Joseph N. Forkey

Bio: Joseph N. Forkey is an academic researcher from University of Pennsylvania. The author has contributed to research in topics: Rayleigh scattering & Total internal reflection fluorescence microscope. The author has an hindex of 18, co-authored 35 publications receiving 3858 citations. Previous affiliations of Joseph N. Forkey include Glenn Research Center & Princeton University.

Myosin V Walks Hand-Over-Hand: Single Fluorophore Imaging with 1.5-nm Localization

Abstract: Myosin V is a dimeric molecular motor that moves processively on actin, with the center of mass moving 37 nanometers for each adenosine triphosphate hydrolyzed. We have labeled myosin V with a single fluorophore at different positions in the light-chain domain and measured the step size with a standard deviation of 1.5 nanometers, with 0.5-second temporal resolution, and observation times of minutes. The step size alternates between 37 2x nm and 37 – 2x, where x is the distance along the direction of motion between the dye and the midpoint between the two heads. These results strongly support a hand-over-hand model of motility, not an inchworm model. Myosin V is a cargo-carrying processive motor

Laser Rayleigh scattering

Abstract: Rayleigh scattering is a powerful diagnostic tool for the study of gases and is particularly useful for aiding in the understanding of complex flow fields and combustion phenomena. Although the mechanism associated with the scattering, induced electric dipole radiation, is conceptually straightforward, the features of the scattering are complex because of the anisotropy of molecules, collective scattering from many molecules and inelastic scattering associated with rotational and vibrational transitions. These effects cause the scattered signal to be depolarized and to have spectral features that reflect the pressure, temperature and internal energy states of the gas. The very small scattering cross section makes molecular Rayleigh scattering particularly susceptible to background interference. Scattering from very small particles also falls into the Rayleigh range and may dominate the scattering from molecules if the particle density is high. This particle scattering can be used to enhance flow visualization and velocity measurements, or it may be removed by spectral filtering. New approaches to spectral filtering are now being applied to both Rayleigh molecular scattering and Rayleigh particle scattering to extract quantitative information about complex gas flow fields. This paper outlines the classical properties of Rayleigh scattering and reviews some of the new advances in flow field imaging that have been achieved using the new filter approaches.

Three-dimensional structural dynamics of myosin V by single-molecule fluorescence polarization

Abstract: The structural change that generates force and motion in actomyosin motility has been proposed to be tilting of the myosin light chain domain, which serves as a lever arm. Several experimental approaches have provided support for the lever arm hypothesis; however, the extent and timing of tilting motions are not well defined in the motor protein complex of functioning actomyosin. Here we report three-dimensional measurements of the structural dynamics of the light chain domain of brain myosin V using a single-molecule fluorescence polarization technique that determines the orientation of individual protein domains with 20-40-ms time resolution. Single fluorescent calmodulin light chains tilted back and forth between two well-defined angles as the myosin molecule processively translocated along actin. The results provide evidence for lever arm rotation of the calmodulin-binding domain in myosin V, and support a 'hand-over-hand' mechanism for the translocation of double-headed myosin V molecules along actin filaments. The technique is applicable to the study of real-time structural changes in other biological systems.

Demonstration and characterization of filtered Rayleigh scattering for planar velocity measurements

