Super-Resolved Fluorescence Lifetime Imaging of Single Cy3 Molecules and Quantum Dots Using Time-Correlated Single...

Area of Science:

• Biophysics
• Nanotechnology
• Optical Microscopy

Background:

• Single molecule imaging requires high spatial and temporal resolution.
• Distinguishing between different types of emitters like quantum dots (QDs) and organic dyes is crucial for complex biological studies.
• Existing microscopy techniques often struggle to achieve simultaneous super-resolution and accurate lifetime measurements.

Purpose of the Study:

• To develop and demonstrate a super-resolved fluorescence lifetime imaging technique.
• To differentiate and precisely locate individual quantum dots (QDs) and Cy3 dye molecules.
• To assess the potential for investigating molecular interactions like Förster resonance energy transfer (FRET) using this method.

Main Methods:

• Combined time-resolved single molecule localization microscopy (TR-SMLM) with a 2x2 pixel fiber optic array camera.
• Utilized time-correlated single photon counting (TCSPC) for fluorescence lifetime analysis.
• Leveraged blinking and bleaching behaviors of Cy3 and QDs for emitter isolation and localization.

Main Results:

• Achieved super-resolution spatial localization of individual QDs (1-4 nm precision) and Cy3 molecules (2-9 nm precision).
• Successfully distinguished and measured the distance between a single QD and a single Cy3 molecule (∼34 nm apart) with 8 nm precision.
• Obtained fluorescence lifetimes for individual emitters (∼6 ns for Cy3, ∼17 ns for QD).

Conclusions:

• The developed TR-SMLM-TCSPC technique provides high spatial resolution and fluorescence lifetime information at the single-molecule level.
• This method enables precise characterization and differentiation of QDs and organic dyes.
• The technique shows significant potential for studying molecular complexes and developing optical biosensors based on FRET and electron transfer.