Recent advances in targeting regions of interest for In situ cryo-electron tomography of cellular architecture

Cryogenic electron tomography (cryo-ET) enables in situ structural analysis of macromolecular assemblies within their native cellular environments, spanning more than four orders of magnitude in spatial scale, from micrometre-level cellular context accessed through correlative imaging to near-sub-nanometre resolution achieved through subtomogram averaging (STA). This review summarises recent advances in mapping cellular architecture, encompassing membrane-bound organelles, cytoskeletal networks, adhesion complexes, and discrete cellular subsystems such as cilia and the nuclear pore complex (NPC). We discuss the principal challenges associated with cellular cryo-ET, including specimen thickness and electron transparency limitations, structural heterogeneity, the transient nature of many assemblies, restricted targeting precision, unreliable molecular identification, preparation-induced artefacts, and labelling constraints. Recent strategies developed to address these challenges are reviewed, with particular emphasis on innovations in sample preparation and their integration with cryo-focused ion beam milling (cryo-FIB), cryo-correlative light and electron microscopy (cryo-CLEM), STA, and complementary volume-imaging approaches such as cryo-scanning transmission electron tomography (cryo-STET) and cryo-soft X-ray tomography (cryo-SXT). We further highlight emerging density-based modelling strategies that enable molecular interpretation when sufficient resolution is achieved, as well as two-dimensional (2D) template-matching approaches. Collectively, these developments position cryo-ET as a central framework for interrogating cellular ultrastructure in its native context.