Counting single photoactivatable fluorescent molecules by photoactivated localization microscopy (PALM)

Area of Science:

• Biophysics
• Microscopy
• Molecular Biology

Background:

• Accurate protein quantification is crucial in molecular biology.
• Photoactivated localization microscopy (PALM) enables single-molecule resolution.
• Photoactivatable fluorescent proteins (e.g., mEos2, Dendra2) are used in PALM but suffer from blinking, causing overcounting errors.

Purpose of the Study:

• To develop a robust single-molecule counting method for photoactivated localization microscopy (PALM).
• To address overcounting and undercounting errors inherent in using photoactivatable fluorescent proteins.
• To compare the suitability of Dendra2 and mEos2 as molecular counting tags in PALM.

Main Methods:

• Developed a kinetic model to characterize protein blinking behavior.
• Implemented a photoactivation scheme to minimize simultaneous molecule activation.
• Quantified and minimized total counting error by balancing over- and undercounting.

Main Results:

• Dendra2 exhibits significantly less blinking and comparable photobleaching to mEos2, making it a better PALM counting tag.
• The optimized method achieved in vitro counting of up to 200 molecules in a diffraction-limited spot with <2% bias and <6% uncertainty within 10 minutes.
• Successfully applied the counting method for in vivo protein quantification of the bacterial flagellar motor protein FliM fused to Dendra2.

Conclusions:

• Dendra2 is a more effective photoactivatable fluorescent protein for molecular counting applications in PALM compared to mEos2.
• The developed kinetic model and photoactivation strategy significantly improve counting accuracy.
• This method provides a reliable approach for precise protein quantification in both in vitro and in vivo biological systems.