DOI: 10.5061/dryad.nk98sf823
title: 4D light sheet imaging, computational reconstruction, and cell tracking in mouse embryos -- example data (raw .czi and fused .klb light sheet images of mouse E7.5)

description: This dataset contains example .czi (Zeiss Z.1 Lightsheet format) image
 files and corresponding and multi-view fused .klb dataset, containing 3
 hours of time lapse imaging of a mouse embryo at E7.5 / EHF, for
 processing and analysis. To be published with "4-dimension light
 sheet imaging and cell tracking in mouse embryos" in STAR Protocols.
 The dataset is intended for users to rehearse our comprehensive workflow
 for in toto capture, processing, and analysis of multi-view LSFM
 experiments using the ex vivo mouse embryo as a model system of
 development. Our protocol describes imaging on commercial LSFM
 instruments, and recites computational analysis in discrete and accessible
 chunks, using open source software.
description: Imaging was performed on a Lightsheet Z.1 (Zeiss) with incubation
 and dual pco.edge 4.2 cameras (PCO), with 20X/1.0 plan apochromat
 water-dipping detection objective (RI=1.34–1.35) and dual 10X/0.2
 illumination objectives. The specimen tank was filled with culture medium
 and maintained at 37°C and 5% CO2. Mouse post-gastrulation embryos were
 subjected to 3 hours of LSFM imaging at 18-minute intervals, using our
 Zeiss Lightsheet Adaptive Position System (ZLAPS), linked in the key
 reagent table. The resulting .czi files were compressed with bzip2 and
 uploaded to Dryad. Following the protocol as published in
 "4-dimension light sheet imaging and cell tracking in mouse
 embryos" in ___ (2024), fused .klb dataset is included as
 well.
description: # 4D light sheet imaging, computational reconstruction, and cell tracking
 in mouse embryos -- example data (raw .czi and fused .klb light sheet
 images of mouse E7.5) |
 [https://doi.org/10.5061/dryad.nk98sf823](https://doi.org/10.5061/dryad.nk98sf823) | | :--------------------------------------------------------------------------------- | Images obtained on Lightsheet Z.1 (Zeiss) with incubation and dual pco.edge 4.2 cameras (PCO), with 20X/1.0 plan apochromat water-dipping detection objective (RI=1.34–1.35) and dual 10X/0.2 illumination objectives. The specimen tank was filled with culture medium and maintained at 37°C and 5% CO2. Mouse post-gastrulation embryos were subjected to 3 hours of imaging at 18-minute intervals, imaged from the ventral aspect in two views offset by 100, using our Zeiss Lightsheet Adaptive Position System (ZLAPS), linked below. The .czi files were compressed with bzip2 and presented here. Also included is the fused .klb dataset,which is the result of deconvolving, filtering, registering the two views, and image fusion. ## Dataset contains two components: * Example raw .czi images for mouse embryo dataset at E7.5 / EHF, captured in two frontal-lateral oblique views (100° offset). Channel 1 acquired with 488nm laser and GFP emission filter, representing Smarcd3-F6-nGFP. Channel 2 acquired with 561nm laser and RFP emission filter, representing Mesp1-Cre lineage through RCL-H2B-mCherry reporter. * Filename structure: **LSFM_000T.czi.bz2**, where **T** represents timepoint number. Timepoints separated by 18 minutes. These raw .czi files contain multiple views and two channels as described above. * To decompress to native .czi format: `bunzip2 LSFM_000X.czi.bz2` OR `bunzip2 *.bz2` * Fused .klb files for mouse example dataset above. These images were produced by processing images in .czi format with the protocol in our STAR Protocols article, registering them in 4D, and fusing the multiple views for each channel/timepoint combination together. * Filename structure: **t0000T_s0C.klb**, where **T** represents timepoint number, and **C** represents channel number. * These files can be opened individually in Fiji, can be subjected to tracking with (F-)TGMM, or further analyzed/processed using the protocol in our STAR Protocols article. ## Sharing/Access information All software utilized to handle images, generate and process tracking solutions, and export data tables for analysis are deposited at Github, and are publicly available. Repositories for each package are listed and linked below. Custom scripts, configuration files, lookup tables (LUT), intermediate data files, and other resources that were used to carry out the protocol on the example dataset are deposited in the Github repository TrackingFiles linked below. ## Code/Software that should be used to process and analyze this dataset | Fiji (base ImageJ v1.53f) | [https://github.com/fiji/fiji](https://github.com/fiji/fiji) | | :---------------------------------------------------------------------------------- | :----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------- | | ZLAPS (ZEN lightsheet adaptive positioning system) | [https://github.com/mhdominguez/ZLAPS](https://github.com/mhdominguez/ZLAPS) | | F-TGMM v2.5 | [https://github.com/mhdominguez/F-TGMM](https://github.com/mhdominguez/F-TGMM) | | TGMM2SVF and SVF2MaMuT | [https://github.com/mhdominguez/SVF](https://github.com/mhdominguez/SVF) | | LSFM Processing Scripts | [https://github.com/mhdominguez/LSFMProcessing](https://github.com/mhdominguez/LSFMProcessing) | | PSF Generator | [http://bigwww.epfl.ch/algorithms/psfgenerator/](http://bigwww.epfl.ch/algorithms/psfgenerator/) | | Parallel Spectral Deconvolution | [https://sites.google.com/site/piotrwendykier/software/deconvolution/parallelspectraldeconvolution](https://sites.google.com/site/piotrwendykier/software/deconvolution/parallelspectraldeconvolution) | | BigStitcher | [https://github.com/mhdominguez/multiview-reconstruction](https://github.com/mhdominguez/multiview-reconstruction) | | KLB file format | [https://github.com/JaneliaSciComp/keller-lab-block-filetype/](https://github.com/JaneliaSciComp/keller-lab-block-filetype/) | | MaMuT | [https://github.com/mhdominguez/MaMuT](https://github.com/mhdominguez/MaMuT) | | MaMuT script library | [https://github.com/mhdominguez/MaMuTLibrary](https://github.com/mhdominguez/MaMuTLibrary) | | TrackingFiles for handling example data with LSFMProcessing, F-TGMM, SVF, and MaMuT | [https://github.com/mhdominguez/Dominguez-Protocols-2024-TrackingFiles](https://github.com/mhdominguez/Dominguez-Protocols-2024-TrackingFiles) |