Light Sheet Fluorescence Microscopy (LISH-M) is a true fluorescence optical sectioning technique, first described by Heinrich Siedentopf in 1902 under the name of Ultramicroscopy. Light sheet microscopy utilises a plane of light to optically section samples. This allows deep imaging within transparent tissues and whole organisms. As tissues are exposed to only a thin plane of light, photobleaching and phototoxicity are minimized compared to wide-field fluorescence, confocal or multiphoton microscopy. Additionally, the use of a light sheet reduces drastically out-of focus light and improves the signal-to-noise ratio of the images. Light Sheet Fluorescence Microscopy combines the advantages of wide-field methods (speed, flexibility, and dynamic range) with those of a confocal arrangement (optical sectioning).
The last ten years have witnessed several implementations of this technology constructed by different laboratories under various acronyms: OPFOS, SPIM, TSLIM, MISERB…. Most of them share the orthogonal design but there are variations in illumination type or add-ons employed. Moreover, Light Sheet Microscopy requires three dimensional (3D) advanced sample preparation techniques to fully visually access most biological samples, ranging from single yeasts to growing Arabidopsis thaliana. Finally, the multiview capability of Light Sheet Fluorescence Microscopy provides users with multiple large stacks of images that must be combined and fused. This is an important limitation as image processing of large datasets (up to 1Tb per experiment) is a challenge especially when fusion is required.
This book chapter will provide the reader with a comprehensive view on this emerging technology. We will also discuss the two associated issues of 3D sample preparation and image processing. Finally, we provide a glossary and a list of useful literature in regard to the different implementations and the various models of Light Sheet Microscope.