Preservation of Fluorescence Signal and Imaging Optimization for Integrated Light and Electron Microscopy

Area of Science:

• Cell biology
• Microscopy
• Biochemistry

Background:

• Accurate molecular localization within cells and tissues is crucial for life science research.
• Fluorescent microscopy offers high-resolution localization but lacks ultrastructural context.
• Correlative light and electron microscopy (CLEM) bridges this gap by combining both imaging techniques.

Purpose of the Study:

• To present an optimized protocol for correlative light and electron microscopy (CLEM).
• To enable simultaneous visualization of fluorescent signals and ultrastructure in resin-embedded samples.
• To facilitate rapid acquisition of CLEM data for biological research.

Main Methods:

• Adopted a protocol preserving genetically-encoded and antibody-derived fluorescent signals in resin-embedded samples.
• Performed high-resolution electron microscopy imaging on the same thin sections.
• Optimized scanning electron microscopy (SEM) imaging parameters for varying sample thicknesses.

Main Results:

• Successfully preserved both fluorescent signals and ultrastructural details in the same samples.
• Enabled high-resolution imaging of fluorescently labeled molecules within their ultrastructural context.
• Demonstrated suitability for dedicated CLEM instruments and adaptability for 3D EM.

Conclusions:

• The developed protocol enables efficient CLEM analysis by preserving fluorescent signals.
• This method provides crucial ultrastructural context for molecular localization studies.
• The protocol is adaptable for advanced 3D electron microscopy applications.