title: Nanoscale protein diffusion by STED-based pair correlation analysis.
abstract: We describe for the first time the combination between cross-pair correlation function analysis (pair correlation analysis or pCF) and stimulated emission depletion (STED) to obtain diffusion maps at spatial resolution below the optical diffraction limit (super-resolution). Our approach was tested in systems characterized by high and low signal to noise ratio, i.e. Capsid Like Particles (CLPs) bearing several (>100) active fluorescent proteins and monomeric fluorescent proteins transiently expressed in living Chinese Hamster Ovary cells, respectively. The latter system represents the usual condition encountered in living cell studies on fluorescent protein chimeras. Spatial resolution of STED-pCF was found to be about 110 nm, with a more than twofold improvement over conventional confocal acquisition. We successfully applied our method to highlight how the proximity to nuclear envelope affects the mobility features of proteins actively imported into the nucleus in living cells. Remarkably, STED-pCF unveiled the existence of local barriers to diffusion as well as the presence of a slow component at distances up to 500-700 nm from either sides of nuclear envelope. The mobility of this component is similar to that previously described for transport complexes. Remarkably, all these features were invisible in conventional confocal mode.
authors: Nanophysics, IIT-Italian Institute of Technology, Genoa, Italy.
authors: Paolo
authors: P
authors: Bianchini
authors: Center for Nanotechnology Innovation @NEST, Istituto Italiano di Tecnologia, Pisa, Italy.
authors: Francesco
authors: F
authors: Cardarelli
authors: NEST, Scuola Normale Superiore and Istituto Nanoscienze - CNR, Pisa, Italy.
authors: Mariagrazia
authors: M
authors: Di Luca
authors: Nanophysics, IIT-Italian Institute of Technology, Genoa, Italy.
authors: Alberto
authors: A
authors: Diaspro
authors: Nanophysics, IIT-Italian Institute of Technology, Genoa, Italy; NEST, Scuola Normale Superiore and Istituto Nanoscienze - CNR, Pisa, Italy; Istituto di Biofisica - CNR, Pisa, Italy.
authors: Ranieri
authors: R
authors: Bizzarri
mesh_terms: D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004058:Diffusion; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008856:Microscopy, Fluorescence / Q000379:methods; D021381:Protein Transport; D012680:Sensitivity and Specificity; D014758:Viral Core Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D014758:Viral Core Proteins; D049452:Green Fluorescent Proteins