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Optofluidics, Microfluidics and Nanofluidics

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Laser switching contrast microscopy to monitor free and restricted diffusion inside the cell nucleus

Franz-Josef Schmitt
  • Corresponding author
  • Technical University of Berlin, Institute of Chemistry PC 14, Straße des 17. Juni 135, D- 10623 Berlin, Germany
/ Cornelia Junghans
  • Corresponding author
  • Technical University of Berlin, Institute of Chemistry PC 14, Straße des 17. Juni 135, D- 10623 Berlin, Germany
/ Matthias Sturm
  • Corresponding author
  • Technical University of Berlin, Institute of Optics and Atomic physics ER1-1, Straße des 17. Juni 135, D-10623 Berlin, Germany
/ Csongor Keuer
  • Corresponding author
  • Technical University of Berlin, Institute of Chemistry PC 14, Straße des 17. Juni 135, D- 10623 Berlin, Germany
/ Hans Joachim Eichler
  • Corresponding author
  • Technical University of Berlin, Institute of Optics and Atomic physics ER1-1, Straße des 17. Juni 135, D-10623 Berlin, Germany
/ Thomas Friedrich
  • Corresponding author
  • Technical University of Berlin, Institute of Chemistry PC 14, Straße des 17. Juni 135, D- 10623 Berlin, Germany
Published Online: 2016-04-22 | DOI: https://doi.org/10.1515/optof-2016-0001


A novel microscopic technique termed laser switching contrast microscopy (LSCM) allows for the imaging of the dynamics of optically switchable proteins in single cell compartments. We present an application for the monitoring of diffusive properties of single molecules of the photo-switchable fluorescent protein Dreiklang (DRK). LSCM in the cell nucleus of Chinese hamster ovary (CHO) cells cytoplasmically expressing DRK unravels quick diffusive equilibration of the DRK molecules inside the whole cytoplasm and inside the cell nucleus within seconds. The nuclear membrane is also highly permeable for DRK. Inside the nucleus entirely distinct regions are found that only partially enable diffusive protein redistribution with mean square displacement proportional to time while in other regions the mobility of the proteins seems to be restricted. After photo-switching string like patterns of light DRK molecules are observed in the cell nucleus. In addition a fraction of these DRK molecules appears immobile. The findings support recent theories of the cell interior described as a random obstacle model with an additional immobile fraction of DRK. Numerical simulations show that at different illumination intensity and different distance from the laser focus similar patterns for fluorescence recovery might be obtained in spite of strongly varying diffusion constants.

Keywords: Fluorescence microscopy; superresolution microscopy; Dreiklang; fluorescent protein; diffusion constant; photo-switchable molecules; random walk; nanofluidics


  • [1] S. W. Hell, J. Wichmann, Breaking the diffraction resolution limit by stimulated emission: stimulated-emission-depletion fluorescence microscopy. In: Optics letters Bd. 19, 1994, 11, 780

  • [2] M. Andresen, M.C. Wahl, A.C. Stiel, F. Gräter, L.V. Schäfer, S. Trowitzsch, G. Weber, C. Eggeling, H. Grubmüller, S.W. Hell, S. Jakobs, Structure and mechanism of the reversible photoswitch of a fluorescent protein. PNAS, 102, 2005, 13070

  • [3] M. Andresen, A.C. Stiel, S. Trowitzsch, G. Weber, C. Eggeling, M.C. Wahl, S.W. Hell , S. Jakobs, Structural basis for reversible photoswitching in Dronpa. PNAS, 104, 2007, 13005 [Web of Science]

  • [4] M. Hofmann, C. Eggeling, S. Jakobs, S.W. Hell, Breaking the diffraction barrier in fluorescence microscopy at low light intensities by using reversibly photoswitchable proteins. PNAS, 102, 2005, 17565

  • [5] P. Dedecker, J. Hotta, C. Flors, M. Sliwa, H. Uji-i, M. Roeffaers, R. Ando, H. Mizuno, A. Miyawaki, J. Hofkens, Subdiffraction imaging through the selective donut-mode depletion of thermally stable photoswitchable fluorophores: numerical analysis and application to the fluorescent protein Dronpa. J Am Chem Soc, 129, 2007, 16132 [Web of Science]

  • [6] C. Eggeling, M. Hilbert, H. Bock, C. Ringemann, M. Hofmann, A. Stiel, M. Andresen, S. Jakobs, A. Egner, A. Schönle, S.W. Hell Reversible photoswitching enables single-molecule fluorescence fluctuation spectroscopy at high molecular concentration. Microsc Res Tech, 70, 2007, 1003 [Crossref]

  • [7] T. Brakemann, A. Stiel, G. Weber, M. Andresen, I. Testa, T. Grotjohann, M. Leutenegger, U. Plessmann, H. Urlaub, C. Eggeling, M.C. Wahl, S.W. Hell, S. Jakobs, A reversibly photoswitchable GFP-like protein with fluorescence excitation decoupled from switching. Nat Biotechnol, 29, 2011, 942 [Web of Science]

