A workflow for 3D-CLEM investigating liver tissue.

Area of Science:

• Cell Biology
• Microscopy Techniques
• Biophysics

Background:

• Correlative Light and Electron Microscopy (CLEM) integrates high-resolution ultrastructural data from electron microscopy (EM) with specific fluorescent signals from light microscopy (LM).
• Advancements in volume EM, like SBF-SEM, enable efficient 3D reconstruction, reducing the resolution gap between LM and EM.
• Complex CLEM workflows necessitate clear protocols and sharing of best practices for successful application.

Purpose of the Study:

• To present a detailed workflow for 3D CLEM on mouse liver tissue.
• To demonstrate the combination of high-resolution confocal microscopy with SBF-SEM for accurate 3D correlation.
• To provide a guide for applying 3D CLEM in dense cellular tissues.

Main Methods:

• Utilized a 3D CLEM workflow combining confocal microscopy and SBF-SEM on mouse liver samples.
• Employed near-infrared laser branding marks as fiducial landmarks for navigating between LM and EM.
• Incorporated intrinsic tissue landmarks for precise overlaying of LM and EM datasets.

Main Results:

• Achieved accurate 3D CLEM overlays of liver tissue, successfully correlating fluorescent signals with ultrastructural details.
• Demonstrated the utility of fiducial markers for efficient 3D navigation and correlation.
• Generated a comprehensive dataset combining fluorescent information with high-resolution ultrastructure.

Conclusions:

• The presented workflow enables precise 3D correlation of fluorescent signals with ultrastructural details in dense cellular tissues.
• This method enhances the understanding of cellular organization by bridging the resolution gap between LM and EM.
• The workflow serves as a valuable guide for researchers conducting 3D correlative microscopy studies.