Lightsheet fluorescence lifetime imaging microscopy with wide-field time-correlated single photon counting

Area of Science:

• Biophotonics
• Microscopy techniques
• Cell biology

Background:

• Fluorescence Lifetime Imaging Microscopy (FLIM) provides functional and molecular information.
• Traditional FLIM methods can be limited by speed or phototoxicity.
• Lightsheet illumination offers reduced photobleaching and scattering for deep tissue imaging.

Purpose of the Study:

• To develop and validate a wide-field time-correlated single photon counting (TCSPC)-based FLIM system.
• To combine the benefits of lightsheet illumination with high-resolution FLIM.
• To demonstrate the system's capability for 3D imaging of biological samples.

Main Methods:

• Utilized a pulsed diode laser for excitation.
• Employed a crossed delay line anode image intensifier for single-photon detection.
• Integrated TCSPC with lightsheet illumination for picosecond time resolution.
• Achieved wide-field data collection with low illumination intensity (microwatts).

Main Results:

• Successfully implemented wide-field TCSPC-FLIM with lightsheet illumination.
• Demonstrated high-resolution 3D FLIM imaging.
• Applied the technique to visualize dye gradients in human cancer cell spheroids.
• Showcased utility in imaging C. elegans.

Conclusions:

• The developed system offers a powerful tool for advanced 3D FLIM.
• Combines low phototoxicity with high spatial and temporal resolution.
• Suitable for studying complex biological structures and processes.