Dual-color single-molecule localization microscopy with a sub-nanometer channel misalignment and sub-1% color...

Area of Science:

• Biophysics
• Microscopy
• Molecular Biology

Background:

• Multicolor single-molecule localization microscopy (SMLM) offers nanoscale resolution crucial for studying subcellular interactions.
• Accurate colocalization analysis of nanometer-sized proteins requires sub-nanometer precision, which is hindered by existing nanometer-scale channel registration errors in SMLM.
• Current super-resolution techniques face limitations in achieving sub-nanometer precision due to residual channel misalignment.

Purpose of the Study:

• To develop a dual-color SMLM method that overcomes nanometer-scale channel registration errors.
• To achieve sub-nanometer precision in colocalization analysis for biomolecular interactions.
• To enhance the reliability of SMLM for studying protein interactions at the subcellular level.

Main Methods:

• Proposed a dual-color single-molecule localization imaging method utilizing periodic trigger synchronization.
• Employed an optical chopper for alternating CW laser illumination of samples at millisecond intervals with synchronous signal collection.
• Validated the method using fluorescent microsphere model experiments and dual-channel imaging of single-color labeled samples.

Main Results:

• Reduced channel misalignment to sub-nanometer levels (<1 nm).
• Increased the Spearman correlation coefficient by 18%-24% and the Manders coefficient by 4%-7% compared to sequential imaging.
• Maintained a color crosstalk rate below 1%.

Conclusions:

• The proposed method achieves sub-nanometer channel registration accuracy, significantly improving upon existing nanometer-level accuracy.
• This advancement offers a robust approach to enhance the reliability of sub-nanometer colocalization analysis in multicolor SMLM.
• The technique is expected to advance the study of biomolecular interactions requiring high spatial precision.