4D Single-particle tracking with asynchronous read-out single-photon avalanche diode array detector

Area of Science:

• Biophysics
• Microscopy
• Cell Biology

Background:

• Single-particle tracking (SPT) is crucial for understanding biological particle dynamics.
• Existing SPT methods face trade-offs in resolution, complexity, and data acquisition.
• There is a need for advanced microscopy techniques with improved spatiotemporal precision.

Purpose of the Study:

• To enhance a confocal laser scanning microscope for superior single-particle tracking.
• To integrate fluorescence lifetime measurement with real-time particle localization.
• To overcome limitations of current SPT techniques for biological investigations.

Main Methods:

• Augmented a confocal laser scanning microscope with an asynchronous single-photon avalanche diode array detector.
• Implemented a real-time feedback system to maintain particle centering within the excitation volume.
• Combined single-particle tracking with fluorescence lifetime measurements using photon detection.

Main Results:

• Achieved 40 nm lateral and 60 nm axial localization precision with 100 photons.
• Enabled sub-millisecond temporal sampling for real-time tracking, refining to microsecond scale offline.
• Successfully tracked fluorescent beads and lysosomes in living cells, measuring membrane protein fluorescence lifetime.

Conclusions:

• The enhanced microscope system offers significant improvements in spatiotemporal resolution for SPT.
• Correlative tracking and fluorescence lifetime measurements provide richer biological insights.
• This advanced microscopy approach is expected to facilitate new correlative imaging and tracking studies.