title: Diffusion Tensor Analysis by Two-Dimensional Pair Correlation of Fluorescence Fluctuations in Cells.
abstract: In a living cell, the movement of biomolecules is highly regulated by the cellular organization into subcompartments that impose barriers to diffusion, can locally break the spatial isotropy, and ultimately guide these molecules to their targets. Despite the pivotal role of these processes, experimental tools to fully probe the complex connectivity (and accessibility) of the cell interior with adequate spatiotemporal resolution are still lacking. Here, we show how the heterogeneity of molecular dynamics and the location of barriers to molecular motion can be mapped in live cells by exploiting a two-dimensional (2D) extension of the pair correlation function (pCF) analysis. Starting from a time series of images collected for the same field of view, the resulting 2D pCF is calculated in the proximity of each point for each time delay and allows us to probe the spatial distribution of the molecules that started from a given pixel. This 2D pCF yields an accurate description of the preferential diffusive routes. Furthermore, we combine this analysis with the image-derived mean-square displacement approach and gain information on the average nanoscopic molecular displacements in different directions. Through these quantities, we build a fluorescence-fluctuation-based diffusion tensor that contains information on speed and directionality of the local dynamical processes. Contrary to classical fluorescence correlation spectroscopy and related methods, this combined approach can distinguish between isotropic and anisotropic local diffusion. We argue that the measurement of this iMSD tensor will contribute to advance our understanding of the role played by the intracellular environment in the regulation of molecular diffusion at the nanoscale.
authors: NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro, Pisa, Italy; Center for Nanotechnology Innovation @NEST, Istituto Italiano di Technologia, Piazza San Silvestro, Pisa, Italy. Electronic address: carmine.dirienzo@sns.it.
authors: Carmine
authors: C
authors: Di Rienzo
authors: Center for Nanotechnology Innovation @NEST, Istituto Italiano di Technologia, Piazza San Silvestro, Pisa, Italy.
authors: Francesco
authors: F
authors: Cardarelli
authors: NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro, Pisa, Italy.
authors: Mariagrazia
authors: M
authors: Di Luca
authors: NEST, Scuola Normale Superiore and Istituto Nanoscienze-CNR, Piazza San Silvestro, Pisa, Italy; Center for Nanotechnology Innovation @NEST, Istituto Italiano di Technologia, Piazza San Silvestro, Pisa, Italy.
authors: Fabio
authors: F
authors: Beltram
authors: Department of Biomedical Engineering, University of California, Irvine, Irvine, California. Electronic address: egratton@uci.edu.
authors: Enrico
authors: E
authors: Gratton
mesh_terms: D000818:Animals; D016466:CHO Cells; D002470:Cell Survival; D006224:Cricetinae; D003412:Cricetulus; D004058:Diffusion; D007091:Image Processing, Computer-Assisted; D008856:Microscopy, Fluorescence; D056004:Molecular Dynamics Simulation; D009068:Movement; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism
chemical_list: D016283:Proto-Oncogene Proteins p21(ras)