Adaptive-optics lattice light-sheet microscopy enables correlative single-molecule imaging of endogenous proteins in native environments

Malivert, M.; Galizia, C. C.; Willems, B. A.; Mondin, M.; Lemoigne, C.; Lecompte Saint-Jean, P.; Demeer, B.; Marcelino, M.; Crippa, D.; Thoumine, O.; Harms, F.; Sainlos, M.; Choquet, D.; Ducros, M.; Getz, A. M.

doi:10.64898/2026.10.10.758183

bioRxiv · 10.64898/2026.10.10.758183

Adaptive-optics lattice light-sheet microscopy enables correlative single-molecule imaging of endogenous proteins in native environments

Malivert, M.·Galizia, C. C.·Willems, B. A.·Mondin, M.·Lemoigne, C.·Lecompte Saint-Jean, P.·Demeer, B.·Marcelino, M.·Crippa, D.·Thoumine, O.·Harms, F.·Sainlos, M.·Choquet, D.·Ducros, M.·Getz, A. M.

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Abstract

Resolving molecular organization and dynamics in native environments remains challenging, as single-molecule imaging requires high sensitivity and spatiotemporal resolution while preserving 3D cellular context. Here, we present ALPINE (Adaptive-optics Lattice light-sheet microscopy for endogenous Protein Imaging in Native Environments), a modular framework for quantitative single-molecule imaging into complex biological samples. ALPINE combines adaptive-optics lattice light-sheet microscopy, programmable PSF shaping and acquisition stabilization, an engineered cell-specific labelling toolkit for endogenous cell-surface proteins and analysis pipelines assigning molecular localizations and trajectories to 3D cellular compartments. Labelling endogenous AMPA receptors in intact brain tissue, we demonstrate aberration-corrected 3D single-molecule localization and nanoscale organization within neuronal compartments. Importantly, fast optical sectioning of lattice light-sheet illumination further enables 3D single-molecule tracking in live tissue and quantification of molecular dynamics within native cellular environment. Together, ALPINE provides a modular approach to investigate how endogenous molecules are organized and dynamically regulated in complex biological systems, with components adaptable to diverse biological questions and imaging modalities.

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Malivert, M., Galizia, C. C., Willems, B. A., Mondin, M., Lemoigne, C., Lecompte Saint-Jean, P., Demeer, B., Marcelino, M., Crippa, D., Thoumine, O., Harms, F., Sainlos, M., Choquet, D., Ducros, M., Getz, A. M.. 2026-10-16. Adaptive-optics lattice light-sheet microscopy enables correlative single-molecule imaging of endogenous proteins in native environments. https://doi.org/10.64898/2026.10.10.758183

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