کد مقاله | کد نشریه | سال انتشار | مقاله انگلیسی | نسخه تمام متن |
---|---|---|---|---|
5513429 | 1541205 | 2017 | 22 صفحه PDF | دانلود رایگان |
- Review of the available light optical super-resolution approaches to study nuclear nanostructure.
- Nuclear genome structure studied at the single cell/single molecule level by Spectral Precision Distance/Position Determination Microscopy (SPDM), a variant of localization microscopy.
- Single Molecule Localization Microscopy (SMLM) using conventional fluorescent proteins, single standard organic fluorophores, or conventional DNA-binding dye molecules.
- Imaging and quantitative analyses of nuclear genome organization in individual cells down to few tens of nanometer (nm) of structural resolution.
- “Molecular optics” approaches to study the nuclear landscape and the functional genome architecture directly in individual cells down to the single molecule level.
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. 200Â nm). 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.
Journal: Methods - Volume 123, 1 July 2017, Pages 11-32