Fast volumetric fluorescence lifetime imaging of multicellular systems using single-objective light-sheet microscopy

Area of Science:

• Biomedical Optics
• Microscopy
• Cell Biology

Background:

• Fluorescence Lifetime Imaging Microscopy (FLIM) is crucial for functional and multiplexed bioimaging in living systems.
• Current FLIM applications are limited by slow acquisition speeds and high phototoxicity, hindering live specimen studies.
• The lifetime of autofluorescence or sensors provides physiologically relevant information.

Purpose of the Study:

• To develop a faster and gentler FLIM technique for live specimen imaging.
• To overcome the limitations of speed and phototoxicity in conventional FLIM.
• To enable high-speed, volumetric, and time-lapse FLIM on live multicellular specimens.

Main Methods:

• Integration of single-objective light-sheet microscopy with pulsed excitation and time-resolved detection using a novel SPAD array detector.
• Implementation of scanned and static light-sheet modalities for benchmarking.
• Quantitative agreement assessment compared to confocal FLIM.

Main Results:

• Achieved 10-100-fold acceleration in FLIM acquisition speed compared to confocal microscopy, with image acquisition times as low as 100 ms.
• Demonstrated volumetric FLIM in 3D using lifetime-based multiplexing on live multicellular specimens.
• Successfully performed time-lapse FLIM of tension probes on living embryonic organoids.

Conclusions:

• The developed FLIM method significantly enhances imaging speed and reduces phototoxicity, making it suitable for live specimen investigation.
• This technique enables advanced applications like 3D multiplexed imaging and time-lapse studies of dynamic biological processes.
• The findings facilitate the integration of advanced FLIM capabilities into various light-sheet microscopy setups.