Plenary Lecture

Super-Resolution Light Microscopy of Nuclear Genome Nanostructure

Professor Christoph Cremer
Institute of Molecular Biology (IMB)
Institute for Pharmacy and Molecular Biotechnology (IPMB)
University Heidelberg & Kirchhoff-Institute for Physics (KIP)
Department of Physics
University Mainz (JGU)

Abstract: A major challenge in the Biosciences is to link the knowledge gained by molecular methods to cellular nanostructure. Until recently, however, “molecular optics” (MO) approaches at the individual cell level using far field light microscopy were substantially limited by the conventional resolution of about 200 nm in the object plane and 600 nm along the optical axis (“Abbe/Rayleigh-limit”). These limits have been overcome by various super-resolution fluorescence microscopy (SRM) methods, such as Stimulated Emission Depletion (STED), Photoactivated Localization Microscopy (PALM), Structured Illumination Microscopy (SIM), or Stochastic Optical Reconstruction Microscopy (STORM)1. Here we report on a complementary SRM approach for “Molecular Optics” (MO) 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, allowing to use the same laser frequency for both photoswitching and fluorescence read out2. As an example for the application of this SRM-MO approach, results on nuclear nanostructure elucidation will be presented: Presently, this approach allows us to optically resolve nuclear structures in individual cells down to few tens of nanometer, and to perform quantitative MO analyses of individual small chromatin domains; of the nanoscale distribution of histones, chromatin remodeling proteins, and transcription, splicing and repair related factors. In addition, it has become possible to combine localization microscopy (SPDM) of nuclear DNA distribution, positioning up to several million individual DNA-bound fluorophore signals in an optical section through a 3D intact cell nucleus, with simultaneous measurements of the spatial positions of individual epigenetic histone marker molecules, Pol II, or newly replicated DNA. The experimental results support recent models of functional nuclear structure3. As a translational application, using dual-color SPDM, it became possible to monitor in mouse myocardial cells quantitatively the effects of ischemia conditions on the chromatin nanostructure (DNA)4. These novel SRM-MO approaches open an avenue to study the molecular landscape directly on the individual cell level at unprecedented resolution. 1C. Cremer, B.R. Masters (2013) Resolution enhancement techniques in microscopy.Eur. Phys. J. H, Eur. Phys. J. H 38: 281–344. 2C. Cremer et al. (2011) Superresolution Imaging of Biological Nanostructures by Spectral Precision Distance Microscopy (SPDM), Biotechnology Journal 6: 1037 – 1051. 3T. Cremer et al. (2015) The 4D nucleome: Evidence for a dynamic nuclear landscape based on coaligned active and inactive nuclear compartments. FEBS LettersFEBS Letters 589: 2931–2943. 4 I.Kirmes et al. (2015) A transient ischemic environment induces reversible compaction of chromatin. Genome Biology 16:246.

Brief Biography of the Speaker: Christoph Cremer studied Physics at the University of Munich (LMU) and obtained a Ph.D. (1976) in Biophysics and Genetics as well as a Dr. med. habil. degree in General Human Genetics and Experimental Cytogenetics (1983) at the University of Freiburg/Germany. Since 1983 he is a Professor for Applied Optics and Information Processing at the University of Heidelberg. Since 2011 he is group leader (super-resolution microscopy) at the Institute of Molecular Biology (IMB) and (since 2013) Honorary Professor (Physics) at the University of Mainz (JGU). Since 2015 he is also a Research Associate of the Max Planck Institute for Chemistry, Mainz. The methodological focus of the Cremer-Lab is the development and application of methods of super-resolving fluorescence light microscopy. Contributions to this field include focused nanoscopy, structured illumination, and various types of localization microscopy. As the main application field, these techniques are applied to study the nuclear nanostructure in various cell types and organisms.

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