The application of fluorescent proteins in live cells has greatly improved our ability to study molecular mobility, which both reflects molecular function in live cells and reveals the properties of the local environment. Although measuring molecular mobility with fluorescent fusion proteins is powerful and convenient, certain experiments still require exogenous macromolecules to be loaded into cells. Cell viability provides a rough gauge of cellular damage following membrane permeabilization, but it is unknown how permeabilization will affect intracellular mobility. We have used fluorescence correlation spectroscopy to measure the intracellular dynamics of the enhanced green fluorescent protein (EGFP) in living human embryonic kidney (HEK) cells under conditions where the EGFP is either expressed or loaded using streptolysin O (SLO) permeabilization to determine how permeabilization effects mobility. We found that purified EGFP loaded with SLO has the same mobility as the expressed EGFP, while the mobility of the expressed EGFP after SLO permeabilization treatment becomes slightly slower. Our results indicate that SLO permeabilization is often accompanied by the loss of cellular soluble proteins to the surrounding medium, which explains the apparent decrease in diffusion rates following treatment. These measurements are also relevant to the role of molecular crowding in the intracellular mobility of proteins.
KEYWORDS: Fluorescence correlation spectroscopy, Luminescence, Fluorescence spectroscopy, Spectroscopy, Point spread functions, 3D modeling, Objectives, Data modeling, Statistical modeling, 3D metrology
Information recovery in fluorescence fluctuation spectroscopy requires accurate models both for the physical dynamics observed and for the effective size and shape of the sample region from which fluorescence signals are measured. In both one- and two-photon fluctuation spectroscopy, the so called observation volume is assumed to be well approximated by a three dimensional Gaussian (3DG) function. Here, we present wave optics computations that provide an accurate representation of the true profile for the fluorescence measurement with two-photon excitation. Fluorescence correlation spectroscopy (FCS) curves are computed for these true profiles for a variety of optical configurations, and we demonstrate that under most illumination conditions the 3DG based FCS models do provide reasonable approximations to the measured FCS curves.
Fluorescence correlation spectroscopy (FCS) and related distribution analysis techniques have become extremely important and widely used research tools for analyzing the dynamics, kinetics, interactions, and mobility of biomolecules. However, it is not widely recognized that photophysical dynamics can dramatically influence the calibration of fluctuation spectroscopy instrumentation. While the basic theories for fluctuation spectroscopy methods are well established, there have not been quantitative models to characterize the photophysical-induced variations observed in measured fluctuation spectroscopy data under varied excitation conditions. We introduce quantitative models to characterize how the fluorescence observation volumes in one-photon confocal microscopy are modified by excitation saturation as well as corresponding models for the effect of the volume changes in FCS. We introduce a simple curve fitting procedure to model the role of saturation in FCS measurements and demonstrate its accuracy in fitting measured correlation curves over a wide range of excitation conditions.
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