ExCel: Super-Resolution Imaging of C. elegans with Expansion Microscopy.

Area of Science:

• Cell Biology
• Microscopy
• Developmental Biology

Background:

• Super-resolution imaging in C. elegans is crucial for understanding biological processes.
• Conventional methods like STORM, PALM, SR-SIM, and STED have limitations in imaging depth and accessibility for C. elegans studies.
• There is a need for advanced imaging techniques that can resolve structures throughout the entire C. elegans organism.

Purpose of the Study:

• To present a versatile toolbox of Expansion Microscopy (ExCel) protocols for super-resolution imaging in C. elegans.
• To enable high-resolution imaging of proteins, RNA, DNA, and general anatomy within the entire C. elegans.
• To provide accessible super-resolution techniques for C. elegans research using conventional confocal microscopes.

Main Methods:

• Development and detailed description of three Expansion of C. elegans (ExCel) protocols: standard, epitope-preserving, and iterative.
• Physical magnification of fixed whole C. elegans animals with high isotropy.
• Utilizing conventional confocal microscopes for imaging after sample expansion.

Main Results:

• The standard ExCel protocol achieves ~70 nm resolution (~3.5× linear expansion) for simultaneous imaging of diverse molecules.
• The epitope-preserving ExCel protocol allows imaging of endogenous proteins using antibodies at ~100 nm resolution (~2.8× linear expansion).
• The iterative ExCel protocol provides ~25 nm resolution (~20× linear expansion) for fluorescent protein imaging.

Conclusions:

• ExCel offers a powerful and accessible method for super-resolution imaging across the entire C. elegans.
• The ExCel toolbox provides researchers with versatile options for high-resolution visualization of various biological targets.
• These protocols significantly advance the capability to study C. elegans at an unprecedented level of detail.