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Probing the architecture, mechanism, and dynamics of genome folding is fundamental to our understanding of genome function in homeostasis and disease. Most chromosome conformation capture studies dissect the genome architecture with population- and time-averaged snapshots and thus have limited capabilities to reveal 3D nuclear organization and dynamics at the single-cell level. Here, we discuss emerging imaging techniques ranging from light microscopy to electron microscopy that enable investigation of genome folding and dynamics at high spatial and temporal resolution. Results from these studies complement genomic data, unveiling principles underlying the spatial arrangement of the genome and its potential functional links to diverse biological activities in the nucleus.