Complementary visualization of mitotic barley chromatin by field-emission scanning electron microscopy and scanning force microscopy

The surface structure of mitotic barley chromatin was studied by field-emis sion scanning electron microscopy (FESEM) and scanning force microscopy (SF M). Different stages of the cell cycle were accessible after a cell suspens ion was dropped onto a glass surface, chemical fixed, and critically point dried. Imaging was carried out with metal-coated specimen or uncoated speci men (only for SFM). The spatial contour of the chromatin could be resolved by SFM correlating to FESEM data. The experimentally determined volume of t he residue chromatin during mitosis was within the range of 65-85 mu m(3). A comparison with the theoretically calculated volume indicated a contribut ion of about 40% of internal cavities, Decondensation of chromosomes by pro teinase K led to a drastic decrease in the chromosome volume, and a 3-D net like architecture of the residue nucleoprotein material, similar to that in the intact chromosome, was obvious. Incubation of metaphase chromosomes in citrate buffer permitted access to different levels of chromatin packing. We imaged intact chromosomes in liquid by SFM: without any intermediate dry ing step. A granular surface was obvious but with an appreciably lower reso lution. Under similar imaging conditions proteinase R-treated chromosomes e xhibited low topographic contrast but were susceptible to plastic deformati ons. (C) 2000 Academic Press.