A simple preparation method for CLEM using pre-embedding immunohistochemistry with a novel fluorescent probe and...

Area of Science:

• Cell Biology
• Microscopy Techniques

Background:

• Correlative light and electron microscopy (CLEM) is crucial for visualizing biomolecule localization.
• Existing CLEM methods can face challenges with image resolution and sample stability.

Purpose of the Study:

• To develop a simplified and improved CLEM workflow.
• To enhance the precision of subcellular localization of fluorescently labeled biomolecules.

Main Methods:

• Combined pre-embedding immunohistochemistry with Fluolid NS Orange and Durcupan™ resin.
• Utilized finder grids for landmark navigation and image alignment.
• Employed high-resolution transmission electron microscopy (TEM) for focused imaging.

Main Results:

• Durcupan™ embedding provided superior stability against electron beam irradiation compared to LR White.
• The developed method successfully superimposed fluorescence and TEM images.
• Achieved high-resolution CLEM images, enabling precise subcellular localization of fluorescence signals.

Conclusions:

• The novel CLEM method offers a simple, robust approach for accurate biomolecule localization.
• The combination of Fluolid NS Orange and Durcupan™ overcomes previous resolution limitations.
• This technique facilitates detailed ultrastructural analysis of cellular components.