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Published on: August 7, 2016
Correlative Light and Electron Microscopy Using Frozen Section Obtained Using Cryo-Ultramicrotomy
Hong-Lim Kim1, Tae-Ryong Riew2, Jieun Park1
1Integrative Research Support Center, College of Medicine, The Catholic University of Korea, Seoul 06591, Korea.
Abstract:
Immuno-electron microscopy (Immuno-EM) is a powerful tool for identifying molecular targets with ultrastructural details in biological specimens. However, technical barriers, such as the loss of ultrastructural integrity, the decrease in antigenicity, or artifacts in the handling process, hinder the widespread use of the technique by biomedical researchers. We developed a method to overcome such challenges by combining light and electron microscopy with immunolabeling based on Tokuyasu's method. Using cryo-sectioned biological specimens, target proteins with excellent antigenicity were first immunolabeled for confocal analysis, and then the same tissue sections were further processed for electron microscopy, which provided a well-preserved ultrastructure comparable to that obtained using conventional electron microscopy. Moreover, this method does not require specifically designed correlative light and electron microscopy (CLEM) devices but rather employs conventional confocal and electron microscopes; therefore, it can be easily applied in many biomedical studies.
Insights
We developed an improved immuno-electron microscopy (Immuno-EM) method for precise molecular target identification. This technique preserves ultrastructure and antigenicity, making it accessible for biomedical research.
Area of Science:
- Cell Biology
- Microscopy Techniques
- Molecular Biology
Background:
- Immuno-electron microscopy (Immuno-EM) is vital for ultrastructural molecular target identification.
- Technical challenges like loss of integrity and antigenicity limit its broad application.
- Existing methods often require specialized equipment, hindering accessibility.
Purpose of the Study:
- To develop an accessible Immuno-EM method overcoming technical barriers.
- To combine light and electron microscopy for enhanced molecular localization.
- To preserve ultrastructural integrity and antigenicity in biological specimens.
Main Methods:
- Utilized Tokuyasu's cryo-sectioning and immunolabeling method.
- Integrated confocal microscopy for initial antigen detection.
- Applied subsequent processing for high-resolution electron microscopy on the same sections.
Main Results:
- Achieved well-preserved ultrastructure comparable to conventional EM.
- Maintained excellent antigenicity for target protein detection.
- Demonstrated successful application using standard confocal and electron microscopes.
Conclusions:
- The developed method enhances Immuno-EM accessibility for biomedical researchers.
- It overcomes key technical limitations, improving ultrastructural detail and antigenicity.
- This technique facilitates correlative light and electron microscopy without specialized devices.
Related Concept Videos
01:28Cryo-electron Microscopy
01:20Preparation of Samples for Electron Microscopy