Improved optical slicing by stimulated emission depletion light sheet microscopy

Three-dimensional microscopy is mandatory for biological investigation. We describe a stimulated emission depletion selective plane illumination microscope (STED-SPIM) that provides both ease of implementation and an efficient optical slicing. This self-aligned system is based on a single diode-pumped solid-state laser and phase masks made of simple cover glass. A three-fold reduction of the light sheet thickness is achieved together with an enhancement of the sheet uniformity. This method is validated by using fluorescent microspheres and thick slices of fixed and clarified mouse brain to provide an enhanced imaging of Alzheimer's disease models.