A time-correlated single photon counting SPAD array camera with a bespoke data-processing algorithm for lightsheet...

Area of Science:

• Biophotonics
• Microscopy
• Spectroscopy

Background:

• Live-cell imaging requires high temporal resolution and sensitivity.
• Fluorescence Lifetime Imaging (FLIM) provides molecular information but is often limited by speed and complexity.
• Traditional time-correlated single-photon counting (TCSPC) methods can be time-consuming for live samples.

Purpose of the Study:

• To develop and demonstrate a novel live-cell fluorescence lifetime imaging microscopy (FLIM) system.
• To integrate a single-photon avalanche diode (SPAD) array camera with TCSPC for enhanced imaging capabilities.
• To validate the system's performance using biological and artificial samples.

Main Methods:

• A wide-field microscope equipped with epi-fluorescence and selective plane illumination was utilized.
• A custom SPAD array camera with integrated time-to-digital converters was employed for photon detection.
• A Monte Carlo algorithm was implemented for correcting systematic delays and nonlinearities in photon arrival times.
• TCSPC principles were applied by histogramming photon arrival times for fluorescence decay analysis.

Main Results:

• The SPAD camera system achieved high accuracy in capturing photon arrival times.
• Fluorescence decays generated by the system were compatible with standard FLIM data processing pipelines.
• Live imaging of unicellular photosynthetic algae demonstrated the system's capability for live-cell FLIM.
• 3D FLIM of artificial lipid vesicles showcased the potential of selective-plane illumination.

Conclusions:

• The developed TCSPC camera-based FLIM microscope enables rapid and accurate live-cell imaging.
• The integration of SPAD technology significantly advances FLIM capabilities for biological research.
• The system offers versatile imaging modes, including epi-fluorescence for live cells and selective-plane illumination for 3D imaging.