Nanoprobes Inc

Combined Fluorescent and Gold Labeling Reagents - Correlative Light and Electron Microscopy (CLEM) Is an Essential Tool for Correlating Structure and Function at the Cellular Level With the Localization and Mechanism of Processes at the Macromolecular Level. New Fluorescence Labeling Technologies Such as Brighter, Sharper Fluorophores, Fluorescent Fusion Proteins and Spectral Deconvolution Afford Highly Multiplexed Mapping, Superresolution Microscopy Has Enabled More Precise Target Localization and Methods Such as Fret Localize Nanometer-range Molecular Interactions. Meanwhile, New Instrument and Workflow Designs and Large-volume Methods Have Greatly Expanded the Scope of Electron Microscopy and Our Ability to Correlative Em and LM Data. However, a Lack of Truly Correlative, Multiplexed Labeling Technologies Has Prevented Realization of Its Full Potential. We Will Address This Unmet Need by Systematically Developing a Set of Reagents That Combine Fluorescence Tags With a Gold Nanoparticle Em Label in a Single Reagent That Can Be Used to Label Any Appropriately Reactive Molecule in One Simple Reaction. We Will Use Monodisperse Biocompatible Macromolecules as Spacers to Separate the Fluorescent and Gold Labels, Thus Minimizing Fluorescence Quenching and Incorporated the Reactive Group for Probe Labeling Into the Gold Nanoparticle to Ensure Highest Possible Em Labeling Precision. Spacers of Different Sizes Will Be Used to Prepare Reagents Using Three Different Gold Nanoparticle Sizes – 1.4, 3, and 5 NM – Each Combined With Different Fluorophores. Reagents Will Be Developed Both With Biorthogonal Reactivity (click, Snap and Halo- Tag) and With Specific Reactivities Towards Functional Groups Such as Thiols and Amines for Conventional Conjugation Reactions With Molecules Such as Peptides, Nucleic Acids, Lipids, Substrate Analogs or Inhibitors. Reagents Will Be Evaluated in Two Experimental Systems: (1) Correlative Fluorescence and Chromatin Electron Microscopy Tomography (chromemt) to Label, Map, and Differentiate the 3-D Distribution of the Three Major Structural Classes of Chromatin: (a) Transcriptionally Active Regions, (B) Transcriptionally Repressed Chromatin, and (C) Bivalent/transitionally Silent Regions (collaboration With DR. Antonio Giraldez and DR. Alice Sherrard of Yale University Medical School). (2) Correlative Fluorescence and Electron Microscopic Labeling of Alpha-synuclein (a-syn) Aggregates to Elucidate Their Uptake by Neurons Using Fret, Fib-sem, Pre and Post-embedding Em and Electron Tomography (consulting Collaboration With DR. Eduardo Rosa-molinar, Washington University in ST. Louis, Center for Cellular Imaging).