Color Coded Optical Nano-Sectioning (COCOS) Reveals Focal Adhesion Dynamics

Abstract

We present a fast imaging technique for performing high contrast microscopy and optical nanosectioning in the sub-100 nm vicinity of a biocompatible metal/dielectric coated substrate. The technique makes use of the distance dependent interactions of excited fluorophors with surface plasmons and polaritons of the coating: The fluorophor's emission spectrum changes with its distance from the substrate and thus allows inferring the molecule-substrate distance with a 50 times higher precision than the diffraction limit.Qualitatively, a COCOS image has a similar appearance to a typical Total Internal Reflection Fluorescence Microscopy (TIRFM) image however contains spectral information that provides an effective axial resolution in the nanometer over the range of the evanescent (surface plasmon polariton) field.The technique is demonstrated for tracking the axial dynamics of GFP-Paxillin and other complexes at the adhesion sites of migrating fibroblasts with approximately 10 nm axial localization precision. Our results for the average separation of the protein from the intercellular matrix are consistent with suggestions from previous studies and show periodic fluctuations in the axial position previously inaccessible. The proposed technique is likely to find a wide range of applications due to its practical simplicity and compatibility with established fluorescence methods.