Improved representation of cell surface structures by freeze substitution and backscattered electron imaging

A surface preparation method for the SEM based on cryofixation is presented suitable for the demonstration of membrane particles on whole cells. LLC-PK1 cells (a renal epithelial cell line in culture) were fast frozen, freeze substituted, critical point dried, shadowed with 2 nm of platinum carbon and stabilized with a carbon backing layer. Membrane bound particles are visualized by the material dependent backscattered electron image mainly originating from the platinum shadow. The surface of the LLC-PK1 cells is almost free of precipitated material indicating that the culture medium is removed during freeze substitution or critical point drying. The apical plasma membrane with microvilli and ciliae is well preserved and differences in particle density can be detected. The feasibility of the coating technique for backscattered electron imaging was tested on the well known hexagonally arranged intramembranous particles of fractured and partially freeze dried yeast. This 16.5 nm periodic structure is clearly demonstrable on the bulk SEM-specimen stablized with the carbon backing layer. Without a carbon layer severe shrinking artifacts occurred.