* Biophysical chemistry with emphasis on measuring macromolecular interactions in living cells using single photon and two-photon variants of image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS)
* Live cell measurement of macromolecular dynamics and clustering phenomena of green fluorescent protein (GFP) integrin constructs to study their role in assembly of cell adhesion structures and in receptor "cross-talk" with other signaling systems in cells.
* Development of new microscopic techniques that extend the capabilities of the ICS and ICCS methods. Development of a combined ICS, ICCS and imaging fluorescence resonance energy transfer microscopy. Applications of nonlinear harmonic microscopy and ICS to measurements of macromolecular mobilities in live cell systems. Application of bio-conjugated quantum dot labels for dynamic ICS measurements in living cells.
* Extension of ICS and ICCS for application to research problems in areas of neuroscience
My research interests lie at the interface between the physical and biological sciences. I am interested in understanding the molecular mechanisms involved in cellular adhesion (how biological cells stick together and to an underlying substrate) and how cells dynamically regulate adhesion receptors to control cellular migration. I am also interested in developing new biophysical methods such as third harmonic generation (THG) microscopy and the use of bioconjugated quantum dots as robust luminescent labels for biophysical imaging applications on live cells.
Cellular adhesion and migration play fundamental roles in the normal development of tissue architecture in organisms and are also known to function abnormally in certain diseases such as the progression of cancers from a single localized growth to dispersed, invasive metastases. Adhesion is mediated by macromolecular complexes that are composed of clusters of plasma membrane receptors called integrins that bind to ligands located in the extracellular matrix outside of cells. The integrins also link to the polymeric cytoskeleton network within the cytoplasm of the cells via intermediary proteins. Adhesion complexes are dynamic structures, not static, and are actively assembled and disassembled by the cells in different locations in the membrane in order to facilitate traction needed for cell migration. Additionally, researchers have demonstrated that the adhesion complexes act as signal centers, integrating inputs from the binding of specific ligands to the integrins which lead to interactions with other proteins within the cell that are in turn linked to biochemical signaling pathways. The emerging theme within the cell is that intermolecular interactions play essential roles in the biochemical mechanisms.
In situ measurement of the physico-chemical properties of the macromolecules involved in regulating the assembly and disassembly of adhesion complexes within living cells will be necessary to achieve a more complete picture of cell migration at the molecular level. My research involves the development and application of novel biophysical microscopic and spectroscopic methods for performing quantitative measurements of the transport properties and interactions of adhesion receptors within the plasma membrane of living cells. The measurements entail using advanced laser-scanning fluorescence microscopy (both single photon confocal and nonlinear two-photon fluorescence laser scanning microscopy) to image the spatial and temporal distribution of fluorescently labeled macromolecules within living cells. The image time series are essentially a record of fluorescence intensity fluctuations that contain complete information on both the absolute molecular concentrations and the dynamics/kinetics of the labeled molecules. Statistical Mechanics provides the theoretical foundation for the extraction of this information by methods known as fluctuation spectroscopy or fluorescence correlation spectroscopy (FCS). I use newly developed imaging variants of FCS: image correlation spectroscopy (ICS) and image cross-correlation spectroscopy (ICCS). The ICS method allows measurement of transport properties (diffusion coefficients and flow speeds) as well as absolute concentration and aggregation state of the fluorescently tagged macromolecules imaged at the cell surface. Two-colour ICCS allows direct measurement of the fraction of interacting macromolecules and the transport dynamics of co-localized macromolecular species if the different macromolecules can be labeled with non-identical fluorophores.
PHYS-200 Space, Time and Matter