Flattening embryos

High-resolution live imaging of embryonic development requires storage and processing of huge amounts of microscopy data, which precludes analysis of a large number of samples. We designed an imaging system that combines SPIM (Selective Plane Illumination Microscopy) with real-time image processing, dedicated to rapid imaging of entire zebrafish embryos. This approach extracts the desired information as images are being acquired and reduces the amount of data by a factor of 240, facilitating data storage, visualization and further analysis. Processing of data in real-time empowers us to obtain a high sample count, opening the doors to high-throughput developmental biology.

HFSP Career Development Award holder Jan Huisken and HFSP Program Grant holder Ingo Roeder and colleagues
authored on Thu, 09 January 2014

Fluorescence microscopes are generally designed to deliver a flat image of a single planar section in the sample. By taking a series of digital snapshots, a three-dimensional image of the specimen is acquired. Such a dataset is by default cuboidal, unnecessarily large and offers no dedicated way of visualizing the tissues in their geometrical context, which makes further processing slow and laborious. We found that image transformations tailored to the shape of the sample can be performed in real time to efficiently compress the data stream from the microscope.

Fig. 1: Cell segmentation (top half) and flow analysis (streamlines; bottom half) show characteristic migration patterns of endodermal cells during gastrulation.

The shape of an early zebrafish embryo can be approximated by a sphere. On its surface, the endodermal cells form a single cell layer. To visualize dynamics of endoderm cells, we exploited the spherical geometry of the embryo to generate a radial maximum intensity projection. Taking insights from cartography, a 2D map of this data is created to visualize the entire tissue in a single image. Our radial projections are computed in real time during acquisition without saving any raw image data, which not only reduces the amount of data from Terabytes to Gigabytes but also offers visualization and evaluation of cell migration in the context of whole embryos.

Fig. 2:  Spatial orientation of a Tg(Sox17:EGFP) embryo in 3D and on the final map projection. Tg(Sox17:EGFP) labels all endoderm cells and DFCs. A, anterior; P, posterior; V, ventral; DFC, dorsal forerunner cells. 

In quantitative developmental biology, high sample count is extremely desirable. Our 4-lens SPIM setup captures two opposite views in one scan of the sample through the light sheet. Combining this with real-time data processing, a panoramic view of the entire embryo is obtained within 10 seconds. This extremely fast and efficient system enables us to image many embryos in parallel, and directly compare embryos of different genotypes. At the same time, merging data from many samples brings forth patterns that are not apparent from single datasets. Using this imaging pipeline, we imaged Tg(Sox17:EGFP) embryos in wildtype and Cxcr4a MO backgrounds. Our results show that loss of Cxcr4a function affects the early distribution and subsequently the end-point position of endodermal cells, without affecting directionality of migration.

In summary, this powerful approach enables us to simultaneously visualize and correlate single cell dynamics and processes at the level of whole tissues. Our study illustrates the requirement for global imaging and multi-sample analysis for quantitative developmental biology.

Reference

High-speed panoramic light-sheet microscopy reveals global endodermal cell dynamics.Schmid B, Shah G, Scherf N, Weber M, Thierbach K, Campos CP, Roeder I, Aanstad P, Huisken J. Nat Commun. 2013 Jul 24;4:2207. doi: 10.1038/ncomms3207.

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