3D Single Molecule Super-Resolution Microscopy of Whole Nuclear Lamina

Area of Science:

• Cell Biology
• Microscopy
• Biophysics

Background:

• Single molecule (SM) super-resolution microscopy achieves nanoscale resolution, surpassing conventional optical limits.
• Optical astigmatism enables 3D imaging with a significant z-range, but requires bright, photoswitching-resilient fluorophores.
• Imaging entire cell volumes in 3D using SM super-resolution presents challenges due to fluorophore limitations.

Purpose of the Study:

• To visualize the complete nuclear lamina in 3D using single molecule super-resolution microscopy.
• To assess the feasibility of imaging deep cellular structures with optical astigmatism and multiplane acquisition.
• To evaluate the performance of Alexa Fluor 647 for extended imaging of nuclear lamina morphology.

Main Methods:

• Employed single molecule (SM) astigmatism combined with multiplane acquisition for 3D imaging.
• Utilized COS-7 and T cells for nuclear lamina visualization.
• Assessed the brightness and photoswitching resilience of Alexa Fluor 647 under continuous high laser power.

Main Results:

• Successfully visualized the entire nuclear lamina of COS-7 and T cells in 3D.
• Alexa Fluor 647 demonstrated viability for over an hour of high laser power exposure.
• Fluorophore brightness allowed detection up to 8 µm deep within the cell.
• Achieved sufficient super-resolution detail for quantifying nuclear dimensions and local membrane features.

Conclusions:

• Single molecule astigmatism with multiplane acquisition enables comprehensive 3D imaging of the nuclear lamina.
• Alexa Fluor 647 is suitable for prolonged, high-power SM super-resolution imaging of deep cellular structures.
• This method provides detailed morphological insights into the nuclear lamina for quantitative analysis.