Feedback localization of freely diffusing fluorescent particles near the optical shot-noise limit

We report near-optimal tracking of freely diffusing fluorescent particles in a quasi-two-dimensional geometry via photon counting and real-time feedback. We present a quantitative statistical model of our feedback network and find excellent agreement with the experiment. We monitor the motion of a single fluorescent particle with a sensitivity of 15 nm/sqrt Hz while collecting fewer than 5000 fluorescence photons/s. Fluorescent microspheres (diffusion coefficient 1.3 μm^2/s) are tracked with a root-mean-square tracking error of 170 nm, within a factor of 2 of the theoretical limit set by photon counting shot noise

Intramolecular fluorescence correlation spectroscopy in a feedback tracking microscope

We derive the statistics of the signals generated by shape fluctuations of large molecules studied by feedback tracking microscopy. We account for the influence of intramolecular dynamics on the response of the tracking system, and derive a general expression for the fluorescence autocorrelation function that applies when those dynamics are linear. We show that tracking provides enhanced sensitivity to translational diffusion, molecular size, heterogeneity and long time-scale decays in comparison to traditional fluorescence correlation spectroscopy. We demonstrate our approach by using a three-dimensional tracking microscope to study genomic $\lambda$-phage DNA molecules with various fluorescence label configurations.Comment: 11 pages, 5 figures, supplemental info: http://minty.stanford.edu/papers/Publications/McHale10aSI.pd

A Game-Based Learning Approach To Enhance Understanding Of Interface Design Principles In Design Education

The design of user interface is an important and challenging topic for student designers to understand and master. The eight principles of good User Interface (UI) design are often taught using primarily cognitive approaches, which can leave room for improvement in students’ ability to apply the principles in a variety of contexts. Game-based learning tools are recognised to be beneficial in university classrooms across a variety of discipline areas and topics due to their capacity to increase engagement. This project presents a first prototype for an instructional tool that leverages constructionism and embodied learning to enhance students’ understanding and application of these principles. This tool takes the form of a board game, thus encouraging peer learning. To test the prototype, three usability tests were carried out. Each user group was unique, the first being internal to the design team, the second having some prior exposure of the subject, and the third, having no prior experience at all. In each sessions, the participants were presented with a series of UI challenges, for which they were asked to construct suitable design solutions. Following the sessions, and where possible, the quality of these solutions were evaluated against a scoring system. This initial study suggests that instructional board games may be flexible enough to support learning outcomes at various stages of knowledge and skills acquisition among different learner groups

Fluctuations in closed-loop fluorescent particle tracking

We present a comprehensive theory of closed-loop particle tracking for calculating the statistics of a diffusing fluorescent particle’s motion relative to the tracking lock point. A detailed comparison is made between the theory and experimental results, with excellent quantitative agreement found in all cases. A generalization of the theory of (open-loop) fluorescence correlation spectroscopy is developed, and the relationship to previous results is discussed. Two applications of the statistical techniques are given: a method for determining a tracked particle’s localization and an algorithm for rapid particle classification based on real-time analysis of the tracking control signal

Identification, characterization, and gene expression analysis of nucleotide binding site (NB)-type resistance gene homologues in switchgrass

Abstract Background Switchgrass (Panicum virgatum L.) is a warm-season perennial grass that can be used as a second generation bioenergy crop. However, foliar fungal pathogens, like switchgrass rust, have the potential to significantly reduce switchgrass biomass yield. Despite its importance as a prominent bioenergy crop, a genome-wide comprehensive analysis of NB-LRR disease resistance genes has yet to be performed in switchgrass. Results In this study, we used a homology-based computational approach to identify 1011 potential NB-LRR resistance gene homologs (RGHs) in the switchgrass genome (v 1.1). In addition, we identified 40 RGHs that potentially contain unique domains including major sperm protein domain, jacalin-like binding domain, calmodulin-like binding, and thioredoxin. RNA-sequencing analysis of leaf tissue from ‘Alamo’, a rust-resistant switchgrass cultivar, and ‘Dacotah’, a rust-susceptible switchgrass cultivar, identified 2634 high quality variants in the RGHs between the two cultivars. RNA-sequencing data from field-grown cultivar ‘Summer’ plants indicated that the expression of some of these RGHs was developmentally regulated. Conclusions Our results provide useful insight into the molecular structure, distribution, and expression patterns of members of the NB-LRR gene family in switchgrass. These results also provide a foundation for future work aimed at elucidating the molecular mechanisms underlying disease resistance in this important bioenergy crop

