Electron microscopy of mammalian cells in the absence of fixing, freezing, dehydration, or specimen coating

Abstract

Human osteoblast-like (bone-forming) cells were imaged using environmental scanning electron microscopy (ESEM). The cells were hydrated, unfrozen, and uncoated. Specimens were cooled to 3°C and imaged in water vapor, with partial pressures varying from saturated conditions to a humidity of approximately 50%, relative to pure water. The ESEM images show the presence of cell nuclei, nucleoli, and cytoplasmic membranes. Comparisons between chemically fixed and unfixed specimens (neither dried nor coated) show that cell morphologies are similar in both cases. These results are compared with a fixed, dried, carbon-coated specimen. Thermodynamic and kinetic arguments are used to show that humidities significantly lower than 100% correspond to metastable states suitable for stabilizing hydrated biological tissues and cells. The ability to perform observations with minimal specimen preparation is potentially useful for studying interactions between mammalian cells and biomaterials that are developed for tissue engineering. The methods employed are equally applicable to the study of specimens in the biological, materials, and physical sciences where careful control over specimen stability is required.