Protocol for intelligent high-content screening of zebrafish embryos on a standard widefield screening microscope
2Institute for Biological Interfaces, Karlsruhe Institute of Technology, Campus North, D-76344 Eggenstein-Leopoldshafen, Germany
3Department of Medical and Molecular Genetics, University of Birmingham, Birmingham B15 2TT, UK
Here we demonstrate a toolset for automated intelligent high-content screening of whole zebrafish embryos at cellular resolution on a standard wide-field screening microscope. Using custom-developed algorithms, predefined regions of interest, such as the brain, are automatically detected. The regions of interest are subsequently imaged automatically at high magnification, enabling rapid capture of cellular resolution data. standardized imaging of internal organs and cellular structures.
A pipeline for automated intelligent high-content screening. (A) Overview of workflow for automated intelligent HCS screening on the Olympus Scan^R screening microscope. The flowchart illustrates the steps carried out to obtain high-resolution widefield screening data (for further details, see “Materials and methods” and “Results and discussion” in the main text, as well as Supplementary Figure S1). The last box depicts optional image restoration techniques to improve experimental data. (B–E) Illustrative example for acquired HCS data; bright-field views are shown. (B) Overview of prescreen data from a single 96-well plate acquired using a 2.5× objective. (C) Single embryo from prescreen data. The bounding box (blue rectangle) indicates the image region matching the chosen template. Red asterisk indicates the pixel coordinates chosen as the center of the field of view for the subsequent high-resolution imaging (see panels D and E). (D) Extended-focus image of 60 z-slices (10-μm slice distance) acquired with a 4× objective. (E) Extended-focus image of 150 z-slices (4-μm slice distance) acquired with a 10× objective. Extended-focus images were generated using Adobe Photoshop CS4. Scale bars: 320 μm (C), 200 μm (D), and 80 μm (E).