Related Experiment Videos
Potency of microwave irradiation during fixation for electron microscopy
P Wild1, M Krähenbühl, E M Schraner
1Institute of Veterinary Anatomy, University of Zürich, Switzerland.
Abstract:
Liver, skeletal muscle, peripheral nerves, pancreas, thyroid and adrenal cortex were prepared for electron microscopy employing microwave energy either during prefixation with glutaraldehyde or instead of prefixation. Microwave irradiation in the presence of glutaraldehyde in Na/K-phosphate or Na-cacodylate containing CaCl2 and MgCl2 led to distinct appearance of membranes, mainly plasma membrane, and membranes of SER, Golgi complex and mitochondria in liver, pancreas and muscle. The area of high quality fixation, however, was limited to the periphery of samples. On the other hand, SER was dilated in cells of the adrenal cortex, and RER markedly vacuolated in thyroid follicular cells. Microwave irradiation in the presence of Na/K-phosphate and subsequent osmication resulted in preservation of the ultrastructure in similar quality as was obtained by osmication without previous immersion in glutaraldehyde. However, the preservation of SER and Golgi complex in liver and pancreas, and of mitochondria in muscle was greatly improved. Small myelin sheaths remained intact whereas large ones showed focal disintegration. We consider that enhancement of fixation by microwave energy may greatly improve preservation of membranes in some tissues. Successful fixation depends on the use of glutaraldehyde during microwave irradiation, the type of buffer, the addition of ions to increase stabilization, the exposure time to heat, and on postosmication.
Insights
Microwave energy improves electron microscopy fixation of biological membranes in tissues like liver and muscle. This method enhances the preservation of cellular structures, offering better insights into tissue ultrastructure.
Area of Science:
- Electron microscopy
- Cell biology
- Histology
Background:
- Traditional electron microscopy fixation methods can be time-consuming and may not optimally preserve delicate cellular structures.
- Microwave energy has emerged as a potential tool to accelerate and enhance fixation processes.
Purpose of the Study:
- To investigate the efficacy of microwave energy in improving the ultrastructural preservation of various tissues for electron microscopy.
- To determine the optimal conditions for microwave-assisted fixation.
Main Methods:
- Tissues including liver, skeletal muscle, and pancreas were prepared using microwave irradiation during or instead of glutaraldehyde prefixation.
- Microwave irradiation was performed in the presence of specific buffers (Na/K-phosphate or Na-cacodylate) with added ions (CaCl2, MgCl2).
- Subsequent osmication was employed, with some samples undergoing microwave irradiation prior to immersion in osmium tetroxide.
Main Results:
- Microwave irradiation with glutaraldehyde enhanced membrane appearance, particularly plasma membrane, SER, Golgi complex, and mitochondria, but fixation quality was limited to sample periphery.
- SER dilation in adrenal cortex and RER vacuolation in thyroid were observed.
- Microwave irradiation followed by osmication yielded preservation quality comparable to traditional methods, with improved preservation of SER, Golgi complex, and mitochondria in specific tissues.
- Small myelin sheaths were preserved, while larger ones showed disintegration.
Conclusions:
- Microwave energy can significantly enhance membrane preservation in electron microscopy for certain tissues.
- Successful microwave-assisted fixation depends on factors including glutaraldehyde use, buffer type, ion addition, irradiation time, and post-osmication.