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A preparation method for observing intracellular structures by scanning electron microscopy.

K Tanaka, A Mitsushima

    Journal of Microscopy
    |February 1, 1984
    PubMed
    Summary

    A revised Osmium-DMSO-Osmium method uses aldehyde fixation for improved cell imaging via scanning electron microscopy (SEM). This technique enhances fine structure preservation and enables cytochemical studies, such as visualizing lysosomal acid phosphatase.

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    Area of Science:

    • Cell Biology
    • Microscopy Techniques
    • Biochemistry

    Background:

    • Scanning electron microscopy (SEM) requires removal of cytoplasmic matrix for observing intracellular structures.
    • The original Osmium-DMSO-Osmium method effectively reveals intracellular details but uses osmium tetroxide, posing disadvantages.
    • Improved methods are needed for better preservation and broader application in cell biology.

    Purpose of the Study:

    • To revise the Osmium-DMSO-Osmium method for enhanced intracellular structure visualization in SEM.
    • To introduce aldehyde fixation as an alternative to osmium tetroxide for initial cell fixation.
    • To demonstrate the utility of the revised method for cytochemical studies using SEM.

    Main Methods:

    • A revised Osmium-DMSO-Osmium protocol was developed using an aldehyde mixture for initial fixation.

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  • Perfusion fixation was employed to achieve better preservation of fine cellular structures.
  • The method was applied to demonstrate acid phosphatase activity in lysosomes.

    • Main Results:

    • The revised method successfully removed excess cytoplasmic matrix, revealing intracellular structures.
    • Aldehyde fixation provided superior preservation of fine structures, particularly in central nervous tissue.
    • Lysosomal acid phosphatase was successfully demonstrated in colored SEM micrographs, validating cytochemical application.

    Conclusions:

    • The revised Osmium-DMSO-Osmium method with aldehyde fixation offers an improved approach for SEM imaging of intracellular structures.
    • This technique enhances structural preservation and is suitable for cytochemical analysis, expanding SEM's utility.
    • The method overcomes limitations of the original protocol, offering advantages for biological research.