6th ESACP Congress, Heidelberg, April 7-11, 1999

A052
SPECTRAL PRECISION DISTANCE MICROSCOPY (SPDM) IN HUMAN GENOME RESEARCH: 3D-NANOSTRUCTURE ANALYSIS OF X-CHROMOSOMAL GENES.
Edelmann P +,*, Esa A +, Dietzel S #, Cremer T #,*, Cremer C +,*

+ Institute of Applied Physics, Research Division Applied Optics and Information Processing, * Interdisciplinary Centre for Scientific Computing University of Heidelberg, Heidelberg, # Institute of Anthropology and Human Genetics, Ludwig-Maximilians-University, Munich, Germany

For the analysis of the 3D-genome nano-structure the precise determination of 3D distances between small specifically labelled chromatin regions in the interior of 3D-conserved cell nuclei is necessary eventually in the living cell. This necessitates the use of far field microscopical methods. A new light mircroscopical approach, Spectral Precision Distance Microscopy (SPDM) was developed which allows the determination of 3D distances far below the resolution limit. Spectral Precision Distance Microscopy (SPDM) is based on: a) the use of point-like objects labelled with different spectral signatures and b), spectrally differential registration of images and c) three- dimensional image analysis. In situ measurements revealed, that 3D distances in intact cell nuclei can be measured with an resolution equivalent of about 50 nm. This analysis tool was applied to study the 3D-positioning of the adenine nucleotide translocase genes ANT2 and ANT3 on both X-chromosome territories (Xa active and Xi inactive) in human female amniotic cell nuclei. ANT2 is transcriptionally active on Xa, but inactive on Xi. ANT3 however escapes X-inactivation. In this first SPDM analysis multicolour fluorescence in situ hybridisation (FISH), confocal laser-scanning microscopy and three-dimensional image analysis was combined and revealed that the relative positions of the ANT2/3 genes correlated with the activation state. On the active X-chromosome (Xa), the active genes ANT2 and ANT3 were located in a peripheral position in the chromosome territory. In the inactive X-chromosome territory (Xi) the active ANT3 gene was also located at a peripheral position, whereas the inactive ANT2 gene was shifted significantly (appr. 200 nm) towards a more interior position in the chromosome territory.