HIGH-RESOLUTION DETECTION OF UNCOATED METAPHASE CHROMOSOMES BY MEANS OF FIELD-EMISSION SCANNING ELECTRON-MICROSCOPY

HeLa metaphase chromosomes were examined by means of ''in lens'' field emission scanning electron microscopy, which permits high resolution detection of uncoated biological samples. By using uncoated chromosome s as a model for comparison we report evidence of how traditional scan ning electron microscopy techniques such as metal coating and conducti ve methods can generate errors in chromosome structure evaluation, sin ce both give rise to morphological artifacts. By comparing the morphol ogy of uncoated chromosomes obtained by two different isolation proced ures, such as that utilized in standard cytogenetics and the polyamine method, we have drawn the following conclusions: (a) the standard cyt ogenetic method gives rise to a chromosome structure consisting of a f lattened network of 10 nm fibers, in which higher order chromatin orga nization is absent. (b) Chromosomes obtained by the polyamine method s how both three-dimensional profile and higher level folding of chromat in fibers, supporting the loop chromosome organization previously sugg ested by scanning electron microscopy observation of hexylene glycol i solated chromosomes.