One step 4× and 12× 3D-ExM enables robust super-resolution microscopy of nanoscale cellular structures.

Area of Science:

• Cell Biology
• Microscopy
• Biophysics

Background:

• Super-resolution microscopy offers nanometer-scale insights into biological systems.
• Traditional methods like electron microscopy have limitations in sample prep and multiplexing.
• Expansion Microscopy (ExM) provides a simpler approach to high-resolution imaging.

Purpose of the Study:

• Introduce optimized 4-fold and 12-fold 3D-isotropic and preserved Expansion Microscopy (3D-ExM) methods.
• Demonstrate the utility of 3D-ExM for nanoscale visualization in cell biology.
• Provide a cost-effective and accessible super-resolution imaging solution.

Main Methods:

• Developed and optimized 4× and 12× 3D-ExM protocols.
• Utilized standard confocal microscopy for imaging expanded samples.
• Applied methods to 2D and 3D cell culture models.

Main Results:

• Achieved robust and reproducible 3D isotropic expansion.
• 12× 3D-ExM with confocal microscopy yielded <30 nm lateral resolution.
• Successfully visualized nanoscale structures like chromosomes, kinetochores, and viral particles.

Conclusions:

• 3D-ExM offers a user-friendly and cost-effective super-resolution microscopy technique.
• The optimized methods are suitable for diverse cell biology research.
• Enables detailed studies of cellular and chromatin architectures at the nanoscale.