SPECTROSCOPY, IMAGING, OTHER TECHNIQUES |
Nano-sizing of specific gene domains in intact human cell
nuclei by Spatially Modulated Illumination (SMI) light
microscopy
Georg Hildenbrand 1, Alexander Rapp 2, Udo Spoeri 1, Christian Wagner 1, Christoph Cremer 1 and Michael Hausmann 3*
1 Kirchhoff-Institute of Physics, University of Heidelberg
2 Institute of Molecular Biotechnology, Jena
3 University Hospital Freiburg
* To whom correspondence should be addressed. E-mail: michael.hausmann{at}uniklinik-freiburg.de.
Submitted on November 22, 2004
Revised on January 20, 2005
Accepted on 3 March 2005
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Abstract |
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Although light microscopy and three-dimensional (3D-)image analysis have made considerable progress during the last decade, it is still challenging to analyse the genome nano-architecture of specific gene domains in 3D cell nuclei by fluorescence microscopy. Here, we present for the first time chromatin compaction measurements in human lymphocyte cell nuclei for three different, specific gene domains using a novel light microscopic approach called Spatially Modulated Illumination (SMI) microscopy. Gene domains for p53, p58, and c-myc were labelled by fluorescence in situ hybridization (FISH) and the sizes of the FISH "spots" were measured. The mean diameters of the gene domains were determined to 103 nm (c-myc), 119 nm (p53), and 123 nm (p58) and did not correlate to the genomic, labelled sequence length. Assuming a spherical domain shape, these values would correspond to volumes of 5.7 x 10-4 µm3 (c-myc), 8.9 x 10-4 µm3 (p53), and 9.7 x 10-4 µm3 (p58). These volumes are about two orders of magnitude smaller than the diffraction limited illumination or observation volume, respectively, in a confocal laser scanning microscope using a high numerical aperture objective lens. By comparison of the labelled sequence length to the domain size, compaction ratios were estimated to 1:129 (p53), 1:235 (p58), and 1:396 (c myc). The measurements demonstrate the advantage of the SMI technique for the analysis of gene domain nano-architecture in cell nuclei. The data indicate that chromatin compaction is subjected to a large variability which may be due to different states of genetic activity or reflect the cell cycle state.
Key Words:
SMI microscopy, gene chromatin compaction, gene region size, genome nano-architecture
