Low pressure mode combined with OsO4 vapor fixation and sputter-coating for the preservation of delicate aerial hyphae and conidia in the ESEM

Abstract

The newer generation of environmental scanning electron microscopes (ESEMs) allows samples to be viewed under a range of different vacuum conditions. No specific sample preparation protocols are required with the ESEMs, as fresh, unfixed samples are used and discarded later. We have worked out a method that preserves aerial hyphae on biltong that closely resemble fresh specimens and may be stored for viewing at a later date. Another advantage is that fixed samples are more resilient to the variable vacuum encountered in the ESEM. When biltong samples with fungal growth were first studied, we observed that vacuum-related artifacts were induced unless vacuum conditions and changes in pressure were carefully controlled. Damage readily occured in conidia and its delicate hyphae. Fresh, unfixed samples are very vulnerable to these artifacts. In addition, biltong proved to be a problematic study sample because of its high salt content, its hygroscopic nature as well as being laden with spices. To eliminate these artifacts, the preservation of specimens by OsO4 vapor fixation combined with a special sputter-coating technique is described. Previous studies confirmed that OsO4 vapor fixation is superior to traditional immersion fixation methods for the examination of hyphae and conidia of various fungi in a conventional SEM. However, both preparation methods induce sample shrinkage. We observed that OsO4 vapor fixation followed by Au coating under strictly controlled vacuum conditions induced fewer artifacts and gave the best images with minimum distortion in low pressure (LP) mode. The proposed method allows samples to be viewed both in ESEM and LP mode. There are however some disadvantages inherent to ESEM mode. Even when viewing fixed, coated samples, care should be taken to maintain a pressure of not lower than 0.2 Torr in the specimen chamber. It is critical that different samples have the same vacuum exposure history, as shrinkage and collapse were found to be directly related to the lowest pressure the samples were subjected to, both in the sputter coater chamber and in the electron microscope specimen chamber.