilibrium. The simplest implementation is fluorescence correlation spectroscopy (FCS), where fluorescence signals from freely diffusing molecules is recorded on a confocal microscope (without image scanning). The resulting time trace is analyzed via autocorrelation, which then informs on diffusion rates and concentration.
Illustration of FCS. Adapted from Schrimpf et al., 2018.
Image correlation spectroscopy extends FFS analysis to imaging data. With Raster ICS (RICS), for example, mobility and concentration information is obtained from confocal laser scanning microscopy images.
Illustration of RICS. Adapted from Schrimpf et al., 2018.
Arbitrary-region ICS (ARICS) even allows mapping molecular properties in e.g. a biological structure, providing detailed insights in the workings of complex biological machineries.
Illustration of ARICS. Adapted from Hendrix et al., 2016.
In our research, we develop continuously refine existing FFS methods and develop novel FFS modalities to extract maximal and quantitative (molecular) information from confocal fluorescence data.
Time-resolved fluorescence and pulsed interleaved excitation
By imaging the fluorescence lifetime and/or time-resolved anisotropy via time-correlated single photon counting (TCSPC), detailed insights can be obtained about the fluorophore’s photophysical properties, which is important for quantitative and absolute interpretations of many fluorescence experiments. In our research we specialize in the development of multicolor TCSPC methods by using nanosecond alternating excitation (aka pulsed interleaved excitation, PIE) (Hendrix et al., 2013 and Hendrix et al., 2014).
Illustration of (A-C) TCSPC and (E-F) PIE. From Hendrix et al., 2014.
For many of our fluorescence lifetime analysis, we use phasor-based analysis.
Illustration of phasor analysis. Adapted from Schrimpf et al., 2018.
Electrophysiology combined with fluorescence
Ion channels are most often studied via electrophysiology, the method by which minute transmembrane currents can be quantified. Fluorescence microscopy can additionally be used to assess their structure, composition (hetero-stoichiometry) and structural dynamics. Combining the two methods on one instrument allows correlating activity and structure, but the patch clamp can also be used to site-specifically deliver molecules.