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

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°.