On-section correlative light and electron microscopy of large cellular volumes using STEM tomography

Area of Science:

• Cell Biology
• Microscopy
• Biophysics

Background:

• Correlative light and electron microscopy (CLEM) combines fluorescence and electron microscopy for advanced cellular imaging.
• Conventional transmission electron microscopy (TEM) tomography is limited to imaging thin sections (~300nm).
• Scanning transmission electron microscopy (STEM) tomography offers enhanced depth penetration for 3D imaging.

Purpose of the Study:

• To integrate STEM tomography into CLEM for enhanced 3D ultrastructural analysis.
• To extend the capabilities of the Kukulski protocol for CLEM.
• To localize fluorescently tagged proteins within cellular ultrastructure in 3D.

Main Methods:

• Human retinal pigment epithelial 1 (RPE1) cells expressing fluorescent fusion proteins were high-pressure frozen and embedded.
• Fluorescently labeled gold nanoparticles were used as fiducials on resin sections for correlative imaging.
• STEM tomography was performed on thicker sections (up to 900nm), and data were overlaid with fluorescence microscopy images using eC-CLEM software.

Main Results:

• Successful integration of STEM tomography into CLEM workflow.
• Demonstrated localization of fluorescently tagged proteins to specific cellular organelles in 3D.
• Achieved 3D ultrastructural localization within volumes of at least 2.7×2.7×0.5μm.

Conclusions:

• The integration of STEM tomography significantly extends the application of CLEM, particularly the Kukulski protocol.
• This advanced CLEM approach allows for precise 3D mapping of protein localization within complex cellular ultrastructures.
• The method provides unprecedented insights into the spatial organization of proteins within cells at high resolution.