Combining pMINFLUX, graphene energy transfer and DNA-PAINT for nanometer precise 3D super-resolution microscopy.

Area of Science:

• Microscopy and Imaging Technologies
• Nanotechnology
• Biophysics

Background:

• Super-resolution microscopy is crucial for complementing ultrastructural techniques in fluorescence imaging.
• Achieving high-resolution 3D imaging with precise localization remains a significant challenge.

Purpose of the Study:

• To develop a 3D super-resolution microscopy technique with nanometric resolution.
• To combine pMINFLUX, graphene energy transfer (GET), and DNA-PAINT for enhanced 3D localization.
• To demonstrate sub-nanometer axial precision and resolve nanoscale structural features.

Main Methods:

• Integration of 2D localization from pMINFLUX with axial information from graphene energy transfer (GET).
• Utilizing single-molecule switching via DNA-PAINT for localization precision.
• Development of local PAINT (L-PAINT) for improved signal-to-background and imaging speed.

Main Results:

• Demonstrated <2 nm localization precision in 3D, with axial precision below 0.3 nm.
• Directly resolved structural features like individual docking strands at 3 nm distances using 3D DNA-PAINT.
• Showcased L-PAINT by imaging a 6 nm triangular structure within seconds.

Conclusions:

• The combination of pMINFLUX and GET offers synergistic advantages for super-resolution imaging, particularly near surfaces.
• The developed technique significantly advances the capability to visualize nanoscale biological structures.
• L-PAINT enhances imaging speed and signal-to-background for localized clusters.