Joseph N. Forkey

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

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.

Laser Rayleigh scattering

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.

CORRECTED AND CALIBRATED I2 ABSORPTION MODEL AT FREQUENCY-DOUBLED ND:YAG LASER WAVELENGTHS

We present a computer model for accurately predicting absorption profiles for molecular iodine cells over the tuning range of frequency-doubled Nd:YAG lasers. The model is compared with experimental data for a number of different cell conditions. This model is intended for use in the design and optimization of absorption filters and for data analysis in applications in which the accuracy of the measurement is related closely to the accuracy with which the filter profile is known.

Filtered Rayleigh scattering measurements in supersonic/hypersonic facilities

Preliminary measurements are presented of flow field properties in Mach 3 and Mach 5 flows using filtered Rayleigh scattering. Filter properties have been characterized by high resolution spectroscopy in order to optimize the selection of laser frequency and filter operating conditions, as well as for the development of an accurate filter modeling program. An optimized filter is used the background suppression feature of this technique to image the boundary layer structure in a Mach 3 high Reynolds number facility and the shock structure in a Mach 5 overexpanded jet. This had been achieved using a visible laser source. By frequency scanning the laser, time-averaged velocity measurements in the Mach 3 and Mach 5 flows are made. Data acquisition at 10 torr and below indicates that this approach can be extrapolated for use in hypersonic flow facilities and is applicable as an in-flight optical air data device for hypersonic vehicles.

Flow field imaging through sharp-edged atomic and molecular `notch' filters

Sharp cut-off atomic and molecular notch filters simultaneously provide high spectral resolution and allow imaging by collecting light over a wide field of view. Many important properties of flow fields can be observed by imaging light elastically scattered from small particles, molecules or electrons. In order to extract information about the flow field from elastic scattering, the spectrum of the scattering must be resolved and the background scattering must be suppressed. Very high resolution, on the order of a few tens of megahertz, is usually required. The spectrum of the scattered light is broadened and shifted by the motion of the scatterers. For particles, which have relatively little thermal or acoustic motion, the spectral shift is only a function of the velocity. For molecules, the scattering spectrum is a function of the temperature, velocity and pressure of the gas as well as its composition. For electrons, the spectrum is a function of the electron temperature and electron number density in a plasma. In this paper, sharp edged notch filters made of rubidium, iodine or mercury vapour are used to image shock wave and boundary layer structure by Rayleigh scattering from particles, to image gas pressure, velocity and temperature by molecular Rayleigh scattering, and to measure electron temperature and electron number density by Thomson scattering. For molecular scattering, filter transmission is generally a function of velocity, temperature and pressure, but, under some circumstances, it is a function of only one or two variables, so a notch filter can provide single-pulse images of a specific flow field parameter.

Observation of a 100-MHz frequency variation across the output of a frequency-doubled injection-seeded unstable-resonator Q-switched Nd:YAG laser.

We report high-resolution measurements of the spatial variation of the optical frequency of an injection-seeded unstable-resonator Q-switched Nd:YAG laser. Images of the second harmonic taken through a molecular-iodine notch filter show frequency variations of as much as 100 MHz (second harmonic) between the center and the edge of the beam.

Control of experimental uncertainties in filtered Rayleigh scattering measurements

Filtered Rayleigh Scattering is a technique which allows for measurement of velocity, temperature, and pressure in unseeded flows, spatially resolved in 2-dimensions. We present an overview of the major components of a Filtered Rayleigh Scattering system. In particular, we develop and discuss a detailed theoretical model along with associated model parameters and related uncertainties. Based on this model, we then present experimental results for ambient room air and for a Mach 2 free jet, including spatially resolved measurements of velocity, temperature, and pressure.

Time-sequenced and spectrally filtered Rayleigh imaging of shock wave and boundary layer structure for inlet characterization

Multiple pulsed Rayleigh imaging and filtered Rayleigh scattering are used to generate images of a complex boundary layer structure, shock wave/boundary layer interactions, and crossing shock waves. Time-sequenced Rayleigh images taken with a visible, double-pulsed laser system show the evolution of boundary layer structure of the internal flow in a generic cross-shock inlet. The images taken in the inlet give insight into 3D effects caused by the inlet geometry and may be used for modeling the complex flows.

Volumetric imaging of supersonic boundary layers using Filtered Rayleigh Scattering background suppression

We demonstrate the use of Filtererd Rayleigh Scattering and a 3D reconstruction technique to interrogate the highly three dimensional flow field inside of a supersonic inlet model. A 3 inch by 3 inch by 2.5 inch volume is reconstructed yielding 3D visualizations of the crossing shock waves and of the boundary layer. In this paper we discuss the details of the techniques used, and present the reconstructured 3D images.

Orientation and Rotational Motions of Single Molecules by Polarized Total Internal Reflection Fluorescence Microscopy (polTIRFM)

In this article, we describe methods to detect the spatial orientation and rotational dynamics of single molecules using polarized total internal reflection fluorescence microscopy (polTIRFM). polTIRFM determines the three-dimensional angular orientation and the extent of wobble of a fluorescent probe bound to the macromolecule of interest. We discuss single-molecule versus ensemble measurements, as well as single-molecule techniques for orientation and rotation, and fluorescent probes for orientation studies. Using calmodulin (CaM) as an example of a target protein, we describe a method for labeling CaM with bifunctional rhodamine (BR). We also describe the physical principles and experimental setup of polTIRFM. We conclude with a brief introduction to assays using polTIRFM to assess the interaction of actin and myosin.