The process of studying dynamic three-dimensional samples has a long history in biological research. Recent advances in hardware and software have made it easier to visualise and record interior detail from multiple focal planes of three-dimensional samples as they change over time (four-dimensional imaging). Once captured, it is possible to watch these events repeatedly and to analyse them in numerous ways. This article discusses the history of and the hardware necessary to perform 4D experiments, the various techniques that make 4D imaging possible, and the applications and various options for 4D-image analysis.