The continuing goal of this multifaceted project is to develop and employ new physics-based methodologies to investigate complex biological structures and materials. Insights gained from studying model materials are used in inquiries of specific phenomena of biomedical import. For example, we previously used computer-based structural modeling, combined with dynamic light scattering (DLS), static light scattering (SLS), and small angle neutron scattering (SANS), to examine conformations of flexible macromolecular complexes, and then applied these techniques to studies of clathrin triskelions in solution. We also developed methods to examine the movement of nanoscopic biological particles through gel-like matrices, and employed them to understand the movements of viruses within vaginal secretions. Recently, we have collaborated on a project to develop a low-cost instrument to perform polarization fluorescence microscopy, and have demonstrated its utility by discerning molecular orientations of probes attached to lipid membranes. Our scheme utilizes a quarter-wave plate, in combination with a liquid crystal variable retarder (LCVR), to provide a tunable method to rotate polarization states of light prior to its being coupled into a fluorescence microscope. To demonstrate the capabilities of this device we measured a series of full-field fluorescence polarization images from fluorescent analogs incorporated in the lipid membrane of Burkitts lymphoma CA46 cells. A spatially-varying contrast in the normalized amplitude was observed on the cell surface when we used molecules whose head groups were labeled with DiI (1,1'-Dioctadecyl 3,3,3',3'-Tetramethylindocarbocyanine) fluorophores, orientated tangentially to the cell membrane. Internally labeled cellular structures showed zero response to changes in incident light polarization, with zero net linear polarization amplitude. This instrument provides a calibrated method that may be coupled to existing fluorescence microscopes to perform investigations of cellular processes that involve changes in molecular orientations. A paper based on this work has been submitted for publication. Another of our activities, also relating to the use of light to investigate biological systems, involves a study of the effects of multiple scattering on the interpretation of fluorescence correlation spectroscopy measurements performed on optically dense samples. Fluorescence correlation spectroscopy (FCS) is widely employed in biological contexts to assess diffusive movements of particles such as protein chimeras containing GFP (or other visual fluorescent moiety) and fluorescently-labeled supramolecular assemblies. The method involves analyzing temporal fluctuations in fluorescent emissions from a small region of an illuminated sample and, because it basically measures number fluctuations, the target material is studied at low concentrations. However, the unique power of this technique is that, in principle, one can focus on the motions of fluorescent entities while signals from non-fluorescent surroundings can be ignored. For this reason, FCS increasingly has been used to study particles moving within complex environments, including molecules on the surfaces of or within biological cells, antibiotics and viruses diffusing in biofilms, and targets located within optically-dense tissues. Typically, time autocorrelation functions are calculated from the fluctuating fluorescent signal and, if the samples are optically clear, interpretation is relatively straightforward. But, if the sample from which the signals are collected demonstrates a high degree of multiple scattering, the dimensions of the illuminated sample volume are distorted, confounding interpretation of the autocorrelation function. By comparing data from various well-defined scattering models, we have been able to obtain insight into the reliability of parameters determined when FCS is used to probe the movement of molecules in highly complex, biological environments. A paper based on this work is in preparation.
