title: Super-resolution microscopy approaches to nuclear nanostructure imaging.
abstract: The human genome has been decoded, but we are still far from understanding the regulation of all gene activities. A largely unexplained role in these regulatory mechanisms is played by the spatial organization of the genome in the cell nucleus which has far-reaching functional consequences for gene regulation. Until recently, it appeared to be impossible to study this problem on the nanoscale by light microscopy. However, novel developments in optical imaging technology have radically surpassed the limited resolution of conventional far-field fluorescence microscopy (ca. 200nm). After a brief review of available super-resolution microscopy (SRM) methods, we focus on a specific SRM approach to study nuclear genome structure at the single cell/single molecule level, Spectral Precision Distance/Position Determination Microscopy (SPDM). SPDM, a variant of localization microscopy, makes use of conventional fluorescent proteins or single standard organic fluorophores in combination with standard (or only slightly modified) specimen preparation conditions; in its actual realization mode, the same laser frequency can be used for both photoswitching and fluorescence read out. Presently, the SPDM method allows us to image nuclear genome organization in individual cells down to few tens of nanometer (nm) of structural resolution, and to perform quantitative analyses of individual small chromatin domains; of the nanoscale distribution of histones, chromatin remodeling proteins, and transcription, splicing and repair related factors. As a biomedical research application, using dual-color SPDM, it became possible to monitor in mouse cardiomyocyte cells quantitatively the effects of ischemia conditions on the chromatin nanostructure (DNA). These novel "molecular optics" approaches open an avenue to study the nuclear landscape directly in individual cells down to the single molecule level and thus to test models of functional genome architecture at unprecedented resolution.
authors.affiliation: Superresolution Microscopy, Institute of Molecular Biology (IMB), Mainz, Germany; Department of Physics, University of Mainz (JGU), Mainz, Germany; Institute for Pharmacy and Molecular Biotechnology (IPMB), and Kirchhoff Institute for Physics (KIP), University of Heidelberg, Heidelberg, Germany. Electronic address: http://www.optics.imb-mainz.de.
authors.forename: Christoph
authors.initials: C
authors.lastname: Cremer
authors.affiliation: Superresolution Microscopy, Institute of Molecular Biology (IMB), Mainz, Germany.
authors.forename: Aleksander
authors.initials: A
authors.lastname: Szczurek
authors.affiliation: Department of Physics, University of Mainz (JGU), Mainz, Germany; Institute for Pharmacy and Molecular Biotechnology (IPMB), and Kirchhoff Institute for Physics (KIP), University of Heidelberg, Heidelberg, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Schock
authors.affiliation: Superresolution Microscopy, Institute of Molecular Biology (IMB), Mainz, Germany.
authors.forename: Amine
authors.initials: A
authors.lastname: Gourram
authors.affiliation: Superresolution Microscopy, Institute of Molecular Biology (IMB), Mainz, Germany; Department of Physics, University of Mainz (JGU), Mainz, Germany; Institute for Pharmacy and Molecular Biotechnology (IPMB), and Kirchhoff Institute for Physics (KIP), University of Heidelberg, Heidelberg, Germany.
authors.forename: Udo
authors.initials: U
authors.lastname: Birk
mesh_terms: D000818:Animals; D002232:Carbocyanines / Q000737:chemistry*; D002467:Cell Nucleus / Q000378:metabolism / Q000648:ultrastructure*; D002843:Chromatin / Q000378:metabolism / Q000648:ultrastructure*; D042002:Chromatin Assembly and Disassembly; D005456:Fluorescent Dyes / Q000737:chemistry*; D016044:Fusion Proteins, bcr-abl / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008856:Microscopy, Fluorescence / Q000295:instrumentation / Q000379:methods*; D032446:Myoblasts / Q000378:metabolism / Q000648:ultrastructure; D032383:Myocytes, Cardiac / Q000378:metabolism / Q000648:ultrastructure; D000071156:Promyelocytic Leukemia Protein / Q000235:genetics / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C569686:Alexa Fluor 647; C581634:BCR-ABL1 fusion protein, human; D002232:Carbocyanines; D002843:Chromatin; D005456:Fluorescent Dyes; D000071156:Promyelocytic Leukemia Protein; C117909:SUMO2 protein, human; D025841:Small Ubiquitin-Related Modifier Proteins; C070576:PML protein, human; D016044:Fusion Proteins, bcr-abl
keywords: (Single molecule) localization microscopy (SMLM); Nanoscopy; Nuclear structure; PALM; SPDM; STORM/dSTORM; Super-resolution light microscopy (SRM)