INTRODUCTIONModern applications in the life sciences are frequently based on in vivo imaging of biological specimens, a domain for which light microscopy approaches are typically best suited. Often, quantitative information must be obtained from large multicellular organisms on the cellular or even subcellular level and with a good temporal resolution. However, this usually requires a combination of conflicting features: high imaging speed, low photobleaching, and low phototoxicity in the specimen, good three-dimensional (3D) resolution, an excellent signal-to-noise ratio, and multiple-view imaging capability. The latter feature refers to the capability of recording a specimen along multiple directions, which is crucial for the imaging of large specimens with strong light-scattering or light-absorbing tissue properties. An imaging technique that fulfills these requirements is essential for many key applications: For example, studying fast cellular processes over long periods of time, imaging entire embryos throughout development, or reconstructing the formation of morphological defects in mutants. Here, we discuss digital scanned laser light sheet fluorescence microscopy (DSLM) as a novel tool for quantitative in vivo imaging in the post-genomic era and show how this emerging technique relates to the currently most widely applied 3D microscopy techniques in biology: confocal fluorescence microscopy and two-photon microscopy.