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. One example is a collaborative project (with personel at the National Institute of Standards and Technology) to develop a new, low-cost optical technique, based on 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. A likely application is a study of the dynamical state of multispecies bacterial microbial colonies present in childhood diseases such as cystic fibrosis and infections of the inner ear. Another of our activities involved 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 moving on the surfaces of, or within, biological cells. Intact extracellular matrix materials present similarly difficult environments for physical characterization. Using well-defined scattering models, we investigated the reliability of parameters determined when FCS is used to probe the movement of molecules in such complex environments. To this end, 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. Monte-Carlo simulations demonstrated 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. Our results thus indicate that multiple scattering does not result in serious measurement artifacts and that single-photon FCS can be a useful technique for measuring probe diffusivity in optically dense media if sufficient signal intensity is attainable. Possible applications are to examine the movement of antibiotics within biofilms, the concentration profiles of morphogens in developing tissues, and the diffusion of proteins in the intra-articular space of cartilage to evaluate structural degeneracies associated with osteoarthritis. We also developed a multi-well polyacrylamide(PA)- based stiffness assay to assess how the mechanical properties of their surroundings of cells might affect their fate, particularly as relating to their responsiveness to therapeutic drugs. Polyacrylamide was chosen due to the ease of manipulating its stiffness and the ability to link that material to constituents of the extracellular matrix. The gels were coated with collagen and used to test the effect of stiffness on cancer cell responsiveness to anti-cancer drugs, in particular their susceptibility to mifrotubule-targeting agents. This multi-well format was chosen in order to facilitate obtaining dose-response curves while testing multiple parameters. A paper describing this work, entitled """"""""Multiwell stiffness assay for the study of cell responsiveness to cytotoxic drugs,"""""""" by S. Zustiak, R. Nossal and D. Sackett, is scheduled to appear in a forthcoming issue of the journal, Biotechnolgy and Bioengineering.
