Computational protocol: Expansion microscopy of zebrafish for neuroscience and developmental biology studies

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Protocol publication

[…] Each figure panel constitutes a single plane from a z-stack, where the area of interest was cropped out of the field of view using Fiji (), or a maximal intensity projection, as indicated in the figure legends. The brightness and contrast of individual channels were adjusted in ImageJ (NIH) after cropping the area of interest. The STED preexpansion images shown in (first and third panels) and used in were deconvolved using Huygens (Scientific Volume Imaging). Tracing of cellular processes (shown in ) was performed using Imaris. This tracing algorithm is intensity-based. First, start and end points are detected, and then these points are connected with traces following the image intensity. is a maximal intensity projection of four stacks acquired separately and stitched together using Fiji’s pairwise stitching plug-in (). The data shown in and were cropped from stacks following illumination correction using CIDRE () and deconvolution using Huygens. Since the illumination model is dictated by the microscope optics, a single illumination model was learned using CIDRE by pooling the datasets together, and then this same model was used for the correction of both pre- and postexpansion datasets. After the application of illumination correction, a dataset-specific threshold was manually set according to the characteristic background noise level. Both pre- and postexpansion datasets were then deconvolved using the exact same procedure and parameters. […]

Pipeline specifications

Software tools ImageJ, Imaris, Stitching, CIDRE
Applications Bright-field microscopy, Microscopic phenotype analysis
Organisms Danio rerio, Drosophila melanogaster
Chemicals Glycine