Preliminary Investigation on the Relationship between Raman Spectra of Beef and Metmyoglobin and Metmyoglobin Reductase Activity.

A hand-held Raman spectroscopic device was used as a rapid nondestructive testing device to predict the metmyoglobin (MetMb) and metmyoglobin reductase activity (MRA) values on the surface layer of fresh beef. Longissimus dorsi muscles were from 10 young bulls (Holstein-Friesian) from two different cattle farms (group A = 5 and B = 5). The Raman spectra of 100 samples were correlated with the MetMb and MRA values using partial least squares regression (PLSR).

Preparation of hemoglobin (Hb)-imprinted poly(ionic liquid)s via Hb-catalyzed eATRP on gold nanodendrites.

Hemoglobin (Hb)-imprinted poly(ionic liquid)s (HIPILs) were prepared on the surface of Au electrode modified with gold nanodendrites (Au/ND/HIPILs). HIPILs were synthesized with 1-vinyl-3-propyl imidazole sulfonate ionic liquids as functional monomers via electrochemically mediated atom transfer radical polymerization (eATRP) catalyzed by Hb. The Au/ND/HIPILs electrode was examined by cyclic voltammetry (CV), scanning electron microscope (SEM), and X-ray photoelectron spectroscopy (XPS).

Single Particle Tracking: From Theory to Biophysical Applications.

After three decades of developments, single particle tracking (SPT) has become a powerful tool to interrogate dynamics in a range of materials including live cells and novel catalytic supports because of its ability to reveal dynamics in the structure-function relationships underlying the heterogeneous nature of such systems. In this review, we summarize the algorithms behind, and practical applications of, SPT. We first cover the theoretical background including particle identification, localization, and trajectory reconstruction.

Optimization of Spectral and Spatial Conditions to Improve Super-Resolution Imaging of Plasmonic Nanoparticles.

Interactions between fluorophores and plasmonic nanoparticles modify the fluorescence intensity, shape, and position of the observed emission pattern, thus inhibiting efforts to optically super-resolve plasmonic nanoparticles. Herein, we investigate the accuracy of localizing dye fluorescence as a function of the spectral and spatial separations between fluorophores (Alexa 647) and gold nanorods (NRs). The distance at which Alexa 647 interacts with NRs is varied by layer-by-layer polyelectrolyte deposition while the spectral separation is tuned by using NRs with varying localized surface plasmon resonance (LSPR) maxima.

Super Temporal-Resolved Microscopy (STReM).

Super-resolution microscopy typically achieves high spatial resolution, but the temporal resolution remains low. We report super temporal-resolved microscopy (STReM) to improve the temporal resolution of 2D super-resolution microscopy by a factor of 20 compared to that of the traditional camera-limited frame rate. This is achieved by rotating a phase mask in the Fourier plane during data acquisition and then recovering the temporal information by fitting the point spread function (PSF) orientations.

Single-Molecule Kinetics of Protein Adsorption on Thin Nylon-6,6 Films.

Understanding and controlling protein adsorption on surfaces is critical to a range of biological and materials applications. Kinetic details that provide the equilibrium and nonequilibrium mechanisms are difficult to acquire. In this work, single-molecule fluorescence microscopy was used to study the adsorption of Alexa 555 labeled α-lactalbumin (α-LA) on two chemically identical but morphologically different polymer surfaces: flat and porous nylon-6,6 thin films.

Generalized recovery algorithm for 3D super-resolution microscopy using rotating point spread functions.

Super-resolution microscopy with phase masks is a promising technique for 3D imaging and tracking. Due to the complexity of the resultant point spread functions, generalized recovery algorithms are still missing. We introduce a 3D super-resolution recovery algorithm that works for a variety of phase masks generating 3D point spread functions.

Adsorption and Unfolding of a Single Protein Triggers Nanoparticle Aggregation.

The response of living systems to nanoparticles is thought to depend on the protein corona, which forms shortly after exposure to physiological fluids and which is linked to a wide array of pathophysiologies. A mechanistic understanding of the dynamic interaction between proteins and nanoparticles and thus the biological fate of nanoparticles and associated proteins is, however, often missing mainly due to the inadequacies in current ensemble experimental approaches. Through the application of a variety of single molecule and single particle spectroscopic techniques in combination with ensemble level characterization tools, we identified different interaction pathways between gold nanorods and bovine serum albumin depending on the protein concentration.

Characterization of Porous Materials by Fluorescence Correlation Spectroscopy Super-resolution Optical Fluctuation Imaging.

Porous materials such as cellular cytosol, hydrogels, and block copolymers have nanoscale features that determine macroscale properties. Characterizing the structure of nanopores is difficult with current techniques due to imaging, sample preparation, and computational challenges. We produce a super-resolution optical image that simultaneously characterizes the nanometer dimensions of and diffusion dynamics within porous structures by correlating stochastic fluctuations from diffusing fluorescent probes in the pores of the sample, dubbed here as "fluorescence correlation spectroscopy super-resolution optical fluctuation imaging" or "fcsSOFI".

Conformational transitions in the glycine-bound GluN1 NMDA receptor LBD via single-molecule FRET.

