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. 1) Energy-driven proton pumps are of major importance for energy transduction in living systems. The respiratory chain of animals, as well as single celled organisms, uses energy released from electron transport to oxygen to form an electrochemical gradient for protons, which is then used to synthesize ATP. In collaboration with Dr. R. Hendler, NHBLI, we have developed optical instrumentation that enables studies on the simple 26000 D, photon-driven, proton pump bacteriorhodopsin (BR) to pinpoint specific molecular transformations that generate the most voltage across the membrane. These studies support the view of the BR photocycle as consisting of two parallel cycles. instead of the single photocycle that is favored by many researchers. Information obtained from this proton pump should help in understanding more complex systems such as mammalian cytochrome oxidase. Combined infra-red and optical spectroscopy has produced kinetic structural information that characterizes each step of the photocycle, and collaborative studies are underway with colleagues at NIST to produce single crystals of BR membrane protein. The ultimate goal of this project is to characterize the BR crystal using similar established approaches in concert with time resolved X-ray diffraction to obtain structural information at the atomic level, thus enabling an understanding of protein conformational changes that result in electrogenic transport of protons across the membrane. We have developed instrumentation to study the optical kinetics of the BR photocycle in approximately 1 microliter samples of membrane fragments. Previously, we positioned the BR between two 600 micron fused silica optical fibers, which carry the visible-spectrum monitoring light, produced by an Oriel xenon arc lamp, and the detection fiber, both orthogonal to a third fiber that carries 532nm, 7ns photolysing pulses generated by a laser. This arrangement enabled us to troubleshoot and optimize the design criteria and control of the detection instrumentation that are described below. We have now replaced the fiber optic cell by one composed of two calcium fluoride windows with the BR sandwiched in-between. This new cell resides in the focal plane of a Bruker infrared microscope that we have adapted to introduce visible monitoring light and to integrate with our visible light detection system by insertion of our data collection (detection) fiber into the camera port of the microscope. This output fiber carries information on the spectral kinetic changes that occur following the photolysis pulse. These changes are identified by positioning the fiber at the entrance slit of an Acton .25m spectrograph, the output of which is monitored by either a Princeton Instruments CCD camera directly or indirectly after intensification by an Opelco-Videoscope image intensifier. This instrumentation acquires 524 successive spectra at 512 wavelengths in the visible spectrum covering the characteristic BR photocycle changes. The spectral changes in the photocycle are not uniform and comprise events ranging from fast events on the sub-microsecond time scale to slower events with millisecond time scale changes. To accomplish this, a staggered time schedule is used with time increments from 5 microseconds to 500 microseconds. Using the CCD directly presents a light limitation at the shorter exposure times, and an image intensifier has been integrated into the system to overcome this limitation and to provide shorter sub-us exposure times. We have spent considerable effort overcoming inherent problems associated with controlling light intensity and exposure in both the CCD camera and the image intensifier. The integration with the Bruker infrared microscope is a parallel collaborative effort with colleagues at NIST at the Center for Advance Research in Biotechnology (CARB), who will collect time-resolved infrared spectroscopic data on the same sample as we collect simultaneous visible data. This combined system will evaluate the feasibility of using this dual approach to study BR membrane crystals that are currently being grown by other colleagues at CARB. We have already been able to collect spectra with an aperture size of 50 microns - a size that approaches the sub 100 micron size of the expected BR crystals. Our development has reached the stage where we are being limited by the inherent noise in the Oriel xenon arc lamp that produces our monitoring light. We have investigated the alternative use of high intensity 405nm LEDs, over-coated with a phosphor that causes them to emit a relatively intense, stable white light emission with additional intensity in the blue-end of the spectrum where critical spectral changes take place in the BR photocycle. By combining this LED with a second LED that has the phosphor removed elevates the signal in the blue region of the spectrum where critical kinetic spectral changes occur in the BR photocycle. A custom 3-D printed mount holds these LEDs and associated optical components to introduce this monitoring light into the microscope condenser and provides an illumination intensity such that attenuation of this light is required to suppress a background turnover of the BR photocycle. We are currently adapting the fiber optic coupling between the microscope and the detection instrumentation to accommodate a larger acceptance fiber that will be relayed to the 600 micron input to the spectrograph by a reducing magnification lens system. This modification will allow flexibility in the size of the BR sample being studied. This work has resulted in two publications 2) In a collaboration with Intelligent Material Solutions, Inc., we are investigating the potential use of infra-red upconverting nanocrystals as labeling agents. The advantage of upconverters over typical fluorescent labeling agents is that background fluorescence that limits the lowest level of detection should be virtually zero. In addition, many detectors have very low sensitivity in the infrared, used to excite the upconverters, thus reducing the need for stringent filtering requirements that are usually required to eliminate the excitation light. We are exploring several possible avenues for biological applications of these upconverters, including labeling and therapeutic agents. One possible avenue that is being explored is the use of these upconverters for basal cell carcinoma screening. 3) Together with colleagues from CIT, who designed and fabricated a visual stimulus, optical imaging, and control apparatus to study neural connectivity in fruit flies, we have collaborated with Dr. Chi-Hon Lee, NICHD, to integrate a near-ir laser into the optical path of the apparatus to provide a feedback stimulus to the fruit fly depending on its response to the visual stimulus. Near infrared is needed to prevent the feedback stimulus from being recognized as a visual stimulus by the fruit fly. Currently, an optical system is being prepared to introduce laser light to a DLP chip such that a variety of images can be presented. 4) In collaboration with Dr. Shawn Chen's group, we are exploring the use of near ir laser irradiation to efficiently raise the temperature of nanoparticles that are associated with tumor masses. Temperature effects are assessed through real time infrared imaging.
|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|
|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|