Abstract
Nuclear texture analysis is a well-established method of cellular pathology. It is hampered, however, by the limits of conventional light microscopy (ca. 200 nm). These limits have been overcome by a variety of super-resolution approaches. An especially promising approach to chromatin texture analysis is single molecule localization microscopy (SMLM) as it provides the highest resolution using fluorescent based methods. At the present state of the art, using fixed whole cell samples and standard DNA dyes, a structural resolution of chromatin in the 50–100 nm range is obtained using SMLM. We highlight how the combination of localization microscopy with standard fluorophores opens the avenue to a plethora of studies including the spatial distribution of DNA and associated proteins in eukaryotic cell nuclei with the potential to elucidate the functional organization of chromatin. These views are based on our experience as well as on recently published research in this field.
Highlights
The linear DNA of the genome provides the information for the production of various types of RNA molecules; besides mRNAs this includes a large variety of small RNA sequences such as silencing RNAs, piRNA, or micro RNAs, which play an important role in the regulation of gene expression, development, metabolism as well as in essentially any cellular process
Using single molecule localization microscopy (SMLM) of human cell nuclei containing histones tagged with fluorescent proteins, light microscopic evidence was obtained for a heterogeneous distribution of chromatin on the nanoscale (Gunkel et al, 2009; Bohn et al, 2010; Markaki et al, 2010)
We focus on visualization of directly labeled DNA, and how such approaches can contribute to a super-resolution analysis e.g., of the architecture of ischemic cells or of nucleoli
Summary
The linear DNA of the genome provides the information for the production of various types of RNA molecules; besides mRNAs this includes a large variety of small RNA sequences such as silencing RNAs (siRNA), piRNA, or micro RNAs (miRNA), which play an important role in the regulation of gene expression, development, metabolism as well as in essentially any cellular process. Electron and enhanced resolution light microscopy (for review see Huang et al, 2009; Cremer and Masters, 2013) have been applied to study the chromatin distribution in mammalian cell nuclei on the nanoscale (Rouquette et al, 2010).
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