Lab for Nanobiology

Welcome to the website of the Dedecker research group! We are located at the Department of Chemistry at the University of Leuven.

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Development of “Smart” Fluorescent Probes

We design new fluorophores based on fluorescent proteins, focusing especially on fluorophores with properties that can be changed through irradiation with light. Rather whimsically we like to call these “smart” fluorophores. We then study their properties and apply these in advanced fluorescence imaging.

The new fluorophores we design are enhanced or modified version of existing fluorescent proteins. The resulting fluorophores include both rational mutations, and mutations developed using directed evolution.

Our aim in this research is both to develop useful tools for imaging, but also to develop a fundamental understanding of the mechanisms underlying the “smart” behaviors responsible for their usefulness.

Improved Fluorescence Imaging

We have a strong interest in fluorescence imaging. In particular we are interested in finding good or even optimal ways to extract more information from the imaging or to image under “difficult” conditions, such as low signal-to-noise ratio, or of fast processes occurring at very small spatial length scales.

One of our main topics recently is the development of stochastic optical fluctuation imaging (SOFI). In SOFI, we make use of spontaneous fluctuations of the fluorophore emission. Since the fluorophore emission is dynamic, we can acquire many complementary images from the same sample and combine these to obtain a single information-enhanced image with a sub-diffraction spatial resolution.

We are particularly interested in SOFI imaging due to the technique's robustness compared to other sub-diffraction imaging techniques. In particular, SOFI imaging does not require that fluorophores are individually resolvable, nor does it require that the excitation light is precisely distributed along a known pattern.


We are also interested in developing fluorescent reporters that combine “smart” photophysical behavior with sensing of the molecular environment. For example, we have recently developed a bimolecular fluorescence complementation (BiFC) pair based on the photochromic fluorescent protein Dronpa. Using this probe we can investigate molecular interactions inside the live system with a sub-diffraction spatial resolution. Currently we are also expanding this to the direct sensing of enzymatic activities.



Our main software package is Localizer. Localizer implements various analyses related to sub-diffraction imaging, including localization microscopy, SOFI imaging, and particle tracking. The advantages of Localizer is that it is typically very fast while also including a complete graphical interface for the Igor Pro software package. Some more information as well as the full source code is available online. Pre-built binaries are available as well.


