Blinking statistics and molecular counting in direct stochastic reconstruction microscopy (dSTORM)

Area of Science:

• Biophysics
• Microscopy
• Computational Biology

Background:

• Single-molecule localization microscopy (SMLM) advances rely on fluorophore photoswitching.
• Stochastic photoswitching complicates precise molecule counting in SMLM.
• Accurate molecule counting is crucial for understanding cellular structures and dynamics.

Purpose of the Study:

• Develop a method to accurately determine the number of molecules in SMLM experiments.
• Address the challenge of precise molecule counting due to fluorophore stochasticity.
• Provide a robust framework for quantitative analysis in SMLM.

Main Methods:

• Modeled fluorophore photoswitching as a continuous-time Markov process.
• Accounted for missed blinks and false positives in localization data.
• Extended the model to arbitrary numbers of molecules in direct stochastic optical reconstruction microscopy (dSTORM).

Main Results:

• Derived the exact probability distribution for localizations from single and multiple photoswitching fluorophores.
• Demonstrated accurate recovery of the unknown number of molecules using posterior mode estimation with training data.
• Quantified adapter protein linker for activation of T cells (LAT) on T-cell immunological synapse surfaces using experimental data.

Conclusions:

• The developed method enables precise molecule counting in SMLM.
• This quantitative approach enhances the biological insights obtainable from super-resolution microscopy.
• The findings have implications for studying molecular interactions in cellular systems.