Analysis of photon trajectories from diffusing single molecules.

Area of Science:

• Biophysics
• Physical Chemistry
• Analytical Chemistry

Background:

• Single-molecule experiments detect photons emitted as molecules pass through a laser spot.
• Only photon bursts provide meaningful data, necessitating careful selection criteria.
• Existing analysis methods may not fully account for burst selection biases.

Purpose of the Study:

• To develop new analytical methods for accurately determining molecular brightness and diffusivity from selected photon bursts.
• To account for biases introduced by burst selection criteria in data analysis.
• To provide robust methods for analyzing photon arrival time data in single-molecule diffusion studies.

Main Methods:

• Derivation of analytical expressions for photon burst distributions (inter-photon times, photon counts).
• Development of Maximum Likelihood (ML) methods for data analysis: burstML, iptML, and pcML.
• Validation using simulated photon trajectories and experimental data (Atto 488 fluorophore).

Main Results:

• New theoretical framework accurately models photon burst statistics, including selection biases.
• Demonstrated accurate determination of photon count rate and diffusion coefficient using ML methods.
• Successful application to both simulated and experimental single-molecule diffusion data.

Conclusions:

• The presented methods provide accurate and bias-corrected analysis of single-molecule diffusion data.
• These tools enhance the quantitative understanding of molecular properties from photon burst analysis.
• The study offers improved methodologies for biophysical and chemical research involving single molecules.