Real-Time 3D Single Particle Tracking: Towards Active Feedback Single Molecule Spectroscopy in Live Cells

Area of Science:

• Biophysics
• Spectroscopy
• Cellular Biology

Background:

• Single molecule fluorescence spectroscopy traditionally requires tethering molecules to a substrate.
• Tethering can perturb biomolecules, like enzymes, by removing them from their native cellular environment.
• This perturbation affects measurements of biomolecular function in non-equilibrium and crowded cellular conditions.

Purpose of the Study:

• To review real-time 3D single particle tracking (RT-3D-SPT) methods for untethered single molecule fluorescence spectroscopy.
• To highlight advancements in detection and excitation schemes for high-speed tracking.
• To discuss the integration of RT-3D-SPT with live-cell imaging.

Main Methods:

• Active feedback mechanisms to lock onto freely diffusing particles.
• High-speed real-time tracking with photon-limited temporal resolution.
• Specialized optical detection and excitation schemes.
• Simultaneous live-cell imaging integration.

Main Results:

• RT-3D-SPT enables continuous measurement of single molecules in their native environment.
• Active feedback allows tracking over large axial ranges with high temporal resolution.
• The reviewed methods facilitate the study of biomolecules without perturbing their function.

Conclusions:

• RT-3D-SPT offers a promising alternative to tethered methods in single molecule fluorescence spectroscopy.
• This technique preserves the native cellular environment, crucial for studying biomolecular function.
• Future developments aim to further enhance tracking capabilities and live-cell imaging integration.