Ultrasensitive Three-Dimensional Orientation Imaging of Single Molecules on Plasmonic Nanohole Arrays Using Second...

Area of Science:

• Nonlinear optics
• Plasmonics
• Super-resolution microscopy

Background:

• Existing super-resolution techniques like STED and STORM are unsuitable for label-free nonlinear microscopy (SHG) due to SHG's properties.
• Single-molecule SHG imaging is challenging due to weak scattering cross-sections and limitations of fluorescence-based localization methods.
• Plasmonic nanostructures offer potential for enhancing SHG signals by concentrating electromagnetic fields.

Purpose of the Study:

• To develop a super-resolution technique for label-free nonlinear microscopy based on second harmonic generation (SHG).
• To achieve simultaneous visualization and 3D orientation imaging of individual molecules using SHG.
• To overcome the limitations of existing methods for single-molecule SHG detection and analysis.

Main Methods:

• Utilized plasmonic silver nanohole arrays to enhance SHG signals.
• Employed a template matching algorithm for determining molecular position and 3D orientation.
• Combined SHG and two-photon fluorescence microscopy with finite-difference time-domain (FDTD) simulations.

Main Results:

• Achieved a nonlinear enhancement factor of ~10^6 at plasmonic hotspots, enabling single-molecule SHG detection.
• Successfully visualized and determined the 3D orientation of individual rhodamine 6G (R6G) molecules.
• Demonstrated the feasibility of SHG-based single-molecule detection and orientation imaging.

Conclusions:

• Developed a novel SHG-based super-resolution microscopy technique for label-free imaging.
• Enabled precise 3D orientation determination of single molecules using plasmon-enhanced SHG.
• This breakthrough has potential applications in nanophotonics, materials science, and super-resolution imaging of biological systems.