The cloning of green fluorescent protein (GFP) 15 years ago revolutionized cell biology by permitting visualization of a wide range of molecular mechanisms within living cells. Though initially used to make largely qualitative assessments of protein levels and localizations, fluorescence microscopy has since evolved to become highly quantitative and high-throughput. Computational image analysis has catalyzed this evolution, enabling rapid and automated processing of large datasets. Here, we review studies that combine time-lapse fluorescence microscopy and automated image analysis to investigate dynamic events at the single-cell level. We highlight examples where single-cell analysis provides unique mechanistic insights into cellular processes that cannot be otherwise resolved in bulk assays. Additionally, we discuss studies where quantitative microscopy facilitates the assembly of detailed 4D lineages in developing organisms. Finally, we describe recent advances in imaging technology, focusing especially on platforms that allow the simultaneous perturbation and quantitative monitoring of biological systems.