Direct-laser writing for subnanometer focusing and single-molecule imaging

Area of Science:

• Nanofabrication
• Optical Microscopy
• Biophysics

Background:

• Two-photon direct laser writing (TPDLW) is a 3D additive fabrication technique.
• TPDLW enables the creation of complex structures with nanometer precision.
• High-resolution optical microscopy requires precise calibration and tracking tools.

Purpose of the Study:

• To fabricate novel 3D optical fiducials using TPDLW.
• To enhance axial discrimination and achieve sub-nanometer 3D focusing in optical microscopy.
• To enable accurate live-cell single-particle tracking and 3D single-molecule localization microscopy.

Main Methods:

• Utilized two-photon absorption of femtosecond laser pulses for polymerization.
• Fabricated unique 3D optical fiducials with controlled position and architecture.
• Employed a standard inverted microscope for imaging and tracking.

Main Results:

• Achieved isotropic sub-nanometer 3D focusing (<0.8 nm) over tens of micrometers.
• Demonstrated improved axial discrimination using the fabricated fiducials.
• Successfully performed 3D single-molecule acquisitions and live-cell tracking with nanometer accuracy.

Conclusions:

• TPDLW is a powerful tool for creating custom 3D optical fiducials.
• These fiducials significantly enhance the precision of 3D single-molecule localization microscopy.
• The developed method allows for high-accuracy live-cell imaging and tracking over cellular volumes.