Superresolution Imaging Using Fluorogen Activating Proteins by Sted Nanoscopy and Equilibrium Localization Microscopy

Abstract

Our center has recently developed the Fluorescence Activating Protein (FAP) technology for genetically targeted fluorescent labeling of proteins in live cells. FAPs are single chain antibodies that can specifically activate the fluorogenic dyes thiazole orange (TO) and malachite green (MG) with nanomolar affinities. When bound to FAPs, the otherwise dark fluorogens generate thousands of fold increase in fluoresce intensity. The fact that FAPs are small in size (12 to 25 kDa) and genetically encoded makes them an ideal fluorescence tag for live cell imaging. Since no appreciable background from the fluorogen alone is detectable, no washing steps are required to remove the excess fluorogen. Moreover, different FAP-fluorogen combinations can result in spectrally resolvable probes for multi-color imaging. We have demonstrated the utility of these probes for both ensemble and single molecule based superresolution methodologies.Because malachite green has a similar quantum yield and spectral characteristic to the STED-efficient dye Atto647N, we tested the feasibility of using MG-FAPs in STED imaging. We show that STED imaging could be performed on both live and fixed and permeabilized cells expressing MG-FAP. When an engineered, cytosolic expressible MG FAP H6.2-MG was fused to the N-terminus of actin, actin filaments with FWHM of 110-122nm were observed, an approximately 3-fold resolution improvement compared to a confocal image.The reversible interaction between the fluorogen and the FAP allows the same FAP to bind and activate fresh fluorogen from solution after one fluorogen dissociates from the FAP. Consequently, multiple binding and unbinding cycles result in a characteristic intermittency, leading to a high photon flux and photobleaching-resistant system. Using low concentrations of the fluorogen gives rise to sparse labeling of objects for localization microscopy with a localization accuracy of <10 nm.