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.
-image- FRET principle and applications
We use FRET at the ensemble (cuvette or droplet) level, but over the years, we’ve also specialized in single molecule implementations of FRET. SmFRET is particularly popular in structural biology studies, as it is the only method that can simultaneously quantify molecular conformation and conformational dynamics.
- Image conformation
Fluorescence fluctuation spectroscopy (FFS)
FFS encompasses a family of methods that allow quantifying molecular properties such as concentrations, mobility, stoichiometry, interaction affinities of diffusing molecules at equilibrium. Combined with imaging FFS allows mapping molecular properties in e.g. a biological structure, providing detailed insights in the workings of complex biological machineries.
- Image Principle of FCS and mapping
Time-correlated single photon counting (TCSPC)
By imaging the fluorescence lifetime and/or time-resolved anisotropy, detailed insights can be obtained in the fluorophore’s photophysical properties, which is important for quantitative and absolute interpretations of any fluorescence experiment. We employ TCSPC almost by default to improve FRET and FFS experiments.
- Image decay, FLIM
Zeiss Elyra PS.1
At UHasselt, we have a top-notch widefield/TIRF microscope at our disposal, the Zeiss Elyra PS.1. This instrument is equipped with structured illumination for super-resolution SIM experiments, but can also be used for off-the-shelf 3D PALM/STORM. The instrument contains a heating unit and CO2 incubation for live-cell experiments, but also a Faraday cage, micromanipulators and perfusion for combined fluorescence-electrophysiology experiments.
- Resolution down to Abbe/10 with PALM/STORM
- Resolution Abbe/2 with SIM
- EM-CCD / sCMOS
We recently equipped the Elyra with an Optosplit for realtime single-molecule FRET experiments of surface-immobilized molecules.
-image- image Zeiss Elyra and Optosplit
Homebuilt dedicated single-molecule microscope
At KU Leuven we have a dedicated state-of-the-art homebuilt 483/635-nm alternating excitation, dual-color dual-polarization detection confocal microscope. This system allows imaging more than 20 parameters per single molecule, making it the ideal system for subnanometer accurate FRET studies.
Homebuilt multicolor FLIM microscope
The lab is embedded in two top-notch imaging facilities (one at UHasselt and one at KU Leuven), so many optical microscopy methods are readily available, such as:
- Live-cell widefield/TIRF and confocal imaging
- Structured illumination microscopy
- PALM/STORM superresolution microsocpy
- Fast Airyscan superresolution microsocpy
- Two-photon microscopy
- TCSPC/FLIM/anisotropy imaging
- Forward/Reflected/Polarized SHG
- Patch clamp fluorometry
- Single-molecule FRET
Additionally, at KU Leuven:
- Confocal single-molecule burst analysis
- Multiparameter (intensity, lifetime, anisotropy) single-molecule imaging
- Nanosecond alternating excitation (ALEX/PIE) imaging
- STED superresolution microsocpy
- Light-sheet microscopy
Patch clamp fluorometry
The goal is to track and analyze the transit of HIV within cytoplasm and nucleus using multi-parametric fluorescence microscopy technique.
In general, we are using single-molecule FRET (smFRET) to study in an accurate way distances and conformational dynamics of biomolecules. We perform smFRET on a home-built multi parameter fluorescence detection setup combined with pulsed interleaved excitation. The robustness of smFRET critically depends on the performance of the employed dyes, yet a systematic and quantitative comparison of different dye pairs is still lacking. In a first project, we assessed the performance of blue (Atto488 and Alexa488) and far-red (Atto647N, Alexa647, StarRed and Atto655) dyes via combined ensemble and single-molecule methods on a rigid dsDNA olignocleotide. In a 2nd project we are studying the E. coli Sec reaction pathway. Although busily studied, important mechanistic details on bacterial protein targeting and secretion are still lacking. Since protein conformation and dynamics seems to be key to the function of translocases, we therefore carried out quantitative studies on the dynamic conformation of SecA in vitro using smFRET. Using FRET restrained structural modeling (FPS), we selected 20 suitable residues for the used FRET pair (Atto488/Alexa647) on SecA, including all the different domains, to perform a dynamic 3D model of SecA. Later on we will me move to an in vitro translocation assay to study the mechanism of substrate docking and –translocation on SecYEG.
Overzicht van PIEFI, ARICS, RSICS…