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

Valentin Dunsing-Eichenauer1,2, Johan Hummert3, Claire Chardès4

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We developed a faster, gentler Fluorescence Lifetime Imaging Microscopy (FLIM) technique for live specimens. This breakthrough enables rapid, high-resolution 3D imaging of cellular dynamics, overcoming previous speed and phototoxicity limitations.

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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.