Linalool enables reversible immobilization for high-resolution in vivo imaging during regeneration in Schmidtea mediterranea.

Live imaging in whole-organism models is often limited in both resolution and repeatability by the lack of relatively non-invasive, reversible immobilization methods. To enable real-time in vivo imaging in the highly regenerative planarian S. mediterranea, we systematically evaluated linalool as a sedation strategy. We identified 2.16 mM linalool as an effective concentration that induces rapid, reversible immobilization without compromising survival or causing detectable stress phenotypes. Acute exposure before amputation or repeated daily exposures during regeneration did not impair blastema growth, stem cell proliferation, or gross brain morphology, demonstrating compatibility with regeneration studies under imaging-relevant conditions. In contrast, continuous exposure throughout regeneration induced stress phenotypes and impaired regenerative outcomes. Together, our findings define operational exposure windows for linalool, establishing a robust, regeneration-compatible immobilization strategy that enables stable, high-resolution imaging across biological scales, from whole-organism organization to cellular dynamics, while remaining compatible with fluorescent dyes and allowing full recovery after imaging.