title: Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development.
abstract: Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics; icha@mpi-cbg.de.
authors.forename: Jaroslav
authors.initials: J
authors.lastname: Icha
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics.
authors.forename: Christopher
authors.initials: C
authors.lastname: Schmied
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics.
authors.forename: Jaydeep
authors.initials: J
authors.lastname: Sidhaye
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics.
authors.forename: Pavel
authors.initials: P
authors.lastname: Tomancak
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics; HHMI Janelia Research Campus; Berlin Institute of Medical Systems Biology of the Max Delbrück Center.
authors.forename: Stephan
authors.initials: S
authors.lastname: Preibisch
authors.affiliation: Max Planck Institute of Molecular Cell Biology and Genetics; norden@mpi-cbg.de.
authors.forename: Caren
authors.initials: C
authors.lastname: Norden
mesh_terms: D000818:Animals; D015509:Developmental Biology; D004625:Embryo, Nonmammalian / Q000196:embryology*; D047108:Embryonic Development; D005123:Eye / Q000196:embryology*; D008027:Light; D008856:Microscopy, Fluorescence / Q000379:methods*; D015027:Zebrafish / Q000196:embryology*