Combination of fluorescence in situ hybridization and scanning force microscopy for the ultrastructural characterization of defined chromatin regions

Abstract

Although the internal arrangement of interphase chromatin is still a matter of conjecture, there exists a large body of evidence for the compartmentalization of chromosomal domains. A study based on combined scanning force and optical microscopy of supramolecular chromatin spreads produced by isotonic lysis of cells suspended in phosphate-buffered saline has been conducted. The ultrastructure of fluorescent labeled chromosomes was resolved with the topographical contrast provided by the scanning force microscope. Fluorescence in situ hybridization was used to label specific DNA sequences. The location of different pericentromeric chromosome regions was determined by fluorescence microscopy and correlated with scanning force microscope topography. Using a single DNA probe, discrimination between labeled chromosome pairs of an aneuploid cell was possible, based on the different intensities of fluorescence signals. The results show that the in situ hybridization technique with fluorescence labeling is compatible with scanning force microscopy. The combination of these methods can be used for the specific identification and lateral localization of DNA sequences in spread chromatin, opening the possibility for the ultrastructural characterization of defined genes in the scanning force microscope.