Tolerance to aberration and misalignment in a two-point-resolving image inversion interferometer.

Image inversion interferometry can measure the separation of two incoherent point sources at or near the quantum limit. This technique has the potential to improve upon current state-of-the-art imaging technologies, with applications ranging from microbiology to astronomy. However, unavoidable aberrations and imperfections in real systems may prevent inversion interferometry from providing an advantage for real-world applications.

Publisher's Note: Estimation of the diffusion constant from intermittent trajectories with variable position uncertainties [Phys. Rev. E 93, 042401 (2016)].

This corrects the article DOI: 10.1103/PhysRevE.93.

Estimation of the diffusion constant from intermittent trajectories with variable position uncertainties.

The movement of a particle described by Brownian motion is quantified by a single parameter, D, the diffusion constant. The estimation of D from a discrete sequence of noisy observations is a fundamental problem in biological single-particle tracking experiments since it can provide information on the environment and/or the state of the particle itself via the hydrodynamic radius. Here, we present a method to estimate D that takes into account several effects that occur in practice, important for the correct estimation of D, and that have hitherto not been combined together for an estimation of D.

Optimal aggregation of FcεRI with a structurally defined trivalent ligand overrides negative regulation driven by phosphatases.

To investigate why responses of mast cells to antigen-induced IgE receptor (FcεRI) aggregation depend nonlinearly on antigen dose, we characterized a new artificial ligand, DF3, through complementary modeling and experimentation. This ligand is a stable trimer of peptides derived from bacteriophage T4 fibritin, each conjugated to a hapten (DNP). We found low and high doses of DF3 at which degranulation of mast cells sensitized with DNP-specific IgE is minimal, but ligand-induced receptor aggregation is comparable to aggregation at an intermediate dose, optimal for degranulation.

Multi-color quantum dot tracking using a high-speed hyperspectral line-scanning microscope.

Many cellular signaling processes are initiated by dimerization or oligomerization of membrane proteins. However, since the spatial scale of these interactions is below the diffraction limit of the light microscope, the dynamics of these interactions have been difficult to study on living cells. We have developed a novel high-speed hyperspectral microscope (HSM) to perform single particle tracking of up to 8 spectrally distinct species of quantum dots (QDs) at 27 frames per second.

ErbB1 dimerization is promoted by domain co-confinement and stabilized by ligand binding.

The extent to which ligand occupancy and dimerization contribute to erbB1 signaling is controversial. To examine this, we used two-color quantum-dot tracking for visualization of the homodimerization of human erbB1 and quantification of the dimer off-rate (k(off)) on living cells. Kinetic parameters were extracted using a three-state hidden Markov model to identify transition rates between free, co-confined and dimerized states.

Determining FcεRI diffusional dynamics via single quantum dot tracking.

Single-particle tracking (SPT) using fluorescent quantum dots (QDs) provides high-resolution spatial-temporal information on receptor dynamics that cannot be obtained through traditional biochemical techniques. In particular, the high brightness and photostability of QDs make them ideal probes for SPT on living cells. We have shown that QD-labeled IgE can be used to characterize the dynamics of the high-affinity IgE Receptor.

Time-resolved three-dimensional molecular tracking in live cells.

We report a method for tracking individual quantum dot (QD) labeled proteins inside of live cells that uses four overlapping confocal volume elements and active feedback once every 5 ms to follow three-dimensional molecular motion. This method has substantial advantages over three-dimensional molecular tracking methods based upon charge-coupled device cameras, including increased Z-tracking range (10 μm demonstrated here), substantially lower excitation powers (15 μW used here), and the ability to perform time-resolved spectroscopy (such as fluorescence lifetime measurements or fluorescence correlation spectroscopy) on the molecules being tracked. In particular, we show for the first time fluorescence photon antibunching of individual QD labeled proteins in live cells and demonstrate the ability to track individual dye-labeled nucleotides (Cy5-dUTP) at biologically relevant transport rates.

Systematic method for the kinetic modeling of temporally resolved hyperspectral microscope images of fluorescently labeled cells.

In this paper we report the application of a novel method for fitting kinetic models to temporally resolved hyperspectral images of fluorescently labeled cells to mathematically resolve pure-component spatial images, pure-component spectra, and pure-component reaction profiles. The method is demonstrated on one simulated image and two experimental cell images, including human embryonic kidney cells (HEK 293) and human A549 pulmonary type II epithelial cells. In both cell images, inhibitor kappa B kinase alpha (IKK(alpha)) and mitochondrial antiviral signaling protein (MAVS) were labeled with green and yellow fluorescent protein, respectively.

Methods for kinetic modeling of temporally resolved hyperspectral confocal fluorescence images.

Elucidating kinetic information (rate constants) from temporally resolved hyperspectral confocal fluorescence images offers some very important opportunities for the interpretation of spatially resolved hyperspectral confocal fluorescence images but also presents significant challenges, these being (1) the massive amount of data contained in a series of time-resolved spectral images (one time course of spectral data for each pixel) and (2) unknown concentrations of the reactants and products at time = 0, a necessary precondition normally required by traditional kinetic fitting approaches. This paper describes two methods for solving these problems: direct nonlinear (DNL) estimation of all parameters and separable least squares (SLS). The DNL method can be applied to reactions of any rate law, while the SLS method is restricted to first-order reactions.

Experimental monitoring and data analysis tools for protein folding: study of steady-state evolution and modeling of kinetic transients by multitechnique and multiexperiment data fusion.

Protein folding is a complex process that can take place through different pathways depending on the inducing agent and on the monitored time scale. This diversity of possibilities requires a good design of experiments and powerful data analysis tools that allow operating with multitechnique measurements and/or with diverse experiments related to different aspects of the process of interest. Multivariate curve resolution-alternating least squares (MCR-ALS) has been the core methodology used to perform multitechnique and/or multiexperiment data analysis.