ADVANCING IN SITU CRYO-ET TO THE NEXT DIMENSION: TOMOGRAPHY ON 3D ORGANOIDS

Interpreting how molecular organization supports cell and tissue function remains a central challenge in biology. In situ cryo-electron tomography (cryo-ET) can help to address this challenge by providing high-resolution, three-dimensional visualization of macromolecules in near-native, minimally disturbed conditions. With its broad resolution, cryo-ET enables us to study the organization and architecture of macromolecules across different spatial scales. Hence, this capacity to bridge molecular and cellular levels offers a unique opportunity to connect structural biology with medical research. However, for a long time, cryo-ET has been mainly applied to simple biological systems, such as viruses, bacteria, and two-dimensional cell cultures. While these systems are technically feasible, they lack the interaction information, resulting in poor translation from cellular to tissue-level biology. To more precisely capture the molecular landscape, ideally, we should extend studies to tissue samples. Yet, they pose significant ethical and methodological challenges. A promising alternative is the use of three-dimensional organoids, which present a balance between physiological relevance and technical capabilities. Organoids recapitulate key aspects of tissue architecture and physiology, thus bridging the gap between cellular and tissue contexts. Additionally, they are simpler and easier to manipulate and access than tissue samples. Combining organoids with in situ cryo-ET creates new opportunities for conducting structural biology in physiologically relevant models. This thesis presents the first applications of cryo-ET to organoid samples, specifically human forebrain organoids and mouse thyroid organoids, enabling in situ structural analysis at subcellular scales within tissue-like environments. To overcome challenges posed by these samples, we developed several methodological advances: (i) Serialized On-grid Lift-In Sectioning for Tomography (SOLIST), an optimized cryo-lift-out technique improving sample stability and lamella production efficiency; (ii) a generalized in situ cryo-ET workflow applicable to a variety of organoids, embedding with or without extracellular matrix; and (iii) a targeted cryo-ET pipeline facilitating precise imaging of defined regions within these organoids. Using these advances, together with other complementary imaging techniques, we successfully resolved the molecular landscape in human forebrain organoids and characterized the follicular organizations in mouse thyroid organoids. By integrating the structural resolution of in situ cryo-ET with the physiological relevance of organoids, this work has established a methodological framework for in-depth investigations at the molecular level in complex and medically relevant systems. The outcome of this study sets the foundations for future approaches, where organoids and patient samples become the preferred choice in structural biology, thereby permitting the validation of simpler model systems and advancing our understanding of research and disease treatment.