Quasi-equilibrium state based quantification of biological macromolecules in single-molecule localization microscopy

Area of Science:

• Cell Biology
• Biophysics
• Microscopy

Background:

• Determining molecular stoichiometry within biological complexes is crucial for understanding cellular function.
• Existing in vitro methods lack precision for in vivo stoichiometry, especially at the single-molecule level needed for heterogeneity analysis.
• Optical microscopy offers limited precision for in vivo stoichiometry, particularly with high fluorophore densities.

Purpose of the Study:

• To develop a precise method for quantifying in vivo molecular stoichiometry using single-molecule localization microscopy (SMLM).
• To overcome limitations of existing techniques in measuring stoichiometry heterogeneity within individual cellular complexes.
• To adapt the method for diverse biological applications, including nuclear pore complexes and chromatin nanodomains.

Main Methods:

• Utilized established single-molecule localization microscopy (SMLM) procedures.
• Focused on quantifying fluorophore detections during the quasi-equilibrium state of the SMLM process.
• Applied the method to analyze stoichiometry in nuclear pore complexes and chromatin nanodomains within cells.

Main Results:

• Achieved a 50-fold improvement in precision for stoichiometry quantification compared to traditional imaging methods at high fluorophore densities.
• Demonstrated accurate stoichiometry estimation in nuclear pore complexes.
• Successfully adapted the method to quantify DNA content in chromatin nanodomains within individual chromosomes.

Conclusions:

• The developed SMLM-based approach enables precise in vivo quantification of molecular stoichiometry in individual complexes.
• This straightforward method significantly enhances measurement accuracy, regardless of fluorophore density.
• The technique is versatile and applicable to various cellular structures, potentially becoming a routine tool for SMLM-based stoichiometry analysis.