Feedback Tracking and Correlation Spectroscopy of Fluorescent Nanoparticles and Biomolecules

The best way to study dynamic fluctuations in single molecules or nanoparticles is to look at only one particle at a time, and to look for as long as possible. Brownian motion makes this difficult, as molecules move along random trajectories that carry them out of any fixed field of view. We developed an instrument that tracks the Brownian motion of single fluorescent molecules in three dimensions and in real-time while measuring fluorescence with nanosecond time resolution and single-photon sensitivity. The apparatus increases observation times by approximately three orders of magnitude while improving data-collecting efficiency by locking tracked objects to a high-intensity region of the excitation laser. As a first application of our technique, we tracked and studied the fluorescence statistics of semiconductor quantum dots. Our measurements were well resolved at 10ns correlation times, allowing measurement of photon anti-bunching on single particles in solution for the first time. We observed variations of (34 ± 16)% in the fluorescence lifetimes and (23 ± 18)% in the absorption cross-sections within an aqueous quantum dot sample, confirming that these variations are real, not artifacts of the immobilization methods previously used to study them. Additionally, we studied quantum dot fluorescence intermittency and its dependence on 2-mercaptoethanol, finding evidence that the chemical suppresses blinking on short time-scales (&#60;1s) by reducing the lifetime of the dark state. Finally, we studied the translational and intramolecular Brownian motion of λ-phage DNA molecules. Our apparatus decouples these motions almost completely, and yielded a translational diffusion coefficient estimate D=(0.71 ± 0.05)μm²/s lying between previous measurements for this molecule under identical solution conditions but with less precise techniques. Our measurements show clear evidence of intramolecular motion of the polymer chain in the form of statistical correlations on time-scales up to 1s, but we have not yet been able to determine the influence of solvent interactions on these dynamics.</p

Evolution: advancing Communities of Practice in naval intelligence

The US Navy is embracing the principles of Knowledge Management (KM). One of the key components of KM is the Community of Practice. Communities of Practice are groups that form to share what they know, and to learn from one another regarding some aspect of their work. Organizations are strengthened through an improved network of contacts and enhanced productivity from their personnel. Personnel benefit through peer-group recognition and continuous learning. This thesis seeks to provide an understanding of how the Naval Intelligence Community, through the implementation of Communities of Practice, can reduce duplication of effort, increase collaboration between its personnel, and better support the resources in its people. In this thesis, we have provided a blueprint for building a successful unclassified Community of Practice for Naval Intelligence. This blueprint is designed to support replication on classified networks.http://archive.org/details/evolutiondvancin10945963Lieutenant, United States NavyApproved for public release; distribution is unlimited

Bayesian Estimation for Species Identification in Single-Molecule Fluorescence Microscopy

In this article we describe a recursive Bayesian estimator for the identification of diffusing fluorophores using photon arrival-time data from a single spectral channel. We present derivations for all relevant diffusion and fluorescence models, and we use simulated diffusion trajectories and photon streams to evaluate the estimator's performance. We consider simplified estimation schemes that bin the photon counts within time intervals of fixed duration, and show that they can perform well in realistic parameter regimes. The latter results indicate the feasibility of performing identification experiments in real time. It will be straightforward to generalize our approach for use in more complicated scenarios, e.g., with multiple spectral channels or fast photophysical dynamics

Quantum Dot Photon Statistics Measured by Three-Dimensional Particle Tracking

We present an instrument for performing correlation spectroscopy on single fluorescent particles while tracking their Brownian motion in three dimensions using real-time feedback. By tracking CdSe/ZnS quantum dots in water (diffusion coefficient ∼ 20 μm^2/s), we make the first measurements of photon antibunching (at ∼10 ns) on single fluorophores free in solution and find fluorescence lifetime heterogeneity within a quantum dot sample. In addition, we show that 2-mercaptoethanol suppresses short time-scale intermittency (1 ms to 1 s) in quantum dot fluorescence by reducing time spent in the off-state