Focal Adhesion Axial Topography by the Z-Phasor Approach in Confocal Microscopy

Abstract

The protein lateral and axial organization within focal adhesions has been studied by state of the art super resolution methods due to its thin structure, well below diffraction limit. However, to achieve high axial resolution, most of the current approaches rely on either sophisticated optics or diligent sample preparation requiring sample fixation that limits their application. In this report we present a phasor-based method that can be applied to fluorescent samples to determine the precise axial position of proteins using a conventional confocal microscope. We demonstrate that when about a total of 4000 photon counts are collected along a z-scan, axial localization precision close to 10 nm is achievable. We show at 10nm resolution that axial localization of paxillin, FAK and talin is similar at different focal adhesion sites, while F-actin shows a sharp increase in height towards the cell center. We further demonstrated that using line scans we can obtain axial resolution of 10 nm with a temporal resolution of less than a second of the changes of topography along the line. With the advantage of simple data acquisition and no special instrument requirement, this approach could have wide dissemination and application potentials.Work supported in part by NIH grants P50 GM076516 and P41 GM103540.