SPECTRAL PRECISION DISTANCE (SPDM) OF NUCLEAR SPATIAL ORGANIZATION

Christoph Cremer 1,2,3

1Institute of Molecular Biology (IMB), Ackermannweg 4, D-55128 , ; 2Kirchhoff Institute for (KIP), and 3Institute for Pharmacy and Molecular Biotechnology (IPMB), University of Heidelberg, Im Neuenheimer Feld, D-69120 Heidelberg, Germany e-mail:[email protected]; [email protected] The spatial organization of the in the interphase nucleus has far reaching functional consequences for gene regulation1. During the last decades, various methods of super- resolution fluorescence microscopy have been developed which made possible to enhance the spatial analysis of biostructures far beyond the conventional limits of about 200 nm in the object plane and 600 nm along the optical axis. Here, the focus will be on quantitative nuclear nanostructure analysis by spectral precision distance microscopy (SPDM), a special variant of fluorescence based far-field localization microscopy2. This nanoscopy approach makes use of conventional fluorescent proteins or standard organic fluorophores in combination with appropriate illumination conditions (typically monochromatic for a given molecule type) and standard (or only slightly modified) specimen preparation conditions. According to the applications envisaged, fluorophores can be applied either in the ‘photostable’ or in the ‘photoswitching’ mode. The SPDM technique was also combined in a custom-built inversed setup which allowed to first perform enhanced resolution imaging of nuclei at low illumination intensities using structured illumination, followed by ‘blinking’ SPDM imaging at higher illumination intensities. Application examples will be presented on the use of such SPDM approaches to perform quantitative analyses, e.g. of the nuclear distribution of individual Fluorescent-Protein tagged histones, chromatin remodeling and repair proteins, splicing factors, and RNA polymerases; or of the nuclear DNA distribution, positioning up to several million individual DNA-bound fluorophore signals in optical sections of various types of mammalian cell nuclei with a localization accuracy in the 10 nm range3. The resulting high structural resolution allowed the ‘nanoimaging’ of small chromatin dense clusters, and to highlight detailed features of the interchromatin compartment. Some perspectives of these novel, quantitative “superresolution” microscopy methods for deciphering the ”4D Nucleome“ will be discussed. 1T. Cremer & C. Cremer (2001) Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nature Reviews 2: 292 – 301. 2C. Cremer et al. (2011) Superresolution Imaging of Biological Nanostructures by Spectral Precision Distance Microscopy (SPDM), Biotechnology Journal 6: 1037 – 1051. 3A. T. Szczurek et al. (2014) Single molecule localization microscopy of the distribution of chromatin using Hoechst and DAPI fluorescent probes. Nucleus 5 (4)1-10.