  • [8] G.J. Kremers, S.G. Gilbert, P.J. Cranfill, M.W. Davidson, D.W. Piston, Fluorescent proteins at a glance, J. Cell. Sci., 124, 2011, 157 [Web of Science]

  • [9] D.M. Chudakov, S. Lukyanov, K.A. Lukyanov, Fluorescent proteins as a toolkit for in vivo imaging, Trends in biotechnology, 23, 2005, 605

  • [10] R.E. Campbell, Fluorescent-protein-based biosensors: modulation of energy transfer as a design principle, Analytical chemistry, 81, 2009, 5972

  • [11] F.-J. Schmitt, E. Maksimov, C. Junghans, J. Weißenborn, P. Hätti, V. Z. Paschenko, S. I. Allakhverdiev, T. Friedrich, Structural Organization and Dynamic Processes in Protein Complexes Determined by Multiparameter Imaging, J. NanoPhoto- BioSciences. Signpost Open Access,1, 2015, 1

  • [12] F.-J. Schmitt, B. Thaa, C. Junghans, M. Vitali, M. Veit and T. Friedrich, eGFP-pHsens as highly sensitive fluorophore for pH determination in cells by fluorescence lifetime imaging microscopy (FLIM), Biochim Biophys Acta. , 1837, 2014, 1581

  • [13] F.-J. Schmitt, G. Renger, T. Friedrich, V. D. Kreslavski, D. A. Los, S. K. Zharmukhamedov, V. V. Kuznetsov, S. I. Allakhverdiev, Re-evaluation of reactive oxygen species: monitoring, generation and role in stress-signaling of phototrophic organisms, BBA-Bioenergetics, 1837, 2014, 835 [Web of Science]

  • [14] S.W. Hell, Strategy for far-?eld optical imaging and writing without diffraction limit. Phys Lett A, 326, 2004, 140

  • [15] J.V. Westphal, S.W. Hell, Nanoscale resolution in the focal plane of an optical microscope. Phys Rev Lett, 94, 2005, 143903 [Crossref]

  • [16] J. V. Westphal, J. Seeger, T. Salditt, S.W. Hell, Stimulated emission depletion microscopy on lithographic nanostructures. J Phys B: At. Mol. Opt. Phys., 38, 2005, 695

  • [17] F.-J. Schmitt, “resolution limits of time- and space-correlated single photon counting”, In: Proceedings of the 2008 international conference on information theory and statistical learning ITSL 2008, editors: M.Dehmer, M.Drmota, F. Emmert- Streib, ISBN: 1-60132-079-5, 2008, 91

  • [18] B. Harke, J. Keller, C.K. Ullal, V. Westphal. A. Schönle, S.W. Hell, Resolution scaling in STED microscopy, Opt. Express, 16, 2008, 4154 [Web of Science] [Crossref]

  • [19] S. Habuchi, R. Ando, P. Dedecker, W. Verheijen, H. Mizuno, A. Miyawaki, J. Hofkens, Reversible single-molecule photoswitching in the GFP-like fluorescent protein Dronpa, PNAS, 102, 2005, 9511

  • [20] S. Habuchi, P. Dedecker, J.-I. Hotta, C. Flors, R. Ando, H. Mizuno, A. Miyawaki, J. Hofkens, Photo-induced protonation/ deprotonation in the GFP-like fluorescent protein Dronpa: mechanism responsible for the reversible photoswitching, Photochem. Photobio. Sci., 5, 2006, 567

  • [21] M. Baum, F. Erdel, M. Wachsmuth, K. Rippe, Retrieving the intracellular topology from multi-scale protein mobility mapping in living cells, Nature communications, 5, 2014, doi:10.1038/ncomms5494 [Crossref]

  • [22] A. Fick, Ueber Diffusion, Pogg. Ann. Phys. Chem. 1705, 1855, 59

  • [23] C.R. Wilke and P. Chang. Correlation of diffusion coeflcients in dilute solutions, A.E.CH.E. Journal, 1, 1955, 264

  • [24] A. L. Magalhăes, P. F. Lito, F. A. Da Silva, C. M. Silva, Simple and accurate correlations for diffusion coeflcients of solutes in liquids and supercritical fluids over wide ranges of temperature and density, The Journal of Supercritical Fluids, 76, 2013, 94

  • [25] Schmitt F.-J., Picobiophotonics for the investigation of pigment-pigment and pigment-protein interactions in photosynthetic complexes, PhD thesis, Berlin, 2011, 230 p

About the article

Received: 2015-11-02

Accepted: 2016-01-08

Published Online: 2016-04-22

Citation Information: Optofluidics, Microfluidics and Nanofluidics, ISSN (Online) 2300-7435, DOI: https://doi.org/10.1515/optof-2016-0001. Export Citation

© 2016 Franz-Josef Schmitt et al.. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License. (CC BY-NC-ND 3.0)

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