Principles of light-sheet-based microscopy. (A) The overall layout of an SPIM. The emission from a laser array (LA) of various wavelengths for exciting fluorescence is combined into a single beam. An acousto-optic tunable filter (AOTF) is used to select the wavelengths and the intensity of the excitation beam. The beam expanding and cylindrical optics (BEO and CO) create the light sheet that illuminates the sample (S). Detection is through a wide-field fluorescence microscope consisting of an objective lens (OL), a filter wheel (FW) that contains filters for rejection of scattered excitation light and background fluorescence, a tube lens (TL), and a camera. (B) Comparison of sample illumination and fluorescence detection in conventional/confocal microscopy and in LSFM. (i) The entire region of interest in the specimen is illuminated in conventional/confocal microscopy, although only a single plane in the specimen is being observed. (ii) In contrast, no photodamage is inflicted outside the in-focus plane of the detection system in the LSFM. (C) Three-dimensional imaging in LSFM is performed by moving the specimen step by step through the light sheet while recording two-dimensional images. In multiple-view imaging, the same volume inside the specimen or even inside the entire specimen is recorded along several angles. The resulting multiple-view information is combined into a single image stack by a fusion algorithm. (B,C, Adapted, with permission, from Keller and Stelzer 2008.)