Browsing by Author "Shuang, Bo"
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Item Adsorption and Unfolding of a Single Protein Triggers Nanoparticle Aggregation(American Chemical Society, 2016) Dominguez-Medina, Sergio; Kisley, Lydia; Tauzin, Lawrence J.; Hoggard, Anneli; Shuang, Bo; Indrasekara, A. Swarnapali D.S.; Chen, Sishan; Wang, Lin-Yung; Derry, Paul J.; Liopo, Anton; Zubarev, Eugene R.; Landes, Christy F.; Link, StephanThe 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. Overall, we found that local changes in protein concentration influence everything from cancer cell uptake to nanoparticle stability and even protein secondary structure. We envision that our findings and methods will lead to strategies to control the associated pathophysiology of nanoparticle exposure in vivo.Item Charge-Dependent Transport Switching of Single Molecular Ions in a Weak Polyelectrolyte Multilayer(American Chemical Society, 2014) Tauzin, Lawrence J.; Shuang, Bo; Kisley, Lydia; Mansur, Andrea P.; Chen, Jixin; de Leon, Al; Advincula, Rigoberto C.; Landes, Christy F.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 cm2/s). Radius of gyration evolution analysis and comparison with simulated trajectories confirmed the coexistence of the two transport modes in the same single molecule trajectories. A mechanistic explanation for the probe and condition mediated dynamics is proposed based on a combination of electrostatics and a reversible, pH-induced alteration of the nanoscopic structure of the film. Our results are in good agreement with ensemble studies conducted on similar films, confirm a previously-unobserved hopping mechanism for charged molecules in polyelectrolyte multilayers, and demonstrate that single molecule spectroscopy can offer mechanistic insight into the role of electrostatics and nanoscale tunability of transport in weak polyelectrolyte multilayers.Item Data Processing for Modern Microscopy: Faster, More Accurate, and More Reproducible(2016-04-22) Shuang, Bo; Landes, Christy F.Modern medicine is currently facing the challenge of improving wellness for the general public, especially the quality of later life that is threatened by aging-associated chronic conditions and diseases. These improvements are heavily dependent on scientific discovery of biological processes occurring on the nanoscale. Single-molecule super-resolution microscopy has made vital advancements to further understand disease mechanisms, revolutionize genome sequencing, and improve drug purification efficiencies. However, single-molecule techniques usually generate large amounts of complex data. Interpretation of this complex data to gather useful information requires sophisticated data processing techniques. In this thesis, several new data processing techniques are presented to extract valuable information in single-molecule data efficiently and accurately. First, maximum likelihood estimators have been proposed to calculate the diffusion coefficient for short single particle tracking trajectories, which can improve the space and time sensitivities of single particle tracking studies. The trade-off between accuracy and precision of different estimation methods is discussed to guide the selection of the developed estimation methods. Secondly, a newly developed single particle tracking package allows users to automatically process large amounts of raw single particle data to produce single-molecule tracking results. This is accomplished by a robust fitting approach that has been developed to localize single emitters to achieve roughly a 10 nm spatial resolution. Moreover, a 3D super-resolution algorithm for general 3D point spread functions has been explored, serving as the first open source program for 3D super-resolution recovery. Finally, an analysis algorithm for single-molecule Förster resonance energy transfer has been created to identify fast step transitions and determine the optimum number of states from single-molecule data. This algorithm outperforms the established cutting-edge algorithm in accuracy and speed. Overall, this thesis offers a broad range of data analysis techniques that benefit the powerful research in single-molecule studies.Item Fast Step Transition and State Identification (STaSI) for Discrete Single-Molecule Data Analysis(American Chemical Society, 2014) Shuang, Bo; Cooper, David; Taylor, J. Nick; Kisley, Lydia; Chen, Jixin; Wang, Wenxiao; Li, Chun Biu; Komatsuzaki, Tamiki; Landes, Christy F.; Rice Quantum InstituteWe 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 t test and group the segments into states by hierarchical clustering. The optimum number of states is determined based on the minimum description length equation. This method provides comprehensive, objective analysis of multiple traces requiring few user inputs about the underlying physical models and is faster and more precise in determining the number of states than established and cutting-edge methods for single-molecule data analysis. Perhaps most importantly, the method does not require either time-tagged photon counting or photon counting in general and thus can be applied to a broad range of experimental setups and analytes.Item Generalized recovery algorithm for 3D super-resolution microscopy using rotating point spread functions(Springer Nature, 2016) Shuang, Bo; Wang, Wenxiao; Shen, Hao; Tauzin, Lawrence J.; Flatebo, Charlotte; Chen, Jianbo; Moringo, Nicholas A.; Bishop, Logan D.C.; Kelly, Kevin F.; Landes, Christy F.