This project involves the design and construction of a polarized evanescent wave fluorescence microscope. This microscope will be used to probe rotational mobilities of protein machines that move within membranes or on DNA. Energy known as evanescent wave radiation penetrates about 100 nanometers from the surface into the rarer medium when light undergoes total internal reflection. Thus, evanescent wave fluorescence microscopy provides a means for excitation of fluorophores from a thin layer, thereby greatly reducing background fluorescence. It has been used to visualize the processivity and nucleotide binding reactions of single motor molecules as they translocate along cytoskeletal polymers. Membrane motors and DNA transcription complexes that translocate more than an order of magnitude slower than cytoskeletal motor proteins provide examples of macromolecular assemblies where measurement of rotational, rather than translational mobilities might be more informative. Measurement of these rotations will be carried our using plane polarized light to excite fluorophores in a defined orientation. Green fluorescent protein fusions and incorporation of synthetic peptides that bind bidentate arsenical fluoresceins with high affinity into the protein sequence of choice provide two technologies that allow specific labeling of proteins in intact cells by rigid fluorescent markers. Fluorophore exited state lifetimes are typically 10 nanoseconds, a time sufficient to allow significant rotational diffusion of single protein molecules and for attenuation of the energy of the excited state by various processes leading to quenching or wavelength shifts of the emitted fluorescence. Polarization will be coupled to evanescent wave illumination to enable selective excitation of membrane bound flagellar motor and chemoreceptor complexes in intact bacteria and of bound T7 polymerase complexes in vitro. Depolarization of fluorescence will be measured by monitoring emission polarized parallel and perpendicular to the exciting light.
A modular system utilizing a single laser source coupled to appropriate filters and detection devices will be constructed to enable execution of these diverse experimental protocols. Time will be devoted to evaluation of the depolarizing effects of the microscope optics, mechanical stability of the instrument and development of software for data analysis. In addition, toxicity, photobleaching and perturbational effects of the designed fluorophores will be assessed as well as strategies for immobilization of the samples on the critically illuminated slide surface.
