ZeBraInspector is a free tool for automatic analysis of 5dpf zebrafish larvae allowing:

  • realignment of the samples for a better comparison between them following screenshot captures ;
  • whole larva and white matter segmentation requesting a lipophilic dye staining ;
  • automatic creation of an excel sheet including results of the volumetric analysis.

Citation: ZeBraInspector, a whole organism screening platform enabling volumetric analysis of zebrafish brain white matter.*
Lempereur S, Machado E, Licata F, Buzer L, Robineau I, Hémon J, Banerjee P, De Crozé N, Léonard M, Affaticati P, Jenett A, Talbot H, Joly JS.
doi: https://doi.org/10.1101/2020.10.26.353656

* If you like and use ZBI, please mention that « This work has benefited from the ZeBraInspector platform of TEFOR PARIS SACLAY (https://tefor.net/portfolio/zebrainspector) »

License: GNU General Public License

Questions and/or inquiries: zbi@tefor.net

NB: A security window might appear when launching the software. If your computer does not run the ZeBraInspector (Fatal error window when launching), please try to install Microsoft Visual C++ 2017.

User guidelines

Please read carefully before starting with ZBI software

Screen resolution: best for 1920×1080 screens. Use large screens.

Configure interface: define numbers of lines and columns. On the right of the top menu bar, select zoom mode. Zoom is performed by clicking on an image.

Snapshot capture: optionnally, tune image parameters by clicking on CH O 1 2 in the bottom menu bar. Click on the snapshot button below each image. Extraction of image may take one minute.

Volumetric analysis results in an excel table: select images with correct segmentations by checking the “selected” checkbox below each image. Click on the volumetry button in the bottom menu bar. During the volumetric analysis, numbers of voxels that are found in each segmentation are counted. Values are stored in a spreadsheet to allow further statistical analysis.

Image format and depth: nd2, mha and tif. Priviledged depth: 12 bit.

Downsampling factor: for rendering, file downsampled four times by default. Better renderings with no downsampling. Adapt for best compromise between sample numbers and sizes and rendering quality.

Segmentation: segmentation must be performed before registration. Select Ref/Dye channel and output channel. Default output: mha. Click tiff if needed.

Image specifications : whole fry needed. 2500X1000X500 voxel images (voxel size around two microns, voxels must be isotropic).  

Optical sections: H, S and T buttons in top menu bar display respectively horizontal, sagittal and transverse views. Moreover, a rotation around the anteroposterior axis can be performed.

Registration: select Ref/Dye channel. Default: samples with depigmented eyes (using H2O2). For pigmentation mutants with native fluorescence, the “pigmented eyes (no H2O2 treatment)” checkbox should be selected.

NB: You can also access the user guide while using the software by clicking on the “?” icon in the main menu.

Additional packages

Two single-channel images (nd2 format) of 5D zebrafish larvae stained with a lipophilic dye using a fast protocol (refer to Fig.2 and Material and Methods in Lempereur et al.)

Ten single-channel images (mha format) of 5D zebrafish larvae used to tune whole-body and white matter segmentation (refer to Fig.6/7 and Material and Methods in Lempereur et al.)

Two dual-channel images (nd2 format) of 5D zebrafish larvae.

Channel 0: lipophilic dye staining of mutant larvae depleted of pigments on the body but with pigmented eyes. No depigmented protocol is applied to these larvae. Hence native fluorescent is preserved, but eyes remain pigmented. To register these images, the “pigmented eyes” option should be selected.

Channel 1: native RFP fluorescent staining in vessels (kdlr promoter). Refer to protocol preserving native fluorescence in Lempereur et al.

Two dual-channel images (nd2 format) of 5D zebafish larvae.

Channel 0: lipophilic dye staining.

Channel 1: anti-HuC antibody staining.  Fish are depigmented by a chemical treatment. Refer to Hu-C immunolabelling protocol in Lempereur et al.

Two three-channel images of 5D zebrafish larvae.

Channel 0: vessels stained in green.

Channel 1: lipophilic dye.

Channel 2: injected red Sindbis virus particle.

To register these images, Channel 1 should be selected as reference channel. Refer to anti-GFP and anti RFP protocol in Material and Methods in Lempereur et al.

A collection of 48 monocanal pictures (DiI staining) to estimate time for loading such big data, for registration, segmentation of whole-body and brain white-matter, and brain volumetry computation.