Fluorescence axial localization with nanometer accuracy and precision.

Area of Science:

• Nanotechnology and Biophysics
• Single-molecule imaging and spectroscopy

Background:

• Accurate measurement of nanoscale object location and orientation is crucial for understanding molecular interactions and functions.
• Existing techniques often lack the required precision for sub-nanometer axial localization.
• The orientation of biomolecules like DNA provides insights into their behavior and interactions with surfaces.

Purpose of the Study:

• To introduce and validate a novel technique, standing wave axial nanometry (SWAN), for high-accuracy axial imaging of nanoscale objects.
• To demonstrate the capability of SWAN in precisely measuring the orientation of single DNA molecules.
• To explore the influence of DNA length and surface functionality on molecular orientation using SWAN.

Main Methods:

• Standing wave axial nanometry (SWAN) utilizes a standing wave generated by an atomic force microscope tip and a focused laser beam.
• Fluorescence excitation is achieved through the standing wave.
• Axial position is determined by analyzing the phase of the emitted fluorescence intensity.

Main Results:

• SWAN achieves sub-nanometer accuracy and 3.7 nm precision in imaging the axial location of single nanoscale fluorescent objects.
• The technique was successfully applied to measure the orientation of individual DNA molecules.
• Measurements were performed on DNA molecules of varying lengths and grafted onto surfaces with different functionalities.

Conclusions:

• Standing wave axial nanometry (SWAN) is a powerful new tool for high-precision axial localization of nanoscale objects.
• SWAN enables detailed studies of single biomolecule orientation and surface interactions.
• This technique has significant potential for advancing nanoscale metrology and molecular biophysics.