The present application is a request for renewal of funding on a two-year Biological Instrumentation Grant for the construction and testing of a Confocal Differential Polarization Scanning Microscope. The basic optical and electronic design and building of this instrument has been completed in our laboratory. This microscope has already shown its enormous potential as an analytical tool in the study of complex Biological systems in-situ. Using this instrument, we have been able, for the first time, to resolve spatially the long-range chiral domains in which the Light Harvesting Complex II is organized in the chloroplast grana. We request funding for an additional period of three years to extend the capabilities of the present instrument to carry out: a) Microspectro-polarimetric measurements at a given location in the object. Presently, the microscope can spatially resolve the optical (linear and circular) anisotropy of the object to within the diffraction limits of the light, at a fixed wavelength of illumination. The building of the proposed instrumentation will allow us to locate a microscopic domain of interest in the studies object and to obtain the Circular Dichroism and linear Dichroism spectra of that localized domain: b) Luminescence Depolarization Imaging. Using the advantages of confocal epi-dillumination optics and the extended sensitivity of polarization modulation and synchronous demodulation, we propose to develop the instrumentation to spatially resolve the molecular dynamics of luminescence-labelled species. This instrumentation will be applied first to imaging the rotational dynamics of IgE receptors in mast cells.
