The continuing goal of this multifaceted project is to develop and employ physics-based methodologies to investigate complex biological structures and materials. Insights gained from studying model materials may be used in inquiries of specific phenomena of biomedical import. Recently, we collaborated on two projects aimed at developing new, low-cost, optical imaging methods for biological samples. One involved constructing an instrument to perform rapid, full-field polarization fluorescence microscopy. 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. The efficacy of this method was demonstrated by observing localized molecular orientations of probes attached to lipid membranes. In principle, one can use such instrumentation to study any cellular process that involves changes in molecular orientation, as long as appropriate fluorescently labels can be found. The second activity was undertaken to support the development and use of hyperspectral imaging to characterize the spatial organization of complex, multispecies cellular communities. Microarray phantoms containing multiple dyes were fabricated and used to test image analysis algorithms for this process. Another of our activities involves a study of the effects of multiple scattering on the interpretation of fluorescence correlation spectroscopy (FCS) measurements performed on optically dense samples. A unique power of FCS is that, in principle, it can detect the motions of fluorescent entities while signals from non-fluorescent surroundings can be ignored. For this reason, FCS increasingly is used to study particles moving in complex environments, examples being molecules on the surfaces of or within biological cells, antibiotics and viruses diffusing in biofilms, and growth factors moving within embryos. Using well-defined scattering models, we have investigated the reliability of parameters determined when FCS is used to probe the movement of molecules in such complex environments. For instance, we measured FCS autocorrelation functions of Atto 488 dye molecules diffusing in solutions of polystyrene beads which acted as scatterers. A scattering-linked increase in the illuminated volume, as much as two fold, resulted in only a minimal increase in apparent diffusivity. To analyze the illuminated beam profile, we employed Monte-Carlo simulations, which indicated a larger broadening of the beam along the axial than the radial directions, and a reduction of the incident intensity at the focal point. The broadening of the volume in the axial direction has only negligible effect on the measured diffusion time, since intensity fluctuations due to diffusion events in the radial direction are dominant in FCS measurements. Collectively, our results indicate that multiple scattering does not result in serious measurement artifacts and thus, when sufficient signal intensity is attainable, single-photon FCS can be a useful technique for measuring probe diffusivity in optically dense media. A paper based on this work has been submitted. Finally, we have used FCS to investigate the diffusion of macromolecules and other targets within the water phase of polymer gels and concentrated polymer solutions. Again making use of the ability of FCS to distinguish the movements of fluorescent particles from those of a non-fluorescent background, we have been establishing and studying various physical properties of such systems, including the link between particle size, diffusion, and the degree of crosslinking of the polymer-chain constituents. The ultimate goal of this and related studies (e.g., an earlier project to examine the movement of charged macromolecules through crowded polymer solutions) is to understand the physics underlying transport and viscoelastic properties of complex milieu, such as those found within biological cells.
