Flurogen Activating Peptides for Single Molecule Localization based Superresolution

Abstract

Fluorgen Activating Peptides (FAPs) operate using an expressible dye binding peptide and a concentration of dye molecules that, upon binding to the receptor, have increased fluorescence excitation cross-sections by factors of hundreds to thousands [1] (see abstract by Qi Yan etal. for single-molecule characterization.) Depending on dye/receptor combination, affinities range from nanomolar to micromolar, corresponding to bound lifetimes up to 10s. The same receptor can repeatedly bind and activate new dye molecules, resulting in resistance to photobleaching when suitable concentration of unbleached dye remains and the FAP module hasn't been photodamaged. Binding rates can be controlled by dye concentration whereas fluorescent to dark state transitions can occur from unbinding and photobleaching. Adjusting dye concentration and excitation intensity allows tuning to maximize dyes localized per second per area. The following combined properties make the FAP system ideal for localization-based superresolution: 1)Expressible binding regions allow live cell studies; 2)Dye replenishment allows unlimited receptor position measurements, therefore arbitrary localization accuracy; 3)Only one excitation wavelength required; 4)Dye specific receptors allow multi-color superresolution.We demonstrate FAP superresolution by imaging live and fixed cells expressing beta-2 adrenergic receptor labeled with an extra-cellular FAP. Cell treatments show protein clustering details not apparent in diffraction limited images. Superresolution images are generated by placing Gaussian blobs at the found location of each activated dye molecule. Dye locations are found using a recently developed, iterative method that performs a maximum likelihood parameter estimation of the background count rate, dye location and dye emission rate. The blob widths are calculated from the Cramer-Rao Lower Bound (CRLB) corresponding to combined estimation of background, position and emission rate. Localization and CRLB are performed on GPU hardware using NVIDIA's CUDA architecture, achieving up to 10^5 combined fits and CRLB calculations per second.1. Szent-Gyorgyi et al, Nature Biotechnology, 2008. 26(2):p235-p240.