Nano-sizing of specific gene domains in intact human cell nuclei by spatially modulated illumination light microscopy.

Abstract:

:Although light microscopy and three-dimensional image analysis have made considerable progress during the last decade, it is still challenging to analyze the genome nano-architecture of specific gene domains in three-dimensional 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 microscopy. Gene domains for p53, p58, and c-myc were labeled by fluorescence in situ hybridization and the sizes of the fluorescence in situ hybridization "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, labeled sequence length. Assuming a spherical domain shape, these values would correspond to volumes of 5.7 x 10(-4) microm(3) (c-myc), 8.9 x 10(-4) microm(3) (p53), and 9.7 x 10(-4) microm(3) (p58). These volumes are approximately 2 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 labeled 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.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Hildenbrand G,Rapp A,Spöri U,Wagner C,Cremer C,Hausmann M

doi

10.1529/biophysj.104.056796

subject

Has Abstract

pub_date

2005-06-01 00:00:00

pages

4312-8

issue

6

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(05)73480-9

journal_volume

88

pub_type

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