Problems in preparation of chromosomes for scanning electron microscopy to reveal morphology and to permit immunocytochemistry of sensitive antigens.

Abstract

Although much information about chromosome structure and behaviour has been obtained using light microscopy, greater resolution is needed for a thorough understanding of chromosome organisation. Scanning electron microscopy (SEM) can provide valuable data about these three-dimensional organelles. The introduction of methods using osmium impregnation of methanol-acetic acid-fixed chromosome spreads revolutionised matters, producing life-like images of chromosomes. Nevertheless, it became clear that osmium impregnation introduced various artefacts, although the resulting images were still useful. Methanol-acetic acid-fixed chromosomes are, in fact, flattened on the glass substratum, and the 3-dimensional appearance obtained after osmium impregnation is the result of swelling during this process. At the same time, the fibrous substructure of the chromosomes becomes much coarser. More recently a number of alternative methods have become available for studying chromosomes by SEM. Isolated chromosomes, that have not been allowed to dry during preparation, retain a 3-dimensional appearance without osmium impregnation, and the same is true of methanol-acetic acid-fixed chromosomes that have been treated with 45% acetic acid and processed without drying; however, these methods do not permit the routine production of intact metaphase spreads. Use of cytocentrifuge preparations obviates the use of acetic acid fixation and osmium impregnation, produces intact metaphase spreads, and permits the immunocytochemical detection of antigens that are easily destroyed by routine fixation procedures.