Investigating Spatial Heterogeneity of Nanoparticles Movement in Live Cells with Pair-Correlation Microscopy and Phasor Analysis

Abstract

How nanoparticles distribute in living cells and overcome cellular barriers are important criteria in the design of drug carriers. Pair-correlation microscopy is a correlation analysis of fluctuation in the fluorescence intensity obtained by a confocal line scan that can quantify the dynamic properties of nanoparticle diffusion including the number of mobile nanoparticles, diffusion coefficient, and transit time across a spatial distance. Due to the potential heterogeneities in nanoparticle properties and the complexity within the cellular environment, quantification of averaged auto- and pair-correlation profiles may obscure important insights into the ability of nanoparticles to deliver drugs. To overcome this issue, we used phasor analysis to develop a data standardizing method, which can segment the scanned line into several subregions according to diffusion and address the spatial heterogeneity of nanoparticles moving inside cells. The phasor analysis is a fit-free method that represents autocorrelation profiles for each pixel relative to free diffusion on the so-called phasor plots. Phasor plots can then be used to select subpopulations for which the auto- and pair-correlation analysis can be performed separately. We demonstrate the phasor analysis for pair-correlation microscopy for investigating 16 nm, Cy5-labeled silica nanoparticles diffusing across the plasma membrane and green fluorescent proteins (GFP) diffusing across nuclear envelope in MCF-7 cells.

In-cell NMR: Why and how?

“…Local nanoscale viscosities are similar among cultured mammalian cell lines [1556] and through the cell cycle [1557], except of course for proteins engaged in complexes that vary with the phases of the cell cycle [1558]. Local viscosities are similar in the cytosol and in the nucleus for proteins of average size [1559,1560]. However, important differences have been measured recently at the sub-micrometer scale: a single protein of 28 kDa can show diffusion coefficients of 0.025 x10 -9 m 2 .s -1 in the cytosol except in the few hundreds of nanometers close to actin cytoskeleton bundles, where its diffusion drops by a factor two; moreover, its diffusion coefficients in the nucleus were measured to be about 0.02 and 0.007 x10 -9 m 2 .s -1 outside and inside nucleoli, respectively [1561].…”