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Updated: Aug 29, 2026
Electrophysiological Measurements from a Moth Olfactory System
Published on: March 29, 2011
SEM observation of the insect prepared by microwave irradiation
Daisuke Koga1, Masaki Ueno, Shohei Yamashina
1Department of Molecular Cell Biology, Graduate School of Medicine, Kitasato University, Sagamihara, Kanagawa 228-8555, Japan. dk1224@med.niigata-u.ac.jp
Abstract:
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 degrees C was attained, followed by intermittent irradiation to maintain a temperature of 75 degrees 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 degrees C indicated that the fixation effect of MWI was due to its heat generation in addition to some unknown mechanism.
Insights
Microwave irradiation (MWI) optimizes fixation for fragile fly organs like the ptilinum and compound eye. Optimal conditions involve 5% glutaraldehyde with MWI, followed by ethanol dehydration and t-butyl alcohol substitution for superior scanning electron microscopy samples.
Area of Science:
- Morphology
- Microscopy
- Biotechnology
Background:
- Fragile biological specimens, such as the ptilinum and compound eye of flies, present challenges for conventional morphological sample fixation.
- Scanning electron microscopy (SEM) requires high-quality sample preparation to preserve delicate structures.
Purpose of the Study:
- To identify optimal fixation conditions for fragile fly organs using microwave irradiation (MWI) for SEM.
- To evaluate the impact of various MWI parameters on sample preservation.
Main Methods:
- Systematic examination of fixation parameters including fixatives, temperature, concentration, duration, dehydration, and substitution using MWI.
- Optimization involved 5% glutaraldehyde with MWI (350 W, 5 min) reaching 75°C, followed by 24h fixation, ethanol dehydration, t-butyl alcohol substitution, and freeze-drying.
Main Results:
- Optimal fixation achieved with 5% glutaraldehyde and MWI at 75°C.
- MWI enhanced fixative permeation uniformly throughout the sample.
- Freeze-drying using t-butyl alcohol yielded the best results.
Conclusions:
- Microwave irradiation provides an effective method for fixing fragile biological samples for SEM.
- MWI's efficacy is attributed to heat generation and potentially other mechanisms.
- The optimized protocol significantly improves the preparation of delicate insect structures for high-resolution imaging.