Abstract: Filtered Rayleigh scattering is an optical diagnostic technique that allows for simultaneous planar measurement of velocity, temperature, and pressure in unseeded flows. An overview of the major components of a filtered Rayleigh scattering system is presented. In particular, a detailed theoretical model is developed and discussed with associated model parameters and related uncertainties. Based on this model, results for two experimental conditions are presented: ambient room air and a Mach 2 freejet. These results include two-dimensional, spatially resolved measurements of velocity, temperature, and pressure derived from time-averaged spectra. ILTERED Rayleigh scattering (FRS), a recently developed flow diagnostic technique,1'2 achieves large suppression of background scattering allowing planar flowfield visualization and obtains quantitative measurements of velocity, temperature, and density in unseeded gaseous flows. This technique makes use of Rayleigh scattering from molecules in the flow and is driven by a high-power, narrow linewidth, tunable, injection seeded laser. When imaging the scattered light onto a charge-coupled device (CCD) camera, unwanted background scattering from stationary objects may be filtered out by tuning the frequency of the narrow linewidth laser to coincide with an atomic or molecular absorption line and by placing a cell containing the atomic or molecular species between the camera and the flow. This cell acts as a notch filter, absorbing all background scatter at the laser frequency. Scattered light that is Doppler shifted, however, passes through the filter and is imaged on the camera. Quantitative measure of flow properties is achieved by measuring the total intensity, Doppler shift, and spectral profile of the Rayleigh scattered light. The total intensity is directly proportional to density; the Doppler shift is directly proportional to velocity; and the spectral profile is a function of temperature and pressure. The scattering intensity, Doppler shift, and spectral profile are determined by passing the scattered light through the notch absorption filter and then by imaging it onto an intensified CCD camera. Because the filter absorbs light in a narrow frequency band, it converts the spectral information contained in the Doppler shift and Rayleigh profile into intensity information at the camera. By collecting data (camera pixel intensity) for varying conditions, v, T, and P may be determined. Previous work has concentrated on the use of this technique for background suppression when visualizing flows and for the measurement of velocity. The background suppression feature of FRS has been used to image flowfields that otherwise would be completely obscured by the strong scattering from wind-tunnel surfaces. The authors have used this technique to image the flowfield inside a Mach 3 inlet and to generate volumetric images of the crossing shocks and boundary layer.3 Elliott et al.4 have also used this technique to observe structures in compressible mixing layers. The use of FRS to measure velocity was initially demonstrated using scattering

疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A

Abstract: 抗原变异可使得多种致病微生物易于逃避宿主免疫应答。表达在感染红细胞表面的恶性疟原虫红细胞表面蛋白1（PfPMP1）与感染红细胞、内皮细胞、树突状细胞以及胎盘的单个或多个受体作用，在黏附及免疫逃避中起关键的作用。每个单倍体基因组var基因家族编码约60种成员，通过启动转录不同的var基因变异体为抗原变异提供了分子基础。

Imaging intracellular fluorescent proteins at nanometer resolution.

Abstract: We introduce a method for optically imaging intracellular proteins at nanometer spatial resolution. Numerous sparse subsets of photoactivatable fluorescent protein molecules were activated, localized (to approximately 2 to 25 nanometers), and then bleached. The aggregate position information from all subsets was then assembled into a superresolution image. We used this method--termed photoactivated localization microscopy--to image specific target proteins in thin sections of lysosomes and mitochondria; in fixed whole cells, we imaged vinculin at focal adhesions, actin within a lamellipodium, and the distribution of the retroviral protein Gag at the plasma membrane.

Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM).

Abstract: We have developed a high-resolution fluorescence microscopy method based on high-accuracy localization of photoswitchable fluorophores. In each imaging cycle, only a fraction of the fluorophores were turned on, allowing their positions to be determined with nanometer accuracy. The fluorophore positions obtained from a series of imaging cycles were used to reconstruct the overall image. We demonstrated an imaging resolution of 20 nm. This technique can, in principle, reach molecular-scale resolution.

Biosensing with plasmonic nanosensors

Abstract: Recent developments have greatly improved the sensitivity of optical sensors based on metal nanoparticle arrays and single nanoparticles. We introduce the localized surface plasmon resonance (LSPR) sensor and describe how its exquisite sensitivity to size, shape and environment can be harnessed to detect molecular binding events and changes in molecular conformation. We then describe recent progress in three areas representing the most significant challenges: pushing sensitivity towards the single-molecule detection limit, combining LSPR with complementary molecular identification techniques such as surface-enhanced Raman spectroscopy, and practical development of sensors and instrumentation for routine use and high-throughput detection. This review highlights several exceptionally promising research directions and discusses how diverse applications of plasmonic nanoparticles can be integrated in the near future.

Principles of nano-optics

Abstract: 1. Introduction 2. Theoretical foundations 3. Propagation and focusing of optical fields 4. Spatial resolution and position accuracy 5. Nanoscale optical microscopy 6. Near-field optical probes 7. Probe-sample distance control 8. Light emission and optical interaction in nanoscale environments 9. Quantum emitters 10. Dipole emission near planar interfaces 11. Photonic crystals and resonators 12. Surface plasmons 13. Forces in confined fields 14. Fluctuation-induced phenomena 15. Theoretical methods in nano-optics Appendices Index.