Nanostructure of specific chromatin regions and nuclear complexes

Abstract

Spatially Modulated Illumination (SMI) microscopy is a method of widefield fluorescence microscopy featuring interferometric illumination, which delivers structural information about nanoscale features in fluorescently labeled cells. Using this approach, structural changes in the context of gene activation and chromatin remodeling may be revealed. In this paper we present the application of SMI microscopy to size measurements of the 7q22 gene region, giving us a size estimate of 105 ± 16nm which corresponds to an average compaction ratio of 1:324. The results for the 7q22 domain are compared with the previously measured sizes of other fluorescently labeled gene regions, and to those obtained for transcription factories. The absence of a correlation between the measured and genomic sizes of the various gene regions indicate that a high variability in chromatin folding is present, with factors other than the sequence length contributing to the chromatin compaction. Measurements of the 7q22 region in different preparations and at different excitation wavelengths show a good agreement, thus demonstrating that the technique is robust when applied to biological samples.

Measurement of replication structures at the nanometer scale using super-resolution light microscopy

“…SMI microscopy is limited to sizes in the range from ∼40 nm to 200 nm. The measurements of objects <40 nm are imprecise, and sizes approximately >220 nm cannot be measured since no further correlation between the axial modulation and the size of the object can be detected (15), resulting in a truncation of the distribution at this value. However, we estimate that in the present study only a small number (<<5%) of foci were outside of the above range; inclusion of these structures would be unlikely to significantly affect the results.…”

High-precision structural analysis of subnuclear complexes in fixed and live cells via spatially modulated illumination (SMI) microscopy

“…SMI microscopy is now an established method for the analysis of topological arrangements of mammalian genome structure. In combination with novel approaches for fluorescence labelling, the SMI Fnanosizing_ technique has proved its applicability for a wide range of biological questions when using fixed cell preparations (Martin et al 2004, Hildenbrand et al 2005, Mathée et al 2006. Nevertheless, the samples that have been analysed so far were fixed specimens and the influence of fixation procedures on the overall cell structure and in particular on the nanostructure of the genome has not yet been clarified (Hendzel & Bazett-Jones 1997, Kozubek et al 2000.…”

The histochemistry and cell biology vade mecum: a review of 2005–2006

“…These data suggest possible diVerences in genome regions replicated by these two types of replication factories. Mathee et al (2006) analyzed the nanostructure of speciWc chromatin regions and nuclear complexes. For this, they applied spatially modulated illumination (SMI) microscopy, which is a method of wideWeld Xuorescence microscopy featuring interferometric illumination.…”