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Cell and organelle shrinkage during preparation for scanning electron microscopy: effects of fixation, dehydration
Abstract:
The critical point drying method of preparing samples for scanning electron microscopy is associated with a variable amount of specimen shrinkage. We studied the causes of this phenomenon is isolated mouse hepatocyte nuclei and in human erythrocytes and found that the critical point drying process itself caused most of the shrinkage that we observed (a 25-30% reduction in diameter in both specimens). Glutaraldehyde fixation and ethanol dehydration caused only minimal size reduction, prior to critical point drying. Substitution of an inert (ethylene glycol-ethylene glycol monethyl ether) dehydration technique did not alter the final result. Previous studies in our laboratory using high resolution SEM and correlative transmission microscopy of isolated nuclei have demonstrated that the shrinkage represents a miniaturization of the organelles in which all structural components retain their usual relationships.
Insights
Critical point drying causes significant specimen shrinkage in cell nuclei and erythrocytes. This size reduction is primarily due to the drying process itself, not fixation or dehydration steps.
Area of Science:
- Cell Biology
- Microscopy Techniques
Background:
- Critical point drying is a common method for preparing biological samples for scanning electron microscopy (SEM).
- Specimen shrinkage during critical point drying can affect the accuracy of morphological analysis.
Purpose of the Study:
- To investigate the causes of specimen shrinkage during critical point drying.
- To quantify the contribution of different preparation steps to overall shrinkage.
Main Methods:
- Studied isolated mouse hepatocyte nuclei and human erythrocytes.
- Compared shrinkage after glutaraldehyde fixation, ethanol dehydration, and critical point drying.
- Utilized an alternative dehydration technique with ethylene glycol-monomethyl ether.
Main Results:
- Critical point drying caused 25-30% diameter reduction in both cell types.
- Glutaraldehyde fixation and ethanol dehydration resulted in minimal shrinkage prior to critical point drying.
- Alternative dehydration did not alter the final shrinkage.
Conclusions:
- The critical point drying process is the primary cause of observed specimen shrinkage.
- Shrinkage represents a uniform miniaturization, preserving structural relationships within organelles.