Nanoscale Protein Diffusion by Sted-Based Spatiotemporal Fluorescence Correlation Spectroscopy

Abstract

Fluorescence Correlation Spectroscopy (FCS) represents an established technique to recover single-molecule diffusion and binding properties in cells. Recently, scanning microscopy imaging was applied to add a spatial dimension to the classic, purely temporal, FCS modality: spatiotemporal FCS (stFCS) provides details about the routes that diffusing particles or molecules follow in the specimen.[1] We report on combining spatiotemporal fluorescence correlation spectroscopy (stFCS) and stimulated emission depletion (STED) to monitor intracellular protein diffusion at a spatial resolution below the optical diffraction limit (super-resolution)[2,3]. We validated our method both in vitro and at intracellular level, studying the diffusion of fluorescent nanocapsids and of GFP bound to SV40 Nuclear Localization Signal (NLS), respectively. NLS-GFP represents a well-known model of actively nuclear-imported protein that has been the subject of intense research.[4] We demonstrated how relevant is our approach discovering persistent complexes between nucleo-cytoplasmic transporters and NLS-GFP at distances >500 nm from the nuclear envelope. Such a phenomenon would be otherwise invisible at the best resolution of conventional confocal imaging mode. We should stress that, in principle, the photophysics of the fluorescent reporter in STED conditions is the only limiting the resolution in stFCS diffusional maps.[5]1. M. A. Digman and E. Gratton, Annu Rev Phys Chem 62, 645-668 (2011).2. F. Cardarelli, R. Bizzarri, M. Serresi, L. Albertazzi, and F. Beltram, J Biol Chem 284(52), 36638-36646 (2009).3. A. Diaspro, Nanoscopy and Multidimensional Optical Fluorescence Microscopy‎, 448 (Chapman and Hall/CRC, Boca Raton, USA, 2010).4. S. Galiani, B. Harke, G. Vicidomini, G. Lignani, F. Benfenati, A. Diaspro, and P. Bianchini, Opt Express 20(7), 7362-7374 (2012).5. S. W. Hell, Science 316(5828), 1153-1158 (2007).