Time-resolved molecule counting by photon statistics across the visible spectrum.

Area of Science:

• Biophysics
• Spectroscopy
• Analytical Chemistry

Background:

• Quantification of fluorescently labeled biomolecules is increasingly important.
• Counting by photon statistics relies on analyzing photon detection events to determine fluorophore number and brightness.
• Accurate measurement of photon statistics is critical for reliable quantification.

Purpose of the Study:

• To establish a quantitative relationship between fundamental fluorophore properties and experimental conditions.
• To enable quantitative evaluation of experimental settings and analysis parameters for photon counting.
• To enhance the robustness of counting by photon statistics for absolute emitter quantification.

Main Methods:

• Combined simulations and experimental approaches.
• Related fluorophore molecular brightness and photostability to excitation power and acquisition time.
• Evaluated brightness and photostability of 16 organic dyes across the visible spectrum.

Main Results:

• Developed a method to quantitatively evaluate experimental settings for photon counting.
• Demonstrated that counting by photon statistics is a robust method for absolute quantification within diffraction-limited volumes.
• Showed time-resolution of counting varies with fluorophore brightness (10-100 ms) and assessed the range of suitable fluorophores.

Conclusions:

• Optimized experimental and analysis parameters improve the accuracy of counting by photon statistics.
• This method provides a robust approach for absolute quantification of single molecules.
• Facilitates absolute stoichiometry analysis in dynamic multi-component complexes and aids in selecting appropriate fluorophores for single-molecule spectroscopy.