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.? ? A digital video, EEG imaging system with seizure detection for multiple small animals, has been developed initially in collaboration with NINDS detects and record sseizures of small animals by monitoring their EEGs together with documenting the animals' accompanying behavior via video recording. The instrumentation consists of animal cages, swivel connectors connected to the head of each animal, EEG analog amplifiers, computer control with movie maker and graphics hardware, and software detection of spikes and seizure patterns. The system has been assembled and will visually display and record EEG data along with video of multiple small animals. The instrument has been tested on small animals and modifications have been made to the mechanical apparatus to allow for more flexible animal movement. Through consultation with the National Naval Medical Center, a system based on the same principles will be developed for research purposes.? An e laser flash photolysis apparatus was developed in collaboration with Dr. Fred Friedman, NCI, and Dr. Stanley Smith (University of Mississippi)to measure the kinetics of carbon monoxide (CO) binding to cytochromes P450 in liver microsomes from rats treated with various drugs and carcinogens. Since numerous forms of P450 contribute to the overall reaction, a difference kinetic method was used to distinguish the kinetic behavior of individual P450s. This method entails analysis of the difference between the kinetic profiles in the absence and presence of a specific P450 effector, and successfully yielded kinetic parameters for individual P450s involved in drug and carcinogen metabolism. Specifically, various polycyclic hydrocarbons differentially accelerated CO binding to the P450 1A1, which metabolizes these carcinogens in a size- and shape-dependent manner. Wavelet analysis and maximum entropy software tools will be evaluated to determine if a signal processing enhancement of the data, results. Wavelet analysis allows selection of critical short time scale regions, which cannot be performed using fast Fourier transform analysis, where high frequency information predominates and provides information on the early events associated with the cytochrome P450 binding kinetics. Additionally, use of the maximum entropy technique will improve the probability distribution of the data.? ? In collaboration with Dr. Steven Stanhope, NICHD, a time reaction reflex movement system has been duplicated to test a person's reaction time in attempting to follow a laser beam directed at the floor. Signals generated in a computer system change the position of the light and indicate when a particular light stimulus is activated and deactivated. The computer correlates signals from force plates in the floor with foot placement. ? ? 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), is 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. The optical system has been realigned to incorporate both the high-speed multichannel analyzer and the infrared spectrometer 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, an optical instrument has been designed to accommodate a charge-coupled device (CCD) camera with an attached spectrograph. The CCDs photon detector contains 1024 rows of 512 pixels thus enabling collection of spectra of 512 wavelengths at 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.? ? 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.
