The structural studies of macromolecules in their physiological context, particularly in tissue, is constrained by the bottleneck of sample preparation. In this study, we present a practical pipeline for preparing multicellular samples for cryo-electron tomography. The pipeline comprises sample isolation, vitrification, and lift-out-based lamella preparation using commercially available instruments. We demonstrate the efficacy of our pipeline by visualizing pancreatic β cells from mouse islets at the molecular level. This pipeline enables the determination of the properties of insulin crystals in situ for the first time, using unperturbed samples.
