Preparation and observation by SEM of hemopoietic cells cloned in soft agar

A method is described to prepare clones of hemopoietic cells grown in soft agar for scanning electron microscopy (SEM). A critical modification of the otherwise quite standard SEM processing procedure for biological samples involved the use of silver micropore disks as an adherent substrate to support the highly labile, deformable agar slabs. This support allows maintenance of the normal flat pancake shape of the specimen through the thiocarbohydrazide osmium ligand binding steps, dehydration, and critical point drying. With this support and careful dissection of the surface agar with a fine steel needle using a stereomicroscope, selected areas and depths within the colony can be exposed and examined by SEM. Surface topography of cloned cells can be correlated with intracellular cytological features by excising areas of interest and directly embedding them in plastic for thin-section preparation and viewing by transmission electron microscopy (TEM). The dried-specimen-teasing method appears useful, because of the ease of preparation of the specimens, its reproducibility, and the degree of visibility and preservation of cell surface structures and intraclonal relationships. Our initial observations, using combined EM techniques, indicate that clonal cell topography is highly variable and that this variability appears to be related both to the relative age and proliferative status of the colony. Based on work to date, we suggest that topographical and spatial analysis, in vitro of cloned, agar-embedded hemopoietic stem cells is possible with simple modifications of conventional SEM preparative techniques.