Experimental approach to extend the range for counting fluorescent molecules based on photon-antibunching

Area of Science:

• Spectroscopy
• Microscopy
• Biophysics

Background:

• Single-molecule fluorescence spectroscopy uses photon antibunching to identify single fluorophores.
• Current methods for counting fluorophores in confocal microscopes are limited to approximately 3 molecules due to calibration curve saturation.

Purpose of the Study:

• To experimentally realize and validate a novel theoretical framework for counting multiple emitting molecules using photon distributions.
• To extend the capability of fluorophore counting beyond the limitations of existing techniques.

Main Methods:

• Utilized a confocal microscope setup equipped with four single-photon detectors.
• Employed novel multi-channel photon-counting electronics.
• Experimentally tested with DNA constructs labeled with five fluorophores (ATTO647N).

Main Results:

• Demonstrated a clear correlation between estimated fluorophore numbers and bleaching steps for DNA probes with five labels, achieving ~20% error.
• Successfully acquired data for up to 15 fluorophores, indicating the simultaneous presence of three DNA probes.
• Validated the effectiveness of analyzing photon distributions from four detection channels for multi-fluorophore counting.

Conclusions:

• The developed method effectively counts fluorescently labeled molecules in aggregates and clusters.
• This technique has potential applications in molecular and cell biology for quantitative analysis.
• Suitable for time-resolved analysis of multi-chromophoric compounds in material sciences.