Nanoscale Imaging of Fish Probe Binding to Metaphase Chromosomes

Abstract

Fluorescence in situ hybridization (FISH) is widely used for diagnosis of genetic abnormalities. We have developed multimodal, nanoscale imaging methods using a combination of fluorescence and atomic force microscopy to determine the precise binding location of FISH probes. The use of high resolution imaging methods is important to validate and improve the binding efficiency for small single copy FISH probes that are being developed for cytogenetic analysis at higher genomic resolution than is possible with current commercial FISH probes. Chromosome topography is imaged by AFM and superimposed on FISH probe location determined by confocal or epifluorescence microscopy. Image processing procedures are used to quantify probe context, relative to neighboring 30 nm chromatin fiber bundles imaged by AFM, and to quantify probe binding to homologs within the same cell. We have correlated optical and topographic images of multicopy centromeric probe for chromosome 17 and have examined the spatial relationship between centromeric DNA and the formation of kinetochore structures and sister chromatid segregation. Interestingly, segregation of sister chromatids is not coordinated with the formation of these structures within individual homologous chromosomes, occurring after the formation of these structures. We hypothesize that asynchronous segregation of previously replicated sister chromosomes may account for differences in the hybridization patterns for probes in different cells from the same individual.