Impact of Molecule Concentration, Diffusion Rates and Surface Passivation on Single-Molecule Fluorescence Studies in...

Area of Science:

• Biophysics
• Analytical Chemistry
• Spectroscopy

Background:

• Single-molecule studies demand extremely low molecule concentrations (picomolar range) for sensitivity.
• Accurate and stable molecule concentrations are crucial for applications like single-molecule Förster resonance energy transfer (smFRET) and two-color coincidence detection (TCCD).
• Surface passivation of cover slides is critical for maintaining concentration stability during measurements.

Purpose of the Study:

• To discuss and present approaches for controlling and measuring absolute molecule concentrations in solution.
• To introduce a method for calculating the probability of chance coincidence events.
• To emphasize the necessity of strict concentration limits for meaningful smFRET measurements.

Main Methods:

• Development of techniques to control and measure absolute molecule concentrations.
• Introduction of a novel approach for calculating chance coincidence probabilities.
• Experimental validation of concentration control and measurement methods.

Main Results:

• Demonstrated methods for precise control and measurement of picomolar molecule concentrations.
• Validated an approach to quantify the likelihood of random molecular coincidences.
• Established that stringent maximal concentration limits are essential for reliable smFRET data.

Conclusions:

• Accurate control and measurement of molecule concentration are paramount for successful single-molecule experiments.
• Understanding and mitigating chance coincidence events are critical for data interpretation.
• The presented methods facilitate more robust and reproducible smFRET and TCCD studies.