SEM observation of the insect prepared by microwave irradiation

The ptilinum of the fly and the compound eye are among the most fragile organs encountered during conventional procedures of morphological sample fixation. In order to identify a fixative suitable for preparing such samples for scanning electron microscopy, we examined various fixation conditions using microwave irradiation (MWI). The conditions examined were: (i) fixatives; (ii) temperature; (iii) concentration; (iv) duration; (v) dehydration; and (vi) substitution. The identified optimal conditions were 5% glutaraldehyde with MWI (350 W, 5 min). The MWI was continued until the maximal temperature of 75°C was attained, followed by intermittent irradiation to maintain a temperature of 75°C. After irradiation, the sample was left at room temperature for 24 h in the fixative and then dehydrated in increasing concentrations of ethanol. Each step in the ethanol series lasted for 24 h. The final absolute ethanol step included three solution changes, with each incubation lasting 1 h. A subsequent stepwise substitution of t-butyl alcohol for ethanol was conducted by reducing the ratio of 100% ethanol to t-butyl alcohol from 2:1 to 1:1 and then 1:2 (24 h each). The substitution was completed by three solution changes using 100% t-butyl alcohol, 30 min each. The best results were obtained by freeze-drying samples using t-butyl alcohol. The use of MWI improved fixative permeation, which occurred at a uniform rate throughout the sample. Comparison with temperature in a water bath at 75°C indicated that the fixation effect of MWI was due to its heat generation in addition to some unknown mechanism.