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Proceedings Paper

Multiscale diffusion of a molecular probe in a crowded environment: a concept
Author(s): Megan Currie; Chang Thao; Randi Timerman; Robb Welty; Brenden Berry; Erin D. Sheets; Ahmed A. Heikal
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Paper Abstract

Living cells are crowded with macromolecules and organelles. Yet, it is not fully understood how macromolecular crowding affects the myriad of biochemical reactions, transport and the structural stability of biomolecules that are essential to cellular function and survival. These molecular processes, with or without electrostatic interactions, in living cells are therefore expected to be distinct from those carried out in test tube in dilute solutions where excluded volumes are absent. Thus there is an urgent need to understand the macromolecular crowding effects on cellular and molecular biophysics towards quantitative cell biology. In this report, we investigated how biomimetic crowding affects both the rotational and translation diffusion of a small probe (rhodamine green, RhG). For biomimetic crowding agents, we used Ficoll-70 (synthetic polymer), bovine serum albumin and ovalbumin (proteins) at various concentrations in a buffer at room temperature. As a control, we carried out similar measurements on glycerolenriched buffer as an environment with homogeneous viscosity as a function of glycerol concentration. The corresponding bulk viscosity was measured independently to test the validity of the Stokes-Einstein model of a diffusing species undergoing a random walk. For rotational diffusion (ps–ns time scale), we used time-resolved anisotropy measurements to examine potential binding of RhG as a function of the crowding agents (surface structure and size). For translational diffusion (μs–s time scale), we used fluorescence correlation spectroscopy for single-molecule fluctuation analysis. Our results allow us to examine the diffusion model of a molecular probe in crowded environments as a function of concentration, length scale, homogeneous versus heterogeneous viscosity, size and surface structures. These biomimetic crowding studies, using non-invasive fluorescence spectroscopy methods, represent an important step towards understanding cellular biophysics and quantitative cell biology.

Paper Details

Date Published: 31 August 2015
PDF: 16 pages
Proc. SPIE 9584, Ultrafast Nonlinear Imaging and Spectroscopy III, 95840E (31 August 2015); doi: 10.1117/12.2188746
Show Author Affiliations
Megan Currie, Univ. of Minnesota, Duluth (United States)
Chang Thao, Univ. of Minnesota, Duluth (United States)
Randi Timerman, Univ. of Minnesota, Duluth (United States)
Robb Welty, Univ. of Minnesota, Duluth (United States)
Brenden Berry, Univ. of Minnesota, Duluth (United States)
Erin D. Sheets, Univ. of Minnesota, Duluth (United States)
Ahmed A. Heikal, Univ. of Minnesota, Duluth (United States)

Published in SPIE Proceedings Vol. 9584:
Ultrafast Nonlinear Imaging and Spectroscopy III
Zhiwen Liu, Editor(s)

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