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Dynamic Bioimaging Lab


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Dynamic Bioimaging Lab

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RESEARCH

We want to unravel molecular structure-function relations and use fluorescence as our research tool. 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.

  

 

 

 

 

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. In our research we characterize different FRET dye pairs, continuously refine our FRET analysis methods and continuously try to increase the accuracy/precision of structure measurements and the time scales of conformational dynamics that we can quantify

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. Combined with imaging it allows mapping molecular properties in e.g. a biological structure, providing detailed insights in the workings of complex biological machineries.

 

 

 

 

 

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).

Electrophysiology

to be added

Zeiss Elyra PS.1

Top-notch widefield/TIRF microscope at our disposal at UHasselt-BIOMED. 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.

 

 

 

 

 

 

 

 

 

 

 

 

Microscope

  • Zeiss Elyra PS.1
  • different low/high NA and magnification objective lenses
  • temperature and CO2 control
  • axial drift control

Excitation

  • 405/488/594/633-nm CW lasers
  • widefield, total internal reflection and structured illumination
  • framewise alternating excitation

Detection

  • PCO Edge 4.2 CMOS 1280x1280 camera
  • Andor iXon DU-897 512x512 EM-CCD
  • Optosplit module for simultaneous two-color detection, for e.g. smFRET experiments

Applications

  • live-cell, time-lapse and large sample tile scans
  • videorate imaging
  • TIRF-based temporal image correlation spectroscopy (TICS)
  • ~150 nm and 3D superresolution imaging via structured illumination (SIM)
  • down to 20 nm and 3D superresolution imaging via PALM/STORM
  • single-molecule and smFRET
  • combined electrophysiology-fluorescence experiments

Homebuilt dedicated single-molecule microscope

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. 

 

 

 

 

 

 

 

 

 

 

 

 

Microscope

  • Olympus body
  • UPLSAPO 60xW NA1.2 objective lens

Excitation

  • 483/635-nm picosecond pulsed lasers
  • underfilled excitation (large PSF for single molecule imaging)
  • PIE
  • linear polarization

Detection

  • 75-um pinhole
  • blue side: two Laser components COUNT BLUE detectors
  • red side: two Perkin Elmer APDs

Applications

  • FCS
  • single-molecule FRET 
  • single molecule fluorescence

Zeiss LSM880

State of the art confocal microscope equipped with multiphoton excitation, FLIM, SHG, spectral detection and automated large sample aqcuisitioning.

 

 

 

 

 

 

 

 

 

 

 

Microscope

  • Zeiss LSM880
  • different low/high NA and magnification objective lenses
  • temperature and CO2 control
  • axial drift control

Excitation

  • SpectraPhysics MaiTai DeepSee 100 fs pulsed TiSaff 690-1050 nm
  • 488-nm pulsed laser diode
  • 458/488/514/543/633-nm CW lasers
  • rotatable polarization control at the excitation side

Detection

  • 34-channel combined GaAsP spectral QUASAR and PMT detector
  • 32-element spatially resolved Airyscan detector
  • Big2 polarization-sensitive (non-) & descanned detectors
  • Becker-Hickl hybrid detector
  • Becker-Hickl SPC830 time-correlated single photon counting

Applications

  • confocal laser scanning microscopy
  • live-cell, time-lapse 3D via z-stacking and large sample tile scans
  • two/multiphoton/deep imaging
  • fluorescence lifetime imaging microscopy (FLIM)
  • fluorescence correlation spectroscopy (FCS) and image correlation spectroscopy (ICS)
  • label-free imaging via second harmonic generation (SHG)
  • 1.7x superresolution imaging via Airyscan

Homebuilt multicolor FLIM microscope

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Microscope

  • Olympus body
  • UPLSAPO 60xW NA1.2 objective lens
  • axial drift control
  • Till Photonics Yanus IV galvo
  • widefield LED triband epifluorescence
  • xenon lamp for transmission

Excitation

  • 405/485/560/635-nm picosecond pulsed quadband
  • 440/510/560/635-nm picosecond pulsed quadband
  • focused overfilled excitation (confocal)
  • PIE/nsALEX
  • linearly polarized

Detection

  • 1-Airy unit pinhole
  • polarization sensitive
  • Picoquant tauSPAD
  • Picoqant PMA hybrid
  • Picoquant Hydraharp 400 4-channel TCSPC device

Applications

  • confocal imaging
  • time-resolved fluorescence (ps - µs lifetimes)
  • FLIM and FLIM-FRET
  • time-resolved anisotropy
  • FCS, PIE-FCCS, ICS, RICS, PIE-ccRICS, tICS...
  • (STED via available phase mask)
  • single-molecule fluorescence

Other instruments

The lab is embedded in two top-notch imaging facilities (Advanced Optical Microscopy Centre at UHasselt and the Molecular Imaging and Photonics division at KU Leuven), so many optical microscopy methods are readily available, such as STED, light sheet, SOFI, correlative light-EM.

 

At the core of our activities are a number of dedicated projects described in more detail below. Through collaborations with different research groups at Hasselt University, KU Leuven or elsewhere, however, we are continuously involved in different research projects as well.  

Correlative fluorescence & electrophysiology

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. In our research we focus on the Glycine receptor alpha-3, and in particular try to understand the function of an intracellular unstructured loop

Single-virus imaging

Starting with in vitro and cell-culture based fluorescence correlation spectroscopy investigations of the human immunodeficiency virus (HIV) Integrase protein and the human HIV cofactor LEDGF/p75 more than 10 years ago, currently we are able to fluorescently label and track individual HIV and murine leukemia virus (MLV) virions during the viral replicative cycle, and at the same time assess the quaternary structure of the IN protein via Förster resonance energy transfer (for a nice review, see Parveen et al., 2018). On the one hand, we continuously develop novel single virus microscopy tools to interrogate the virions and intracellular viral complexes in more detail, and on the other hand we try to solve timely questions in retrovirology research together with KU Leuven virologist Prof. Zeger Debyser and colleagues.

Dynamic structural biology

Single-molecule based Förster resonance energy transfer can be used to measure in real time the distance between two fluorescent probes that are attached to a biomolecule with near Ångstrom precision. The main advantages of this approach are that the biomolecule does not need to be crystallized, does not need to be purified in large amounts, and can be assessed in real time in solution. On the one hand, we use this method to gain mere structural insights into proteins, and on the other hand, we use it to provide detailed insights into the intrinsic structural dynamics that some proteins possess.

Figuur van SecA met van die lijntjes op
Figuur van EFTu

Multiparameter Fluctuation imaging

Advanced image correlation spectroscopy allows extracting maximal information from live samples containing diffusing molecules. We combined pulsed interleaved excitation with raster image correlation spectroscopy, which allows exploiting the fluorescence lifetime to discern between different fluorescent species or to remove noise from experimental data. We developed a new image correlation algorithm that allows studying molecular mobility in arbitrarily shaped regions of interest (e.g. the membrane or other organelles). We also developed an algorithm to resolve different diffusing molecules on the basis of their fluorescence spectrum, using array-based spectral detection. In our current research we continuously try to improve the robustness and ease-of-use of different advanced fluctuation methods, and continuously try to increase the number of fluorescence readouts (intensity, lifetime, anisotropy, spectrum) in routine imaging experiments.