Development of unique instrumentation using novel approaches is, in many instances, necessary to the success of biomedical research. Areas of emphasis within our group are summarized below. In collaboration with Dr. Richard Hendler, NHLBI, a modified version of a high speed optical multichannel spectrometer, developed previously, has been enhanced in terms of temporal and signal amplitude resolution. The kinetics of the bacteriorhodopsin (BR) photocycle, initiated with a synchronized laser pulse (532nm, 7ns), are being studied using an optical system that follows the spectral changes associated with the transient intermediates of the photocycle. Complete spectra from approximately 400nm to 700nm are collected with less than 10 microsecond resolution, permitting extraction, though single value decomposition analysis, of the role of the intermediates. These studies support the view that the BR photocycle consists of two parallel cycles instead of a single photocycle favored by other groups. To adapt to the next phase of this project, which entails collecting infrared data, a collaboration has been established with the National Institute of Standards and Technology (NIST). The optical system has been relocated to the Center for Advanced Research in Biology of which NIST is a member. The realigned system incorporates both the high-speed multichannel analyzer and the infrared spectrometer that has permitted capturing a parallel data set of infrared spectral data. Combined optical and spectral kinetic data studies will allow characterization of the structural information for each step in the photocycle. Our ultimate goal is to apply the same approaches to time-resolved X-ray diffraction using BR membrane crystals thus obtaining structural information at the atomic level to visualize protein conformational changes resulting from the electrogenic movement of protons across the membrane. To capture the necessary spectral information across the complete visible spectrum with time resolution under 200ns, an optical instrument has been designed to accommodate a charge-coupled device (CCD) camera, an image intensifier that are attached to a spectrograph. The dispersed spectrographic data is positioned on two rows of the CCDs photon detector (1048 rows of 512 pixels) thus enabling collection of spectra of 524 wavelengths at 512 different points in time. To record points that are staggered in time to account for the different lifetimes of the BR photocycle intermediates a time gated image intensifier will be integrated into the instrument. Time synchronization is achieved through custom electonic hardware and software developed by CIT colleagues. This year we have demonstrated the capability to collect spectral data from sample volumes of 12nl. To achieve this required fabrication of a custom cell incorporating fiber optic technology to carry monitoring light to the sample and deliver through a 200 micron fiber the spectral information to the spectrograph. In lieu of the laser photolysis, we were able to initiate the photocycle through 532 nm carried by a 600 micron fiber. To simulate the laser pulse, a 100 micron pinhole in a wheel rotating at 100 Hz was placed in the fiber optic path using a microscope objective to focus the 5W laser beam through the pinhole. A complete description of the results is presented in Dr. Hendler's annual report. In collaboration with Dr. Janine Smith, NEI, an ocular imaging system for measuring dry eye severity has being developed and is undergoing evaluation for clinical use. The method uses the Oxford Scheme for grading ocular staining in dry eyes using various dyes. The software that processes the ocular images will be tested in the clinic in conjunction with a slit bio-microscope. The image analysis uses a support vector machine algorithm from statistical learning theory. Based on the clinical feedback a subsequent instrument development may be necessary to improve image capture that will enable image enhancement, and the correction of imaging defects. This system will enhance image quality by minimizing the potential effects of eye movement, glare, vignetting, lens distortion, noise, and non-uniform eye lighting. System and monitor calibration techniques for color management and white balance will be developed. It is anticipated that further improvement to image quality would be obtained by standardizing the instillation and timing of dye administration to account for time dilution of dye staining. A database will be developed for storing patient information and history, and to save patient images. Both the instrument and software will be initiated using a semi-automated system with the goal of future development as a fully automatic system. In collaboration with Drs. Dietrich Haubenberger and Mark Hallett, NINDS, a system has been designed for a wireless sensor to be placed on the wrist of Parkinson's disease patients to analyze limb motion. Initial assembly has taken place. It will measure intensity, frequency and duration of tremors due to Parkinsons disease. The system will use a Shimmer mote platform consisting of a microcontroller, transceiver and sensors.
| Hendler, Richard W; Meuse, Curtis W; Smith, Paul D et al. (2013) Further studies with isolated absolute infrared spectra of bacteriorhodopsin photocycle intermediates: conformational changes and possible role of a new proton-binding center. Appl Spectrosc 67:73-85 |
| Hendler, Richard W; Meuse, Curtis W; Braiman, Mark S et al. (2011) Infrared and visible absolute and difference spectra of bacteriorhodopsin photocycle intermediates. Appl Spectrosc 65:1029-45 |
| Kakareka, John W; Smith, Paul D; Pohida, Thomas J et al. (2008) Simultaneous measurements of fast optical and proton current kinetics in the bacteriorhodopsin photocycle using an enhanced spectrophotometer. J Biochem Biophys Methods 70:1116-23 |
