COMBINATION OF FLUORESCENCE IN-SITU HYBRIDIZATION AND SCANNING FORCE MICROSCOPY FOR THE ULTRASTRUCTURAL CHARACTERIZATION OF DEFINED CHROMATIN REGIONS

Although the internal arrangement of interphase chromatin is still a m atter of conjecture, there exists a large body of evidence for the com partmentalization of chromosomal domains. A study based on combined sc anning force and optical microscopy of supramolecular chromatin spread s produced by isotonic lysis of cells suspended in phosphate-buffered saline has been conducted. The ultrastructure of fluorescent labeled c hromosomes was resolved with the topographical contrast provided by th e scanning force microscope. Fluorescence in situ hybridization was us ed to label specific DNA sequences. The location of different pericent romeric chromosome regions was determined by fluorescence microscopy a nd correlated with scanning force microscope topography. Using a singl e DNA probe, discrimination between labeled chromosome pairs of an ane uploid cell was possible, based on the different intensities of fluore scence signals. The results show that the in situ hybridization techni que with fluorescence labeling is compatible with scanning force micro scopy. The combination of these methods can be used for the specific i dentification and lateral localization of DNA sequences in spread chro matin, opening the possibility for the ultrastructural characterizatio n of defined genes in the scanning force microscope. (C) 1996 American Vacuum Society.