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Correlative Light and Electron Microscopy (CLEM) as a Tool to Visualize Microinjected Molecules and their Eukaryotic Sub-cellular Targets
Published on: May 4, 2012
Correlative light and electron microscopy of GFP
1Department of Systems Cell Biology, Max-Planck-Institute for Molecular Physiology, Otto-Hahn-Str. 11, D-44227 Dortmund, North Rhine-Westphalia, Germany.
Methods in Cell Biology
|August 4, 2012
Summary
Correlative microscopy links live-cell imaging with electron microscopy. This method uses green fluorescent proteins (GFPs) and photo-oxidation to visualize specific molecules ultrastructurally.
Area of Science:
- Biophysics
- Cell Biology
- Microscopy
Background:
- Correlative light and electron microscopy (CLEM) integrates live-cell dynamics with high-resolution ultrastructural imaging.
- Green fluorescent proteins (GFPs) and derivatives have transformed live-cell imaging.
- Existing CLEM methods have limitations in correlating specific molecular locations.
Purpose of the Study:
- To review the method of correlative microscopy using green fluorescent proteins (GFPs) and photo-oxidation.
- To highlight the ability to achieve direct ultrastructural visualization of fluorophores.
- To demonstrate the highest level of molecular correlation in CLEM.
Main Methods:
- Utilizes photo-oxidation of diaminobenzidine (DAB) triggered by green fluorescent protein (GFP) illumination.
- Generates oxygen radicals during GFP bleaching to oxidize DAB.
- Forms an electron-dense DAB precipitate for electron microscopy (EM) visualization.
Main Results:
- Achieves direct ultrastructural localization of GFPs within cells.
- Enables visualization of DAB precipitate by routine EM and electron tomography.
- Facilitates 3D ultrastructural analysis of specific molecular targets.
Conclusions:
- Photo-oxidation of DAB via GFPs offers unparalleled molecular correlation in CLEM.
- This technique allows for precise ultrastructural mapping of specific molecules.
- It advances the capability to study dynamic biological processes at the molecular level.
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