Analysis by atomic force microscopy of morphological changes in barley chromosomes during FISH treatment.

We employed atomic force microscopy (AFM) to examine structural changes in barley chromosomes during the four steps of standard FISH processes. Rehydration and dehydration with alcohol accompanying RNase treatment increased chromosome arm width and decreased chromosome height about 50%. Subsequent heat denaturation reduced chromosome height further. These three-dimensional structural changes of the chromosomes were substantial, but the FISH signal produced by the hybridization of fluorescent probes was clear when observed by a fluorescence microscope. In higher-magnification images, we observed granular structures considered to represent the chromatin fiber on the surface of the chromosomes in each FISH protocol step. These our results indicate that FISH treatments result in severe damage of the three-dimensional higher-order structures of the chromosomes, although nano-structures, such as nucleosome and chromatin fibers, remain intact and relatively unaffected.