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Conventional and high resolution scanning electron microscopy of biological sectioned material
1Institute of Clinical Electron Microscopy, S. Orsola Hospital, University of Bologna, Italy.
Scanning Microscopy
|March 1, 1991
Summary
Researchers developed a new method using scanning electron microscopy (SEM) to visualize intracellular structures in biological tissues. This technique enhances 3D imaging of organelles and cell membranes, improving upon traditional microscopy methods.
Area of Science:
- Cell Biology
- Microscopy Techniques
- Histology
Background:
- Conventional microscopy methods have limitations in visualizing the intricate 3D arrangement of intracellular organelles.
- Standard fixation techniques can obscure fine cellular details, hindering accurate observation.
- Previous light microscopy (LM) observations of embedded biological tissues could be further enhanced.
Purpose of the Study:
- To develop and validate advanced scanning electron microscopy (SEM) techniques for observing intracellular structures.
- To improve the 3D visualization of organelles and macromolecular cell membrane structures.
- To overcome limitations of conventional fixation and embedding methods in electron microscopy.
Main Methods:
- Utilized conventional-scanning electron microscopy (C-SEM) and high-resolution scanning electron microscopy (HR-SEM) on glass knife-sectioned biological tissues (rat kidney, myocardium, small intestine).
- Employed osmium maceration to remove excess cytoplasmic matrix, followed by resin removal for 3D C-SEM observation.
- Used an acetone-soluble acrylic resin for embedding to facilitate 3D imaging of intracellular organelles.
Main Results:
- C-SEM provided high-quality 2D secondary electron images (SEI) that integrated and extended LM observations.
- The 3D arrangement of intracellular organelles was successfully visualized using the novel embedding and de-embedding approach.
- De-embedded, osmium-treated sections allowed for smooth chromium film deposition, enabling HR-SEM studies of cell membrane structures.
Conclusions:
- The developed SEM techniques, including osmium maceration and specialized resin embedding, significantly enhance the visualization of intracellular structures.
- This method offers superior 3D morphological insights into organelles and cell membranes compared to conventional approaches.
- The technique provides a valuable tool for detailed ultrastructural analysis in cell biology and histology.