Nanoscale protein diffusion by STED-based pair correlation analysis

• Published in: PloS one Vol. 9; no. 6; p. e99619
• Main Authors: Bianchini, Paolo, Cardarelli, Francesco, Di Luca, Mariagrazia, Diaspro, Alberto, Bizzarri, Ranieri
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 26.06.2014; Public Library of Science (PLoS)
• Subjects: Animals; Biology and Life Sciences; Cells (biology); Chimeras; CHO Cells; Correlation; Correlation analysis; Cricetinae; Cricetulus; Diffusion; Diffusion barriers; Diffusion rate; E coli; Emission analysis; Fluorescence; Function analysis; Green Fluorescent Proteins - chemistry; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; Hepatitis; Microscopy; Microscopy, Fluorescence - methods; Mobility; Nuclei; Nuclei (cytology); Physical Sciences; Protein Transport; Proteins; Sensitivity and Specificity; Signal to noise ratio; Spatial discrimination; Spatial resolution; Spectrum analysis; Stimulated emission; Viral Core Proteins - chemistry; Viral Core Proteins - genetics; Viral Core Proteins - metabolism
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0099619

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.