Super-resolution microscopy approaches to nuclear nanostructure imaging
| Autor: | Florian Schock, Christoph Cremer, Amine Gourram, Udo Birk, Aleksander Szczurek |
|---|---|
| Rok vydání: | 2017 |
| Předmět: |
0301 basic medicine
Fusion Proteins bcr-abl Gene Expression Nanotechnology Promyelocytic Leukemia Protein Biology Genome General Biochemistry Genetics and Molecular Biology Chromatin remodeling Myoblasts Mice 03 medical and health sciences Microscopy Fluorescence microscope medicine Animals Humans Myocytes Cardiac Molecular Biology Fluorescent Dyes Cell Nucleus Super-resolution microscopy Carbocyanines Chromatin Assembly and Disassembly Chromatin Cell nucleus 030104 developmental biology medicine.anatomical_structure Microscopy Fluorescence Small Ubiquitin-Related Modifier Proteins Biophysics Human genome HeLa Cells |
| Zdroj: | Methods. 123:11-32 |
| ISSN: | 1046-2023 |
| DOI: | 10.1016/j.ymeth.2017.03.019 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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