splitSMLM, a spectral demixing method for high-precision multi-color localization microscopy applied to nuclear pore...

Area of Science:

• Biophysics
• Microscopy techniques
• Molecular imaging

Background:

• Single molecule localization microscopy (SMLM) enables multicolor imaging for molecular colocalization.
• Existing methods using dichroic image splitters face limitations in localization precision and chromatic error correction.
• Accurate multicolor SMLM is crucial for understanding complex biological structures.

Purpose of the Study:

• To develop an improved multicolor SMLM method (splitSMLM) with enhanced localization precision and drift correction.
• To compensate for chromatic distortions and optimize fluorophore performance for simultaneous imaging.
• To validate the method's efficacy in multicolor imaging of the nuclear pore complex (NPC).

Main Methods:

• Development of splitSMLM, an image splitter-based multicolor SMLM technique.
• Introduction of SplitViSu, a novel spectral demixing algorithm for precise localization and chromatic error correction.
• Optimization of fluorophore and filter combinations for enhanced multicolor performance.

Main Results:

• splitSMLM achieved significantly improved localization precision and drift correction.
• The SplitViSu algorithm corrected chromatic errors at the nanometer scale with minimal data loss.
• Three-color imaging of the NPC revealed refined positioning of proteins and identified Pom121 clusters as deposition loci.

Conclusions:

• The splitSMLM method offers superior performance for multicolor SMLM, overcoming limitations of classical approaches.
• SplitViSu algorithm provides accurate chromatic aberration correction and preserves localization precision.
• The method's application to NPC imaging demonstrates its strength and broad applicability in cell biology.