New Fluorescence Correlation Spectroscopy Enabling Direct Observation of Spatiotemporal Dependence of Diffusion Constants as an Evidence of Anomalous Transport in Extracellular Matrices

Abstract

The potential of fluorescence correlation spectroscopy (FCS) is extended to enable the direct observation of anomalous subdiffusion (ASD) in inhomogeneous media that are of great importance particularly in many biological systems, such as membranes, cytoplasm, and extracellular matrices (ECMs). Because ASD can be confirmed by monitoring the spatiotemporal dependence of observable diffusion coefficients (D(obs)), the size of the effective confocal volume (V(eff)) for FCS sampling (sampling volume) was continuously changed on a scale of 300-500 nm using a motorized variable beam expander through which an illuminating laser beam passes. This new method, namely, sampling-volume-controlled (SVC)-FCS, was applied to the analysis of hyaluronan (HA) aqueous solutions where the D(obs) of light-emitting solute (Alexa 488) markedly changed, corresponding to the change in V(eff) (220-340 nm in the half-axis), because the network structure of HA of 7-33 nm (nanostructure) interferes with the material transport within it. The results indicate that moderate ASD may occur even in the presence of a small amount ( approximately 0.1 wt %) of HA in ECM. Because the change in D(obs) along with the traveling distance (the mean-square displacement) can be identified even in systems with no deformation of the autocorrelation function, this technique has a great potential for general applications to many biological systems in which ASD shows complex time and space dependences.

Characterizing anomalous diffusion in crowded polymer solutions and gels over five decades in time with variable-lengthscale fluorescence correlation spectroscopy

“…Although the propagator cannot be extracted directly from an experimental ACF, an assumption can be made about its Gaussianity, and, as discussed below, VLS-FCS data can be used to test this assumption. Several strategies have been employed for varying the size of the FCS observation volume, notably decreasing the size of the incoming excitation beam with a variable beam expander 43 or (as done in this study, allowing to cover almost an order of magnitude in lengthscales) with a variable diameter iris 8 . For fluorophores restricted to two-dimensional diffusion, the observation lengthscale can also be changed by placing the diffusion plane slightly out of focus 70 , or by using the information captured in time-lapsed images [71][72][73] .…”

New insights into diffusion in 3D crowded media by Monte Carlo simulations: effect of size, mobility and spatial distribution of obstacles

“…A great deal of information about motion of molecules in living cells has been obtained from intracellular measurements using different experimental techniques 13,[16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35] and from simulations 14,[36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52][53] . Experimental data are usually obtained by fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) techniques.…”

“…In order to give only a few examples, FRAP experiments have revealed anomalous diffusion of dextrans [17][18]23 , of proteins in cytoplasm 19,[21][22]25,33 and of proteins in dextrans 35 . FCS experiments have also shown anomalous diffusion of nanoparticles with different sizes in agarose gel 26 , of dextrans in HeLa cell cytoplasm 28 , of proteins in three-dimensional crowded media 27,31 , of Alexa488 lightemitting particles in extracellular matrices 32 , respective of the inert gold nanoparticles in the cytoplasm and nucleoplasm under various stress conditions 34 .…”

Diffusion in macromolecular crowded media: Monte Carlo simulation of obstructed diffusion vs. FRAP experiments

“…A great deal of information about motion of molecules in living cells has been obtained from intracellular measurements using different experimental techniques [7,[11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] and from simulations [10,[31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46][47][48][49]. Experimental data are usually obtained by fluorescence recovery after photobleaching (FRAP) [12-14, 16, 18, 20, 28, 30], fluorescence correlation spectroscopy (FCS) [11-23, 26-27, 29] and single particle tracking (SPT) [15,19,[24][25] techniques.…”