The N-methyl-D-aspartate receptor (NMDAR) is a member of the glutamate receptor family of proteins and is responsible for excitatory transmission. Activation of the receptor is thought to be controlled by conformational changes in the ligand binding domain (LBD); however, glutamate receptor LBDs can occupy multiple conformations even in the activated form. This work probes equilibrium transitions among NMDAR LBD conformations by monitoring the distance across the glycine-bound LBD cleft using single-molecule Förster resonance energy transfer (smFRET).

Tuning the acoustic frequency of a gold nanodisk through its adhesion layer.

To fabricate robust metallic nanostructures with top-down patterning methods such as electron-beam lithography, an initial nanometer-scale layer of a second metal is deposited to promote adhesion of the metal of interest. However, how this nanoscale layer affects the mechanical properties of the nanostructure and how adhesion layer thickness controls the binding strength to the substrate are still open questions. Here we use ultrafast laser pulses to impulsively launch acoustic phonons in single gold nanodisks with variable titanium layer thicknesses, and observe an increase in phonon frequencies as a thicker adhesion layer facilitates stronger binding to the glass substrate.

Excitonic energy migration in conjugated polymers: the critical role of interchain morphology.

Excitonic energy migration was studied using single molecule spectroscopy of individual conjugated polymer (CP) chains and aggregates. To probe the effect of interchain morphology on energy migration in CP, tailored interchain morphologies were achieved using solvent vapor annealing to construct polymer aggregates, which were then studied with single aggregate spectroscopy. We report that highly ordered interchain packing in regioregular poly(3-hexylthiophene) (rr-P3HT) enables long-range interchain energy migration, while disordered packing in regiorandom poly(3-hexylthiophene) (rra-P3HT), even in aggregates of just a few chains, can dramatically impede the interchain mechanism.

Fast Step Transition and State Identification (STaSI) for Discrete Single-Molecule Data Analysis.

We introduce a step transition and state identification (STaSI) method for piecewise constant single-molecule data with a newly derived minimum description length equation as the objective function. We detect the step transitions using the Student's test and group the segments into states by hierarchical clustering. The optimum number of states is determined based on the minimum description length equation.

Charge-dependent transport switching of single molecular ions in a weak polyelectrolyte multilayer.

The tunable nature of weak polyelectrolyte multilayers makes them ideal candidates for drug loading and delivery, water filtration, and separations, yet the lateral transport of charged molecules in these systems remains largely unexplored at the single molecule level. We report the direct measurement of the charge-dependent, pH-tunable, multimodal interaction of single charged molecules with a weak polyelectrolyte multilayer thin film, a 10 bilayer film of poly(acrylic acid) and poly(allylamine hydrochloride) PAA/PAH. Using fluorescence microscopy and single-molecule tracking, two modes of interaction were detected: (1) adsorption, characterized by the molecule remaining immobilized in a subresolution region and (2) diffusion trajectories characteristic of hopping (D ∼ 10(-9) cm(2)/s).

High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: a single-molecule study.

The retention and elution of proteins in ion-exchange chromatography is routinely controlled by adjusting the mobile phase salt concentration. It has repeatedly been observed, as judged from adsorption isotherms, that the apparent heterogeneity of adsorption is lower at more-eluting, higher ionic strength. Here, we present an investigation into the mechanism of this phenomenon using a single-molecule, super-resolution imaging technique called motion-blur Points Accumulation for Imaging in Nanoscale Topography (mbPAINT).

Unified superresolution experiments and stochastic theory provide mechanistic insight into protein ion-exchange adsorptive separations.

Chromatographic protein separations, immunoassays, and biosensing all typically involve the adsorption of proteins to surfaces decorated with charged, hydrophobic, or affinity ligands. Despite increasingly widespread use throughout the pharmaceutical industry, mechanistic detail about the interactions of proteins with individual chromatographic adsorbent sites is available only via inference from ensemble measurements such as binding isotherms, calorimetry, and chromatography. In this work, we present the direct superresolution mapping and kinetic characterization of functional sites on ion-exchange ligands based on agarose, a support matrix routinely used in protein chromatography.

Troika of single particle tracking programing: SNR enhancement, particle identification, and mapping.

Single particle tracking (SPT) techniques provide a microscopic approach to probe in vivo and in vitro structure and reactions. Automatic analysis of SPT data with high efficiency and accuracy spurs the development of SPT algorithms. In this perspective, we review a range of available techniques used in SPT analysis programs.

Super-resolution mbPAINT for optical localization of single-stranded DNA.

We demonstrate the application of superlocalization microscopy to identify sequence-specific portions of single-stranded DNA (ssDNA) with sequence resolution of 50 nucleotides, corresponding to a spatial resolution of 30 nm. Super-resolution imaging was achieved using a variation of a single-molecule localization method, termed as "motion blur" point accumulation for imaging in nanoscale topography (mbPAINT). The target ssDNA molecules were immobilized on the substrate.

Improved analysis for determining diffusion coefficients from short, single-molecule trajectories with photoblinking.

Two maximum likelihood estimation (MLE) methods were developed for optimizing the analysis of single-molecule trajectories that include phenomena such as experimental noise, photoblinking, photobleaching, and translation or rotation out of the collection plane. In particular, short, single-molecule trajectories with photoblinking were studied, and our method was compared to existing analytical techniques applied to simulated data. The optimal method for various experimental cases was established, and the optimized MLE method was applied to a real experimental system: single-molecule diffusion of fluorescent molecular machines known as nanocars.