Correlation functions quantify super-resolution images and estimate apparent clustering due to over-counting

We present an analytical method to quantify clustering in super-resolution localization images of static surfaces in two dimensions. The method also describes how over-counting of labeled molecules contributes to apparent self-clustering and how the effective lateral resolution of an image can be determined. This treatment applies to clustering of proteins and lipids in membranes, where there is significant interest in using super-resolution localization techniques to probe membrane heterogeneity. When images are quantified using pair correlation functions, the magnitude of apparent clustering due to over-counting will vary inversely with the surface density of labeled molecules and does not depend on the number of times an average molecule is counted. Over-counting does not yield apparent co-clustering in double label experiments when pair cross-correlation functions are measured. We apply our analytical method to quantify the distribution of the IgE receptor (Fc{\epsilon}RI) on the plasma membranes of chemically fixed RBL-2H3 mast cells from images acquired using stochastic optical reconstruction microscopy (STORM) and scanning electron microscopy (SEM). We find that apparent clustering of labeled IgE bound to Fc{\epsilon}RI detected with both methods arises from over-counting of individual complexes. Thus our results indicate that these receptors are randomly distributed within the resolution and sensitivity limits of these experiments.Comment: 22 pages, 5 figure

Analysis of vaginal microbicide film hydration kinetics by quantitative imaging refractometry

We have developed a quantitative imaging refractometry technique, based on holographic phase microscopy, as a tool for investigating microscopic structural changes in water-soluble polymeric materials. Here we apply the approach to analyze the structural degradation of vaginal topical microbicide films due to water uptake. We implemented transmission imaging of 1-mm diameter film samples loaded into a flow chamber with a 1.5×2 mm field of view. After water was flooded into the chamber, interference images were captured and analyzed to obtain high resolution maps of the local refractive index and subsequently the volume fraction and mass density of film material at each spatial location. Here, we compare the hydration dynamics of a panel of films with varying thicknesses and polymer compositions, demonstrating that quantitative imaging refractometry can be an effective tool for evaluating and characterizing the performance of candidate microbicide film designs for anti-HIV drug delivery. © 2014 Rinehart et al

Spatiotemporal fluctuation analysis of molecular diffusion laws in live-cell membranes

A present challenge of membrane biophysics is deciphering the dynamic behavior of molecules, such as lipids and proteins, within the natural environment of a living-cell membrane. Here, a fluorescence fluctuation-based approach will be described, which makes it possible to probe the \ue2\u80\u9cdiffusion law\ue2\u80\u9d of molecules directly from imaging, in the form of a mean square displacement vs time-delay plot (iMSD), with no need for interpretative models. Of note, the presented approach does not require extraction of the molecular trajectories nor the use of bright fluorophores. Conversely, it can be used at high fluorophore density and with relatively dim fluorophores, such as GFP-tagged molecules transiently expressed within cells. The ability of this approach to resolve average molecular dynamic properties well below the diffraction limit will be discussed. Overall, this novel approach is proposed as a powerful tool for the determination of kinetic and thermodynamic parameters over wide spatial and temporal scales