Luminescent metal complex probes for correlative microscopy

DESCRIPTION provided by applicant Novel transition metal complex cluster based probes for correlative light and electron microscopy CLEM and universally adoptable robust protocols for labeling whole tissue mounts will be developed The new probes and protocols will enable simultaneous localization of two or more antigens labeled using a single step labeling procedure Following fluorescence imaging the fluorescently labeled targets with analogs of ruthenium II poly pyridine will be made andquot visibleandquot in the electron microscope by catalyzed deposition of electron dense silver that provides higher contrast and well defined punctuate signal as compared to the photo polymerized andapos diaminobenzidine DAB that results in defuse signal and requires osmium tetroxide OsO The proposed probes have following advantages i higher quantum yields QY than gold probes and comparable QYs to the semiconductor andquot quantum dotsandquot QDs ii smaller hydrodynamic radii less toxicity and better stability in biological buffers than QDs iii long half lives and large Stokes shifts for time resolved imaging iv punctate signal and better signal to noise ratios in the EM following silver deposition as compared to photoconvertible fluorescent proteins and ReAsH reagents that use DAB OsO v possible imaging with some of the andquot super resolutionandquot techniques and vi correlative multiplexing when used with genetically encoded photo convertible and EM tags In Phase I the proposed probes will be used to accurately locate gap junctions at andquot mixed synapsesandquot conjoined electrical and chemical synaptic components and facilitate the unambiguous identification of one or two constituent synaptic proteins found at mixed synapses and determination of the membrane andquot sidednessandquot with correlative light and electron microscopy CLEM Localization of mixed synapses and specific synaptic proteins is problematic because cell membranes and their constituent proteins are below the limit of resolution of light microscopic imaging techniques CLEM will be carried out in collaboration with Dr Eduardo Rosa Molinar Biological Imaging Group University of Puerto Rico Rio Piedras Dr Rosa Molinar has been working on elucidating the spinal motor circuitry controlling the adult male Gambusiaandapos s extremely rapid ms coital behavior Gambusiaandapos s circuitry which is an ideal test system for performing CLEM with the new probes In the longer term we plan to develop reagents and protocols for correlative andquot super resolutionandquot microscopy and serial section electron tomography Serial Block Face dual beam SEM and tilt series TEM PUBLIC HEALTH RELEVANCE This project will provide researchers in cell molecular and structural biology with simple universal reagents for correlative light and electron microscopy to study cellular life processes and disease processes at macromolecular resolution providing structural information at the molecular level