Polarization effect on position accuracy of fluorophore localization

Area of Science:

• Biophysics
• Optical microscopy
• Nanotechnology

Background:

• Single-molecule localization microscopy techniques, like FIONA, are crucial for biophysical studies.
• Accurate position determination relies on fitting emission intensity to a 2D Gaussian model.
• Molecular orientation influences fluorescence emission intensity, potentially affecting localization accuracy.

Purpose of the Study:

• To investigate the impact of molecular orientation on the accuracy of single-molecule position determination using FIONA.
• To evaluate the performance of objectives with varying numerical apertures for FIONA applications.
• To establish guidelines for achieving optimal positional accuracy in single-molecule imaging.

Main Methods:

• Extensive numerical simulations were performed to analyze position determination accuracy.
• The study systematically varied molecular orientation and objective numerical apertures.
• Effective pixel size and anisotropy effects were quantified.

Main Results:

• Positional accuracy is highly dependent on molecular orientation and objective numerical aperture.
• An effective pixel size of 100 nm or less per CCD pixel is recommended for good accuracy.
• High numerical aperture objectives can lead to position errors up to 10 nm due to orientation effects.
• Using objectives with a numerical aperture of 1.2 significantly improves accuracy to < 2.5 nm.

Conclusions:

• Molecular orientation is a critical factor affecting FIONA accuracy.
• Optimizing objective choice and pixel size is essential for precise single-molecule localization.
• Lowering probe anisotropy further reduces positional uncertainty, enhancing measurement reliability.