Four-dimensional multi-particle tracking in living cells based on lifetime imaging

Area of Science:

• Cellular Biology
• Biophysics
• Microscopy

Background:

• Understanding dynamic events in living cells, like intracellular transport, is crucial for cell function.
• The cellular microenvironment can significantly influence the behavior of moving vesicles and biomacromolecules.

Purpose of the Study:

• To develop a method for simultaneously monitoring spatial positions and local environment changes (via fluorescence lifetime) of multiple particles in living cells.
• To enable four-dimensional (4D) multi-particle parallel-tracking for enhanced cellular process research.

Main Methods:

• Utilized double-helix point spread function (DH-PSF) microscopy combined with a streak camera.
• Integrated three-dimensional (3D) localization with fluorescence lifetime imaging by modifying the point spread function (PSF).
• Developed a proof-of-concept 4D tracking system.

Main Results:

• Achieved 3D localization precision of σ(x, y, z) = (26 nm, 35 nm, 53 nm) and fluorescence lifetime precision of σ(τ) = 103 ps.
• Demonstrated an effective depth of field of approximately 4 μm.
• Successfully tracked microspheres during intracellular endocytosis within a 4 μm axial depth.

Conclusions:

• The developed 4D multi-particle parallel-tracking method provides simultaneous physical and chemical information of moving targets in living cells.
• This technique offers a new perspective for studying dynamic cellular processes by revealing microenvironmental influences on molecular behavior.
• The system's capabilities were validated through simulations and experimental tracking of intracellular movement.