Live-cell CLEM of subcellular targets: an optimized procedure for polymer-based imaging substrates

Area of Science:

• Cell Biology
• Microscopy Techniques

Background:

• Live-cell microscopy allows visualization of dynamic biological processes.
• Correlative light and electron microscopy (CLEM) bridges optical and ultrastructural imaging.
• 3D relocalization of cellular compartments after electron microscopy (EM) preparation is challenging.

Purpose of the Study:

• To develop a detailed protocol for live-cell CLEM with straightforward 3D relocalization.
• To enable correlation of live-cell imaging with high-resolution ultrastructural analysis.

Main Methods:

• Utilized TOPAS polymer film as an imaging substrate.
• Employed toner particles for 2D tracking and fiducial markers for 3D relocation.
• Demonstrated the method by laser microirradiating a mitochondrion and analyzing its ultrastructure via transmission electron microscopy (TEM).

Main Results:

• Successfully established a protocol for live-cell CLEM facilitating 3D relocalization.
• Demonstrated the ability to observe ultrastructural changes in targeted organelles, such as mitochondria, after laser treatment.
• The method is compatible with standard inverted optical microscopes.

Conclusions:

• The developed live-cell CLEM protocol simplifies 3D relocalization of intracellular structures.
• This technique allows for the correlation of dynamic live-cell events with detailed ultrastructural changes.
• The method provides a valuable tool for studying cellular responses at the ultrastructural level.