Single-molecule fluorescence detection: autocorrelation criterion and experimental realization with phycoerythrin

Area of Science:

• Biophysics
• Analytical Chemistry
• Optical Physics

Background:

• Single-molecule detection offers unparalleled sensitivity for analyzing biological samples.
• Conventional fluorescence detection methods face limitations in sensitivity and resolution.
• B-phycoerythrin (PE) is a fluorescent protein often used in biological assays.

Purpose of the Study:

• To develop and validate a theoretical framework for single-molecule fluorescence detection.
• To analyze experimental data from B-phycoerythrin solutions using the developed theory.
• To demonstrate the system's capability for ultra-sensitive concentration measurements.

Main Methods:

• Development of a theoretical model for fluorescence detection, considering Poissonian background and fluorophore bursts.
• Analysis of fluorescence count distributions and autocorrelation functions to identify single-molecule events.
• Optimization of experimental parameters, including laser power and transit time, for enhanced detection.

Main Results:

• The developed theory accurately describes the distribution of detected counts and autocorrelation functions.
• Single-molecule detection of B-phycoerythrin was achieved at subpicomolar concentrations (down to 100-200 fM).
• The system demonstrated a mean incremental count rate of 1.5/100 µs for monomers and 3.0/100 µs for dimers.
• A hard-wired system achieved B-phycoerythrin concentration measurements down to 1 fM.

Conclusions:

• The developed theory provides a robust foundation for single-molecule fluorescence detection.
• The system achieves a 3-orders-of-magnitude increase in sensitivity compared to conventional methods.
• This technology enables highly sensitive quantification of fluorescent molecules, with applications in various biological and chemical analyses.