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. A fast deconvolution process generates initial guesses, which are further refined by least squares fitting. Overfitting is suppressed using a machine learning determined threshold. Preliminary results on experimental data show that our algorithm can be used to super-localize 3D adsorption events within a porous polymer film and is useful for evaluating potential phase masks. Finally, we demonstrate that parallel computation on graphics processing units can reduce the processing time required for 3D recovery. Simulations reveal that, through desktop parallelization, the ultimate limit of real-time processing is possible. Our program is the first open source recovery program for generalized 3D recovery using rotating point spread functions.Item High ionic strength narrows the population of sites participating in protein ion-exchange adsorption: A single-molecule study(Elsevier, 2014) Kisley, Lydia; Chen, Jixin; Mansur, Andrea P.; Dominguez-Medina, Sergio; Kulla, Eliona; Kang, Marci; Shuang, Bo; Kourentzi, Katerina; Poongavanam, Mohan-Vivekanandan; Dhamane, Sagar; Willson, Richard C.; Landes, Christy F.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). We observed that the number of functional adsorption sites was smaller at high ionic strength and that these sites had reduced desorption kinetic heterogeneity, and thus narrower predicted elution profiles, for the anion-exchange adsorption of ?-lactalbumin on an agarose-supported, clustered-charge ligand stationary phase. Explanations for the narrowing of the functional population such as inter-protein interactions and protein or support structural changes were investigated through kinetic analysis, circular dichroism spectroscopy, and microscopy of agarose microbeads, respectively. The results suggest the reduction of heterogeneity is due to both electrostatic screening between the protein and ligand and tuning the steric availability within the agarose support. Overall, we have shown that single molecule spectroscopy can aid in understanding the influence of ionic strength on the population of functional adsorbent sites participating in the ion-exchange chromatographic separation of proteins.Item Improved Analysis for Determining Diffusion Coefficients from Short, Single-Molecule Trajectories with Photoblinking(American Chemical Society, 2013) Shuang, Bo; Byers, Chad P.; Kisley, Lydia; Wang, Lin-Yung; Zhao, Julia; Morimura, Hiroyuki; Link, Stephan; Landes, Christy F.; Rice Quantum InstituteTwo 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 with 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.Item Super-Resolution mbPAINT for Optical Localization of Single-Stranded DNA(American Chemical Society, 2013) Chen, Jixin; Bremauntz, Alberto; Kisley, Lydia; Shuang, Bo; Landes, Christy F.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 ﾓmotion blurﾔ point accumulation for imaging in nanoscale topography (mbPAINT). The target ssDNA molecules were immobilized on the substrate. Short, dye-labeled, and complementary ssDNA molecules stochastically bound to the target ssDNA, with repeated binding events allowing super-resolution. Sequence specificity was demonstrated via the use of a control, noncomplementary probe. The results support the possibility of employing relatively inexpensive short ssDNAs to identify gene sequence specificity with improved resolution in comparison to the existing methods.Item Troika of single particle tracking programing: SNR enhancement, particle identification, and mapping(Royal Society of Chemistry, 2014) Shuang, Bo; Chen, Jixin; Kisley, Lydia; Landes, Christy F.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. In addition, we present an example SPT program step-by-step to provide a guide so that researchers can use, modify, and/or write a SPT program for their own purposes.Item Tuning the acoustic frequency of a gold nanodisk through its adhesion layer(Nature Publishing Group, 2015) Chang, Wei-Shun; Wen, Fangfang; Chakraborty, Debadi; Su, Man-Nung; Zhang, Yue; Shuang, Bo; Nordlander, Peter; Sader, John E.; Halas, Naomi J.; Link, Stephan; Laboratory for NanophotonicsTo 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. In addition to an all-optical interrogation of nanoscale mechanical properties, our results show that the adhesion layer can be used to controllably modify the acoustic phonon modes of a gold nanodisk. This direct coupling between optically excited plasmon modes and phonon modes can be exploited for a variety of emerging optomechanical applications.