Imaging nanoscale nuclear structures with expansion microscopy.

Area of Science:

• Cell Biology
• Microscopy
• Molecular Biology

Background:

• Super-resolution light microscopy offers nanoscale insights but faces depth, speed, and cost challenges.
• Expansion microscopy (ExM) addresses these limitations but has been hindered by concerns of uneven nuclear expansion.
• Investigating nuclear processes at the nanoscale is crucial for understanding cellular functions.

Purpose of the Study:

• To demonstrate conditions for isotropic nuclear expansion using ExM.
• To achieve nanoscale resolution (120-130 nm pre-expansion) for nuclear imaging.
• To quantitatively analyze 3D nanoscale organization of DNA damage response (DDR) structures.

Main Methods:

• Optimized Expansion Microscopy (ExM) protocols for achieving isotropic nuclear expansion.
• Utilized super-resolution imaging techniques to capture nanoscale details.
• Applied quantitative analysis to large datasets of DDR structures.

Main Results:

• Established conditions for uniform, isotropic expansion of the cell nucleus.
• Achieved nanoscale resolution, enabling detailed visualization of nuclear architecture.
• Quantitatively characterized the 3D nanoscale organization of over 50,000 DNA damage response structures, including BRCA1, 53BP1, and RAD51.
• Demonstrated simultaneous assessment of four distinct nuclear elements and chromatin-regulated events.

Conclusions:

• Expansion microscopy (ExM) can be reliably applied to achieve high-resolution, 3D nanoscale imaging of nuclear processes.
• ExM overcomes significant limitations of traditional super-resolution microscopy for nuclear studies.
• This approach facilitates the investigation of complex nanoscale nuclear organization and events, such as DNA damage response.