  1. Genetically encoded biosensors for visualizing live-cell biochemical activity at super-resolution
    G. Mo, B. Ross, F. Hertel, P. Manna, X. Yang, E. Greenwald, C. Booth, A. Plummer, B. Tenner, Z. Chen, Y. Wang, E. Kennedy, P. Cole, K. Fleming, A. Palmer, R. Jimenez, J. Xiao, P. Dedecker, J. Zhang
    Nat. Methods 14:427 2017 PDF
  2. Effect of probe diffusion on the SOFI imaging accuracy
    W. Vandenberg, P. Dedecker
    Sci. Rep. 7: 2017 PDF
  3. Complementarity of PALM and SOFI for super-resolution live-cell imaging of focal adhesions
    H. Deschout, T. Lukes, A. Sharipov, D. Szlag, L. Feletti, W. Vandenberg, P. Dedecker, J. Hofkens, M. Leutenegger, T. Lasser, A. Radenovic
    Nat. Commun. 7: 2016 PDF
  4. Field-Controlled Charge Separation in a Conductive Matrix at the Single-Molecule Level: Toward Controlling Single-Molecule Fluorescence Intermittency
    K. Kennes, P. Dedecker, J. Hutchison, E. Fron, H. Uji-i, J. Hofkens, M. Van der Auweraer
    ACS Omega 1:1383 2016 PDF
  5. SOFI Simulation Tool: A Software Package for Simulating and Testing Super-Resolution Optical Fluctuation Imaging
    A. Girsault, T. Lukes, A. Sharipov, S. Geissbuehler, M. Leutenegger, W. Vandenberg, P. Dedecker, J. Hofkens, T. Lasser
    PLOS ONE 11: 2016 PDF
  6. Sparse deconvolution of high-density super-resolution images
    S. Hugelier, J. de Rooi, R. Bernex, S. Duwe, O. Devos, M. Sliwa, P. Dedecker, P. Eilers, C. Ruckebusch
    Sci. Rep. 6: 2016 PDF
  7. Model-free uncertainty estimation in stochastical optical fluctuation imaging (SOFI) leads to a doubled temporal resolution
    W. Vandenberg, S. Duwe, M. Leutenegger, B. Moeyaert, B. Krajnik, T. Lasser, P. Dedecker
    Biomed. Opt. Express 7:467 2016 PDF
  8. RefSOFI for Mapping Nanoscale Organization of Protein-Protein Interactions in Living Cells
    F. Hertel, G. Mo, S. Duwe, P. Dedecker, J. Zhang
    Cell Rep. 14:390 2016 PDF
  9. Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution Microscopy
    S. Duwe, E. De Zitter, V. Gielen, B. Moeyaert, W. Vandenberg, T. Grotjohann, K. Clays, S. Jakobs, L. Van Meervelt, P. Dedecker
    ACS Nano 9:9528 2015 PDF
  10. Molecular Dynamic Indicators of the Photoswitching Properties of Green Fluorescent Proteins
    D. Smyrnova, B. Moeyaert, S. Michielssens, J. Hofkens, P. Dedecker, A. Ceulemans
    J. Phys. Chem. B 119:12007 2015 PDF
  11. Super-resolution mapping of glutamate receptors in C. elegans by confocal correlated PALM
    J. Vangindertael, I. Beets, S. Rocha, P. Dedecker, L. Schoofs, K. Vanhoorelbeeke, J. Hofkens, H. Mizuno
    Sci. Rep. 5: 2015 PDF
  12. Mapping Pixel Dissimilarity in Wide-Field Super-Resolution Fluorescence Microscopy
    C. Ruckebusch, R. Bernex, F. Allegrini, M. Sliwa, J. Holkens, P. Dedecker
    Anal. Chem. 87:4675 2015 PDF
  13. Diffraction-unlimited imaging: from pretty pictures to hard numbers
    W. Vandenberg, M. Leutenegger, T. Lasser, J. Hofkens, P. Dedecker
    Cell Tissue Res. 360:151 2015 PDF
  14. High-Resolution Single-Molecule Fluorescence Imaging of Zeolite Aggregates within Real-Life Fluid Catalytic Cracking Particles
    Z. Ristanovic, M. Kerssens, A. Kubarev, F. Hendriks, P. Dedecker, J. Hofkens, M. Roeffaers, B. Weckhuysen
    Angew. Chem. Int. Ed. 54:1836 2015 PDF
  15. A study of SeqA subcellular localization in Escherichia coli using photo-activated localization microscopy
    J. Mika, A. Vanhecke, P. Dedecker, T. Swings, J. Vangindertael, B. Van den Bergh, J. Michiels, J. Hofkens
    Faraday Discuss. 184:425 2015 PDF
  16. Spatial distribution and temporal evolution of DRONPA-fused SNAP25 clusters in adrenal chromaffin cells
    Y. Antoku, P. Dedecker, P. Pinheiro, T. Vosch, J. Sorensen
    Photochem. Photobiol. Sci. 14:1005 2015 PDF
  17. Combing of Genomic DNA from Droplets Containing Picograms of Material
    J. Deen, W. Sempels, R. De Dier, J. Vermant, P. Dedecker, J. Hofkens, R. Neely
    ACS Nano 9:809 2015 PDF
