Dual Color MIET and FRET for Cell Nanoscopy of Stress Fibers and Focal Adhesions

ORAL

Abstract

We report a novel method, dual color axial nanometric localization by Metal Induced Energy Transfer (dcMIET), and combine it with Förster Resonant Energy Transfer (FRET) for resolving structural details in cells on the molecular level. We demonstrate the capability of this method on cytoskeletal elements and adhesions in human mesenchymal stem cells (hMSCs). Our approach is based on Fluorescence-Lifetime-Imaging Microscopy (FLIM), and allows for precise determination of the 3D architecture of intracellular structures, here in particular, stress fibers anchoring at focal adhesions, thus yielding crucial information to understand cell-matrix mechanics. In addition to resolving nanometric structural details along the z-axis, we use FRET to gain precise information on the distance between actin and vinculin at focal adhesions. Here, we will present data where we use our method to follow the maturation of focal adhesions and acto-myosin fibers in hMSCs over time and show that actin fibers are nearly parallel to the substrate yielding an inclination angle of only 0.2°.

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Presenters

  • Florian Rehfeldt

    Third Institute of Physics - Biophysics, University of Göttingen, 37077 Göttingen, Germany, 3rd Institute of Physics - Biophysics, University of Göttingen

Authors

  • Anna M. Chizhik

    3rd Institute of Physics - Biophysics, University of Göttingen

  • Carina Wollnik

    3rd Institute of Physics - Biophysics, University of Göttingen

  • Daja Ruhlandt

    3rd Institute of Physics - Biophysics, University of Göttingen

  • Narein Karedla

    3rd Institute of Physics - Biophysics, University of Göttingen

  • Alexey I. Chizhik

    3rd Institute of Physics - Biophysics, University of Göttingen

  • Lara Hauke

    3rd Institute of Physics - Biophysics, University of Göttingen

  • Dirk Hähnel

    3rd Institute of Physics - Biophysics, University of Göttingen

  • Ingo Gregor

    3rd Institute of Physics - Biophysics, University of Göttingen

  • Jörg Enderlein

    Göttingen University, 3rd Institute of Physics - Biophysics, University of Göttingen, Third Physical Institute, Georg-August-University Goettingen

  • Florian Rehfeldt

    Third Institute of Physics - Biophysics, University of Göttingen, 37077 Göttingen, Germany, 3rd Institute of Physics - Biophysics, University of Göttingen