Fast volumetric fluorescence lifetime imaging of multicellular systems using single-objective light-sheet microscopy
Fluorescence lifetime imaging (FLIM) is widely used for functional and multiplexed bioimaging. The lifetime of autofluorescence or fluorescent sensors encodes physiologically relevant parameters. Thus, FLIM is especially relevant for the investigation of living systems. However, application of FLIM to live specimen is hampered by its slow speed and high phototoxicity. To enable faster and gentler FLIM, we integrated single-objective light-sheet microscopy with pulsed excitation and time-resolved detection on a novel SPAD array detector. We achieved 10-100-fold acceleration compared to confocal FLIM, down to 100 ms acquisition time per image, with excellent quantitative agreement. The massively enhanced speed enables volumetric FLIM acquisitions on live multicellular specimens, which we demonstrate with lifetime-based multiplexing in 3D and time-lapse FLIM of tension probes on living embryonic organoids. We benchmark both scanned and static light-sheet modalities to facilitate adding FLIM capability to a large variety of light-sheet microscopes.
Authors
- Rainer Erdmann (ORCID: https://orcid.org/0000-0003-2019-2794)
- Ivan Michel Antolović (ORCID: https://orcid.org/0000-0001-6155-6895)
- Jean‐Baptiste Sibarita (ORCID: https://orcid.org/0000-0002-9920-7700)
- Claire Chardès (ORCID: https://orcid.org/0000-0001-7016-1174)
- Thomas Schönau (ORCID: https://orcid.org/0000-0003-1533-7434)
- Felix Koberling
- Valentin Dunsing (ORCID: https://orcid.org/0000-0003-2482-1498)
- Johan Hummert (ORCID: https://orcid.org/0000-0003-3554-713X)
- Pierre‐François Lenne (ORCID: https://orcid.org/0000-0003-1066-7506)
- Max Tillmann (ORCID: https://orcid.org/0000-0001-6935-2803)
- Virgile Viasnoff (ORCID: https://orcid.org/0000-0003-3949-2244)
- Léo Guignard (ORCID: https://orcid.org/0000-0002-3686-1385)
- Rémi Galland (ORCID: https://orcid.org/0000-0001-7117-3281)
- Gianluca Grenci (ORCID: https://orcid.org/0000-0001-9313-6748)
- Corinna Nock
Institutions
- Centre National de la Recherche Scientifique (FR)
- University of Nottingham (GB)
- Université de Bordeaux (FR)
- National University of Singapore (SG)
- Aix-Marseille Université (FR)
- PicoQuant (Germany) (DE)
- Laboratoire d’Informatique et Systèmes (FR)
- Institut de Biologie du Développement Marseille (FR)
- Institut Interdisciplinaire de Neuroscience (FR)
- Living Systems (United States) (US)
- École Polytechnique Fédérale de Lausanne (CH)
- Charité - Universitätsmedizin Berlin (DE)
- University of Birmingham (GB)
Publication Details
- Journal
- Communications Biology
- Published
- 2025-11-29
- DOI
- https://doi.org/10.1038/s42003-025-09221-2
- Primary Topic
- Advanced Fluorescence Microscopy Techniques
- Type
- article
- Field-Weighted Citation Impact
- 0.00
Funders
- Agence Nationale de la Recherche
- Bundesministerium für Bildung und Forschung
- Université de Bordeaux