biostudies-literatureHubert AEuropean Research Council (ERC) under the European Union's Horizon 2020 research innovation programEuropean Research CouncilANRIle de France, PhD fellowshipThis work was supported by grants from Région Ile-de-France23760https://www.ebi.ac.uk/biostudies/studies/S-EPMC12229697biostudies-literatureUnknown15(1)Two-photon light sheet microscopy offers great potential for a range of biological applications. Still, its practical implementation is impeded by the high cost of laser sources, the complexity of construction, and the challenges associated with adapting to existing microscope setups. Here, we release an open-source design that addresses these limitations by providing detailed building instructions for transforming a brightfield microscope into a versatile one- and two-photon light sheet system. Our design incorporates a specially designed broadband hollow core fiber, enabling the simultaneous utilization of visible laser alongside an expansive pulsed laser source from another setup. This integration allows for uncompromised image resolution and speed. Furthermore, the design reduces the cScientific reportsA versatile and open source design for one and two photon light sheet microscopy.PMC12229697715980ARDoC 17012950ANR-21-CE16-0037DIM ElicitBeaudou BMigault GBormuth VPanier TDebregeas GTrentesaux HHubert AfalseA versatile and open source design for one and two photon light sheet microscopy.Two-photon light sheet microscopy offers great potential for a range of biological applications. Still, its practical implementation is impeded by the high cost of laser sources, the complexity of construction, and the challenges associated with adapting to existing microscope setups. Here, we release an open-source design that addresses these limitations by providing detailed building instructions for transforming a brightfield microscope into a versatile one- and two-photon light sheet system. Our design incorporates a specially designed broadband hollow core fiber, enabling the simultaneous utilization of visible laser alongside an expansive pulsed laser source from another setup. This integration allows for uncompromised image resolution and speed. Furthermore, the design reduces the c2025-01-01T00:00:00Z2025 Jul2026-07-15T12:44:40.012Z2026-07-04T03:16:21.257ZS-EPMC122296974061049110.1038/s41598-025-03107-3