Fluorescence lifetime tracking and imaging of single moving particles assisted by a low-photon-count analysis...

Area of Science:

• Biophysics
• Microscopy techniques
• Photon counting

Background:

• Fluorescence lifetime imaging microscopy (FLIM) is crucial for sensing cellular microenvironments due to its specificity and sensitivity.
• Time-correlated single photon counting (TCSPC) is the standard FLIM method but suffers from slow imaging speeds and long data acquisition times.
• Tracking dynamic biological processes requires faster FLIM imaging capabilities.

Purpose of the Study:

• To develop a rapid FLIM technology for tracking and imaging single moving particles.
• To enhance imaging speed and reduce data acquisition time for FLIM.
• To enable quantitative analysis of dynamic biological events at the single-particle level.

Main Methods:

• Introduced single particle tracking FLIM (SPT-FLIM) utilizing feedback-controlled addressing scanning and Mosaic FLIM mode.
• Developed a compressed sensing algorithm (ADCG-FLIM) for analyzing low-photon-count data.
• Validated the ADCG-FLIM algorithm on simulated and experimental datasets.

Main Results:

• ADCG-FLIM demonstrated reliable and accurate fluorescence lifetime estimation with photon counts below 100.
• Reduced photon count requirements from 1000 to 100 per pixel significantly shortened acquisition time and increased imaging speed.
• Successfully obtained lifetime trajectories of moving fluorescent beads using SPT-FLIM.

Conclusions:

• The proposed SPT-FLIM technique offers a powerful tool for fluorescence lifetime tracking and imaging of single moving particles.
• This advancement significantly improves the speed and efficiency of TCSPC-FLIM.
• The developed method is expected to broaden the application of FLIM in dynamic biological research.