  18. HIV Virions as Nanoscopic Test Tubes for Probing Oligomerization of the Integrase Enzyme
    D. Borrenberghs, W. Thys, S. Rocha, J. Demeulemeester, C. Weydert, P. Dedecker, J. Hofkens, Z. Debyser, J. Hendrix
    ACS Nano 8:3531 2014 PDF
  19. Green-to-Red Photoconvertible Dronpa Mutant for Multimodal Super-resolution Fluorescence Microscopy
    B. Moeyaert, N. Bich, E. De Zitter, S. Rocha, K. Clays, H. Mizuno, L. Van Meervelt, J. Hofkens, P. Dedecker
    ACS Nano 8:1664 2014 PDF
  20. Excited state dynamics of the photoconvertible fluorescent protein Kaede revealed by ultrafast spectroscopy
    E. Fron, M. Sliwa, V. Adam, J. Michiels, S. Rocha, P. Dedecker, J. Hofkens, H. Mizuno
    Photochem. Photobiol. Sci. 13:867 2014 PDF
  21. Excited State Dynamics of Photoswitchable Fluorescent Protein Padron
    E. Fron, M. Van der Auweraer, J. Hofkens, P. Dedecker
    J. Phys. Chem. B 117:16422 2013 PDF
  22. Fluorescent Proteins: Shine on, You Crazy Diamond
    P. Dedecker, F. De Schryver, J. Hofkens
    J. Am. Chem. Soc. 135:2387 2013 PDF
  23. Localizer: fast, accurate, open-source, and modular software package for superresolution microscopy
    P. Dedecker, S. Duwe, R. Neely, J. Zhang
    J. Biomed. Opt. 17: 2012 PDF
  24. Structural basis for the influence of a single mutation K145N on the oligomerization and photoswitching rate of Dronpa
    N. Bich, B. Moeyaert, K. Van Hecke, P. Dedecker, H. Mizuno, J. Hofkens, L. Van Meervelt
    Acta Crystallogr., Sect. D: Biol. Crystallogr. 68:1653 2012 PDF
  25. Widely accessible method for superresolution fluorescence imaging of living systems
    P. Dedecker, G. Mo, T. Dertinger, J. Zhang
    Proc. Natl. Acad. Sci. USA 109:10909 2012 PDF
  26. Spectroscopic characterization of Venus at the single molecule level
    C. David, P. Dedecker, G. De Cremer, N. Verstraeten, C. Kint, J. Michiels, J. Hofkens
    Photochem. Photobiol. Sci. 11:358 2012 PDF
  27. Rational Design of Photoconvertible and Biphotochromic Fluorescent Proteins for Advanced Microscopy Applications
    V. Adam, B. Moeyaert, C. David, H. Mizuno, M. Lelimousin, P. Dedecker, R. Ando, A. Miyawaki, J. Michiels, Y. Engelborghs, J. Hofkens
    Chem. Biol. 18:1241 2011 PDF
  28. Fluorescent probes for superresolution imaging of lipid domains on the plasma membrane
    H. Mizuno, M. Abe, P. Dedecker, A. Makino, S. Rocha, Y. Ohno-Iwashita, J. Hofkens, T. Kobayashi, A. Miyawaki
    Chem. Sci. 2:1548 2011 PDF
  29. DNA fluorocode: A single molecule, optical map of DNA with nanometre resolution
    R. Neely, P. Dedecker, J. Hotta, G. Urbanaviciute, S. Klimasauskas, J. Hofkens
    Chem. Sci. 1:453 2010 PDF
  30. Spectroscopic Rationale for Efficient Stimulated-Emission Depletion Microscopy Fluorophores
    J. Hotta, E. Fron, P. Dedecker, K. Janssen, C. Li, K. Mullen, B. Harke, J. Buckers, S. Hell, J. Hofkens
    J. Am. Chem. Soc. 132:5021 2010 PDF
  31. High-Resolution Single-Turnover Mapping Reveals Intraparticle Diffusion Limitation in Ti-MCM-41-Catalyzed Epoxidation
    G. De Cremer, M. Roeffaers, E. Bartholomeeusen, K. Lin, P. Dedecker, P. Pescarmona, P. Jacobs, D. De Vos, J. Hofkens, B. Sels
    Angew. Chem. Int. Ed. 49:908 2010 PDF
  32. Higher resolution in localization microscopy by slower switching of a photochromic protein
    H. Mizuno, P. Dedecker, R. Ando, T. Fukano, J. Hofkens, A. Miyawaki
    Photochem. Photobiol. Sci. 9:239 2010 PDF
  33. Defocused Wide-field Imaging Unravels Structural and Temporal Heterogeneity in Complex Systems
    P. Dedecker, B. Muls, A. Deres, H. Uji-i, J. Hotta, M. Sliwa, J. Soumillion, K. Mullen, J. Enderlein, J. Hofkens
    Adv. Mater. 21:1079 2009 PDF
  34. Photoswitches: Key molecules for subdiffraction-resolution fluorescence imaging and molecular quantification
    M. Heilemann, P. Dedecker, J. Hofkens, M. Sauer
    Laser Photon. Rev. 3:180 2009 PDF
  35. On the use of Z-scan fluorescence correlation experiments on giant unilamellar vesicles
