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

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.