Laboratory for Fluorescence Dynamics
E. Gratton,* W. W. Mantulin,* R. M. Clegg,* T. Hazlett, S. Sanchez, N. Barry, W. Yu, O. Akcakir, T. Ragan, Y. Chen, J. Eid, Q. Ruan, J. Muller, L. Bagatolli, J. Sutin, K. Kispetikova
National Institutes of Health, P41-RR-03155

The Laboratory for Fluorescence Dynamics (LFD), a national biomedical resource, has a dual and equal commitment to foster fluorescence research and to provide service in a user-oriented facility. Fluorescence Research and Development The research goal of the LFD is to develop new fluorescence instrumentation, design new theoretical formulations of fluorescence phenomena, and compile appropriate software, with the aim of advancing basic research and biomedical applications. Examples of current projects include: instrumentation (frequency domain fluorometer with lifetime and spectral resolution, laser heterodyning, lifetime fluorescence microscopy pump probe stimulated emission spectroscopy), software (global analysis of multifrequency data sets), optical imaging (near-infrared images of tissue), and applications (two-photon fluorescence correlation spectroscopy). These advances in fluorescence technology are transferred to the user fluorescence and microscopy laboratories. Fluorescence Laboratory The laboratory serves both the campus research community and visiting scientists. To date, core and collaborative research has stressed macromolecular assembly and dynamics, membrane structure/function relationships, and fluorescence microscopy of cells. The LFD houses a spectropolarimeter for circular dichroism measurements. Fluorescence equipment includes high-sensitivity, photon-counting, scanning fluorometers (with polarization accessory), three laser-based variable multifrequency phase/modulation fluorometers with different excitation wavelength and modulation frequency options, stopped flow and high pressure accessories. Dedicated personal computers assist in data collection and analysis. Ancillary support for biomedical research is housed in a general biochemistry laboratory, which is equipped for biological sample manipulation. Fluorescence Microscopy Development Laboratory (FMDL) FMDL is a technology development laboratory for multiphotonic fluorescence microscopy, which also serves users. It conducts core and collaborative research on a variety of cellular components and systems (membranes, receptors, antibodies, etc.). The instrumentation includes Ti:sapphire lasers, upright and inverted fluorescence microscopes, and correlation systems for photon counting. The multiphotonic techniques under development include: fluctuation correlation spectroscopy, fluorescence lifetime imaging, pump-prove stimulated emission, particle tracking, and single molecular studies.

Membrane Activity of a Secreted Phospholipase A2
T. L. Hazlett,* S. Sanchez
University of Illinois

Phospholipase A2 (PLA2) is an enzyme which hydrolyzes membrane phospholipids. PLA2 is not a 'membrane protein' but works at the membrane-solution interface and is sensitive to the organization of the phospholipid. We are researching the dimeric PLA2 from rattlesnake venom. We are applying fluorescence methods to measure the extent of subunit dissociation in solution and on the membrane. Also, PLA2 binding to membranes is being researched by directly visualizing the enzyme molecules through fluorescence. Long-standing issues on the dependence of PLA2 action by the organization of membrane lipid will be addressed.

Optical Imaging of Thick Tissues
E. Gratton,* M. Filiaci, V. Toronov, M. Wolf, A. Michalos
National Institutes of Health, 1RO1 CA57032

This project explores the use of frequency-domain methods to obtain near-infrared optical images of thick tissues. The use of near-infrared radiation has been proposed as an attractive alternative to obtain information about the oxygenation state of tissues due to the difference in optical spectra of the oxy- and deoxy- form of hemoglobin. Our frequency-domain approach uses the propagation of high-frequency amplitude modulated light. In the frequency-domain, propagation of the AM intensity wave in a highly scattering medium is analogous with wave optics. An object immersed in the medium produces deformation of the propagation wavefront of the amplitude modulated wave and results in an easy identification of absorbing and scattering objects such as blood vessels or bone. Computer algorithms display in real-time the wavefront of the AM wave after traversing the tissue.

Optical Monitors for Vascular Insufficiency in Peripheral Tissue
E. Gratton,* W. W. Mantulin, A. Paunescu, U. Wolf
National Institutes of Health, 5 R01 RR10966

Peripheral vascular disease (PVD), a chronic disease, afflicts diabetics and others with vascular pathologies. The level of tissue oxygenation in extremities is an important parameter for diagnosis of PVD. We have developed a new technology based on near-IR frequency domain spectroscopy that provides quantitative information on the level of tissue oxygenation. The optical signal is derived from penetration of photon density waves in tissue. We have designed and built noninvasive, portable, tissue oxygen saturation monitors. Preliminary tests show that the optical oxygen monitor can be clinically useful by providing the clinician with a quantitative physiological parameter which is a meaningful index for the early detection and treatment of PVD.

Theory and Simulation of Biopolymer Aggregates
K. Schulten,* R. Skeel* (Comput. Sci.), L. Kale* (Comput. Sci.), T. Martinez* (Chemistry), S. Adem, S. Arslan, A. Balaeff (Biophysics), J. Baudry, R. Braun, A. Damjanovic, M. Gau, J. Gullingsrud, B. Isralewitz (Biophysics), D. Kosztin, T. Ritz, M. Sener
University of Illinois

Our research focuses on the structure, dynamics, and function of biopolymer aggregates, e.g., lipids and water forming membrane bilayers, proteins complexing with DNA and regulating gene expression, and proteins involved in complexes with other proteins. The studies require very-large-scale computer simulations and have become possible through the development of statistical mechanical theory, efficient algorithms, graphics, and simulation programs (VMD and NAMD), interactive molecular dynamics (IMD), collaborative environment (Bio CoRe), and the group's powerful computational resources which function as high-performance parallel computers.