Size changes in single muscle fibers during fixation and embedding.

Area of Science:

• Cell Biology
• Microscopy Techniques
• Biochemistry

Background:

• Accurate volume determination of biological samples is crucial for understanding cellular structure and function.
• Electron microscopy sample preparation involves multiple chemical and physical steps that can induce volume changes.
• The osmotic properties of cell membranes during fixation and processing are not fully understood.

Purpose of the Study:

• To quantify the volume changes of single muscle fibers during various stages of electron microscopy preparation.
• To investigate the role of membrane osmotic properties in volume fluctuations during sample processing.

Main Methods:

• Single muscle fibers were subjected to fixation with glutaraldehyde.
• Subsequent steps included rinsing, osmium tetroxide treatment, uranyl acetate staining, dehydration with ethanol and propylene oxide, and embedding with Epon.
• Volume changes were monitored throughout the preparation process.

Main Results:

• A 20% volume shrinkage occurred during initial glutaraldehyde fixation.
• Volume recovered near normal levels after rinsing and osmium tetroxide treatment.
• Further shrinkage of 20% occurred with uranyl acetate staining.
• Dehydration and Epon embedding caused an additional 25% volume reduction.
• Total observed shrinkage reached 45%, nearly double the apparent shrinkage of the myosin lattice.

Conclusions:

• Cellular membranes may retain significant osmotic properties even after fixation and extensive processing.
• The cumulative shrinkage during electron microscopy preparation substantially alters the native volume of muscle fibers.
• These findings have implications for interpreting structural data obtained from electron microscopy of biological tissues.