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Dynamic Bioimaging Lab


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Dynamic Bioimaging Lab

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RESEARCH

Our goal is to provide insights in molecular structure-function relations using fluorescence as a jack-knife. To achieve this goal, we specialize in the development of (often single-molecule) sensitive fluorescence methods and analysis algorithms. We apply these methods to elucidate complex dynamic mechanisms and pathways in the natural sciences.


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.

  • Live-cell
  • 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. 

Zeiss LSM880

Tba 

Homebuilt multicolor FLIM microscope

tba 

Other instruments

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

Overzicht hier

Single-virus imaging

The goal is to track and analyze the transit of HIV within cytoplasm and nucleus using multi-parametric fluorescence microscopy technique.

Conformational dynamics

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.

 Fluctuation imaging

Overzicht van PIEFI, ARICS, RSICS…