Improved localization precision via restricting confined biomolecule stochastic motion in single-molecule...

Area of Science:

• Biophysics
• Microscopy
• Molecular Biology

Background:

• Single-molecule localization microscopy (SMLM) is crucial for resolving nanometer-sized biomolecules beyond the diffraction limit.
• Immobilized biomolecules in SMLM are expected to exhibit confined stochastic motion, but its impact on localization precision is poorly understood.

Purpose of the Study:

• To investigate the detectability and influence of confined stochastic motion on localization precision in SMLM.
• To explore methods for improving localization precision by managing biomolecule motion.

Main Methods:

• Analyzing localization displacements of immobilized Alexa Fluor-647-conjugated oligonucleotides at varying frame intervals.
• Calculating the relationship between biomolecule motion and localization precision.
• Evaluating the effect of biomolecule size and post-crosslinking on precision using cell samples stained for tubulin.

Main Results:

• Biomolecule stochastic motion was detected and found to be confined, with displacements increasing with time intervals before saturating.
• Localization precision was found to be inversely proportional to the degree of confined stochastic motion.
• Enlarging biomolecules decreased precision, while post-crosslinking improved it, as demonstrated in tubulin imaging.

Conclusions:

• Confined stochastic motion of immobilized biomolecules negatively impacts SMLM localization precision.
• Strategies to restrict this motion, such as post-crosslinking, can enhance imaging accuracy in SMLM.