Advantages of environmental scanning electron microscopy in studies of microorganisms

Microorganisms, including bacteria, fungi, protozoa, and microalgae, are composed predominantly of water which prohibits direct observation in a traditional scanning electron microscope (SEM). Preparation for SEM requires that microorganisms be fixed, frozen or dehydrated, and coated with a conductive film before observation in a high vacuum environment. Sample preparation may mechanically disturb delicate samples, compromise morphological information, and introduce other artifacts. The environmental scanning electron microscope (ESEM) provides a technology for imaging hydrated or dehydrated biological samples with minimal manipulation and without the need for conductive coatings. Sporulating cultures of three fungi, Aspergillus sp., Cunninghamella sp., and Mucor sp., were imaged in the ESEM to assess usefulness of the instrument in the direct observation of delicate, uncoated, biological specimens. Asexual sporophores showed no evidence of conidial displacement or disruption of sporangia. Uncoated algal cells of Euglena gracilis and Spirogyra sp. were examined using the backscatter electron detector (BSE) and the environmental secondary electron detector (ESD) of the ESEM. BSE images had more clearly defined intracellular structures, whereas ESD gave a clearer view of the surface E. gracilis cells fixed with potassium permanganate, Spirogyra sp. stained with Lugol's solution, and Saprolegnia sp. fixed with osmium tetroxide were compared using BSE and ESD to demonstrate that cellular details could be enhanced by the introduction of heavy metals. The effect of cellular water on signal quality was evaluated by comparing hydrated to critical point dried specimens.