    E. Gielen, M. Vandeven, A. Margineanu, P. Dedecker, M. Van der Auweraer, Y. Engelborghs, J. Hofkens, M. Ameloot
    Chem. Phys. Lett. 469:110 2009 PDF
  36. Synthesis, Spectroscopy, Crystal Structure, Electrochemistry, and Quantum Chemical and Molecular Dynamics Calculations of a 3-Anilino Difluoroboron Dipyrromethene Dye
    W. Qin, V. Leen, T. Rohand, W. Dehaen, P. Dedecker, M. Van der Auweraer, K. Robeyns, L. Van Meervelt, D. Beljonne, B. Van Averbeke, J. Clifford, K. Driesen, K. Binnemans, N. Boens
    J. Phys. Chem. B 113:439 2009 PDF
  37. Super-Resolution Reactivity Mapping of Nanostructured Catalyst Particles
    M. Roeffaers, G. De Cremer, J. Libeert, R. Ameloot, P. Dedecker, A. Bons, M. Buckins, J. Martens, B. Sels, D. De Vos, J. Hofkens
    Angew. Chem. Int. Ed. 48:9285 2009 PDF
  38. How is cis-trans isomerization controlled in Dronpa mutants? A replica exchange molecular dynamics study
    S. Moors, S. Michielssens, C. Flors, P. Dedecker, J. Hofkens, A. Ceulemans
    J. Chem. Theory Comp. 4:1012 2008 PDF
  39. Dark states in monomeric red fluorescent proteins studied by fluorescence correlation and single molecule spectroscopy
    J. Hendrix, C. Flors, P. Dedecker, J. Hofkens, Y. Engelborghs
    Biophys. J. 94:4103 2008 PDF
  40. Excitation energy migration processes in cyclic porphyrin Arrays probed by single molecule spectroscopy
    J. Yang, M. Park, Z. Yoon, T. Hori, X. Peng, N. Aratani, P. Dedecker, J. Hotta, H. Uji-I, M. Sliwa, J. Hofkens, A. Osuka, D. Kim
    J. Am. Chem. Soc. 130:1879 2008 PDF
  41. Radical polymerization tracked by single molecule spectroscopy
    D. Woell, H. Uji-I, T. Schnitzler, J. Hotta, P. Dedecker, A. Herrmann, F. De Schryver, K. Muellen, J. Hofkens
    Angew. Chem. Int. Ed. 47:783 2008 PDF
  42. Diffraction-unlimited optical microscopy
    P. Dedecker, J. Hofkens, J. Hotta
    Mater. Today 11:12 2008 PDF
  43. Subdiffraction imaging through the selective donut-mode depletion of thermally stable photoswitchable fluorophores: Numerical analysis and application to the fluorescent protein Dronpa
    P. Dedecker, J. Hotta, C. Flors, M. Sliwa, H. Uji-I, M. Roeffaers, R. Ando, H. Mizuno, A. Miyawaki, J. Hofkens
    J. Am. Chem. Soc. 129:16132 2007 PDF
  44. A stroboscopic approach for fast photoactivation-localization microscopy with Dronpa mutants
    C. Flors, J. Hotta, H. Uji-I, P. Dedecker, R. Ando, H. Mizuno, A. Miyawaki, J. Hofkens
    J. Am. Chem. Soc. 129:13970 2007 PDF
  45. Fluorescence of single molecules in polymer films: Sensitivity of blinking to local environment
    J. Clifford, T. Bell, P. Tinnefeld, M. Heilemann, S. Melnikov, J. Hotta, M. Sliwa, P. Dedecker, M. Sauer, J. Hofkens, E. Yeow
    J. Phys. Chem. B 111:6987 2007 PDF
  46. Orientational effects in the excitation and de-excitation of single molecules interacting with donut-mode laser beams
    P. Dedecker, B. Muls, J. Hofkens, J. Enderlein, J. Hotta
    Opt. Express 15:3372 2007 PDF
  47. 3D nanoscopy: Bringing biological nanostructures into sharp focus
    P. Dedecker, C. Flors, J. Hotta, H. Uji-I, J. Hofkens
    Angew. Chem. Int. Ed. 46:8330 2007 PDF
  48. Fast and reversible photoswitching of the fluorescent protein Dronpa as evidenced by fluorescence correlation spectroscopy
    P. Dedecker, J. Hotta, R. Ando, A. Miyawaki, Y. Engelborghs, J. Hofkens
    Biophys. J. 91:L45 2006 PDF
  49. Photo-induced protonation/deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching
    S. Habuchi, P. Dedecker, J. Hotta, C. Flors, R. Ando, H. Mizuno, A. Miyawaki, J. Hofkens
    Photochem. Photobiol. Sci. 5:567 2006 PDF
  50. Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa
    S. Habuchi, R. Ando, P. Dedecker, W. Verheijen, H. Mizuno, A. Miyawaki, J. Hofkens
    Proc. Natl. Acad. Sci. USA 102:9511 2005 PDF

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Dedecker group
Departement Chemie
Celestijnenlaan 200G
3001 Heverlee

+32 16 32 75 26