In situ single-particle Cryo-EM methods: Principle and applications

Cryo-electron microscopy (cryo-EM) is transitioning from determining structures of isolated proteins in vitro to visualizing macromolecular architecture directly in situ. Conventional in situ approaches, primarily relying on cryo-electron tomography combined with subtomogram averaging, are often limited in resolution due to complex workflows, cumulative errors in processing, and low data throughput. Emerging in situ single-particle cryo-EM methods address these limitations by collecting high-dose, untilted images of cellular lamellae. Using high-resolution templates for particle identification and refinement, these methods have significantly advanced both data throughput and achievable resolution. This review systematically outlines the principles, workflow, and advantages of in situ single-particle methods, highlights their key applications, and discusses future perspectives.