The application of cryo‐SEM techniques to the study of the symbiotic association in the Azolla leaf cavity

The preparation of frozen‐hydrated samples for scanning electron microscopy to observe symbionts in Azolla is described and compared to the conventional method of chemical fixation followed by critical‐point drying. The frozen‐hydrated specimens preserve the structure of the associating symbionts and the liquid phase in the Azollaleaf cavity. The leaf cavity structure and the endosymbionts in the frozen‐hydrated specimens were intact and unharmed throughout the fixation. The cyanobiont cells were distributed along the envelope of the leaf cavity, and did not aggregate around the hair cells, as reported for chemical fixation specimens. The mucilage layer removed by the chemical fixation could be observed in the cryo‐fixation specimens, especially at the lower part of the cavity. 1992 Blackwell Science Ltd