Optical microscopy plays an increasing role in studies of 3-dimensional cell cultures as well as whole organisms. Since the sample thickness often exceeds the depth of focus considerably, confocal methods have been developed more than 30 years ago in order to select individual focal planes and to suppress out-of-focus images. After shifting of the focal plane in the axial direction a sharp image of the whole sample can be reconstructed. The potential of confocal microscopy has also been increased by advanced methods, e.g. Airy Scan Microscopy, Stimulated Emission Depletion Microscopy (STED) and related techniques. These techniques often provide super-resolution far below the Abbe criterion. However, wide-field methods, such as Super-localization techniques, Structured Illumination Microscopy (SIM), Light Sheet Microscopy or Total Internal Reflection (TIRF) microscopy gained considerable importance. These are of importance with respect to resolution (Super-localization, SIM), 3D imaging (Light Sheet) and selective detection of cell membranes (TIRF). Due to extremely low light exposure in Light Sheet Microscopy, this skill plays an important role in long-term observation, for example, in developmental biology. Further development of these methods often in combination with spectral or temporal resolution as well as their applications in a broad field of biomedical research, diagnostics or pharmacology is the subject of this JoVE methods collection.