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

A054
SPECTRAL PRECISION DISTANCE MICROSCOPY IN THE GENOME RESEARCH: INVESTIGATION OF THE NANOSTRUCTURE OF CANCER CORRELATED REGIONS
Esa A 1, Edelmann P 1, Trakhtenbrot L 2, Cremer C 1

1) Applied Optics and Information Processing, Institute of Applied Physics, University of Heidelberg, Germany, 2) Institute of Hematology, The Chaim Sheba Medical Center; Tel Hashomer, Israel.

Many human cancers are associated with structural chromosomal aberrations. The Philadelphia chromosome [t(9;22); translocation] is the cytogenetic hallmark of one of the most common human leukemia, Chronic Myelogenous Leukemia (CML). Despite this fact, there is much less information about the mechanism leading to the formation of these chromosomal aberrations. The microscopic analysis of the 3D-nanostructure of these chromatin regions in intact cell nuclei can contribute to an better understanding of the spatial organisation of these regions and thus of the formation of chromosome aberrations induced by ionising radiation. To study the 3D-nanostructure of these cancer correlated regions, it is highly desirable to use far field fluorescent light microscopic techniques, e.g. confocal laser scanning microscopy (CLSM). However, experimental evidence indicates that under biologically relevant conditions by using objects with the same fluorochrome, the spatial resolution of the CLSM is limited to about 750 nm in axial direction and about 300 nm in lateral direction. To overcome this shortcoming of resolution, a recently developed light microscopical approach, Spectral Precision Distance microscopy (SPDM) will be presented. This technique is based on spectrally differential registration of images and quantitative spectral image analysis of spatially neighbouring nuclear sites. In combination with specific labelling of very small chromatin targets with dyes of different spectral signatures by fluorescence in situ hybridisation, SPDM allows to analyse nuclear topology in three dimensionally conserved nuclei with a resolution " equivalent " of about 50 nm. A confocal laser scanning mode of this technique was applied to study the 3D-topology of the BCR-ABL region involved in the formation of the Philadelphia chromosome [t(9;22)] of CML patients.