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Optical tomography complements light sheet microscopy for in toto imaging of zebrafish development.

Bassi A, Schmid B, Huisken J - Development (2015)

Bottom Line: Fluorescently labeled structures can be spectrally isolated and imaged at high resolution in living embryos by light sheet microscopy.We found that the bright-field contrast of unstained specimens in a selective plane illumination microscopy (SPIM) setup can be exploited for in vivo tomographic reconstructions of the three-dimensional anatomy of zebrafish, without causing phototoxicity.We report multimodal imaging of entire zebrafish embryos over several hours of development, as well as segmentation, tracking and automatic registration of individual organs.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden 01307, Germany Politecnico di Milano, Dipartimento di Fisica, Milano 20133, Italy.

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Multimodal imaging. (A-D) Sagittal (A,B) or transverse (C,D) slice of a transgenic Tg(neurog1:GFP)×Tg(kdrl:rasCherry) zebrafish (3.5 dpf) visualized with (A,C) SPIM or (B,D) SPIM (red/green) combined with optical tomography (gray). The dotted lines in the sagittal sections indicate the position of the transverse section and vice versa. (E-F′) Lateral (E) and dorsal (F) views of the sample created with weighted intensity projection. The boxed regions in E,F are enlarged in E′,F′ to illustrate the fine details in the data. Scale bars: 100 µm.
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DEV116970F2: Multimodal imaging. (A-D) Sagittal (A,B) or transverse (C,D) slice of a transgenic Tg(neurog1:GFP)×Tg(kdrl:rasCherry) zebrafish (3.5 dpf) visualized with (A,C) SPIM or (B,D) SPIM (red/green) combined with optical tomography (gray). The dotted lines in the sagittal sections indicate the position of the transverse section and vice versa. (E-F′) Lateral (E) and dorsal (F) views of the sample created with weighted intensity projection. The boxed regions in E,F are enlarged in E′,F′ to illustrate the fine details in the data. Scale bars: 100 µm.

Mentions: To assess the value of the multimodal imaging, we recorded and superimposed optical tomography and SPIM volumes of a Tg(neurog1:GFP)×Tg(kdrl:rasCherry) embryo expressing green fluorescent protein (GFP) in neuronal cells and mCherry in endothelial cells. The fluorescence detected with SPIM was sparse and difficult to assign to any anatomical region (Fig. 2A,C). Only the additional optical tomography information placed the fluorescence data in the proper anatomical context within the zebrafish in all of the reconstructed sections (Fig. 2B,D; supplementary material Fig. S3) and in the entire volume (Fig. 2E,F; supplementary material Movie 4). Registration of the two modalities was straightforward, as both datasets were acquired using the same detector in short succession (supplementary material Fig. S4). Only the relative z-position of the reconstructed volume had to be determined by registration (supplementary material Fig. S5). We conclude that the multimodal system provides a comprehensive visualization of the specimen, making it well suited for in vivo localization analysis of gene expression in the entire zebrafish.Fig. 2.


Optical tomography complements light sheet microscopy for in toto imaging of zebrafish development.

Bassi A, Schmid B, Huisken J - Development (2015)

Multimodal imaging. (A-D) Sagittal (A,B) or transverse (C,D) slice of a transgenic Tg(neurog1:GFP)×Tg(kdrl:rasCherry) zebrafish (3.5 dpf) visualized with (A,C) SPIM or (B,D) SPIM (red/green) combined with optical tomography (gray). The dotted lines in the sagittal sections indicate the position of the transverse section and vice versa. (E-F′) Lateral (E) and dorsal (F) views of the sample created with weighted intensity projection. The boxed regions in E,F are enlarged in E′,F′ to illustrate the fine details in the data. Scale bars: 100 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4352980&req=5

DEV116970F2: Multimodal imaging. (A-D) Sagittal (A,B) or transverse (C,D) slice of a transgenic Tg(neurog1:GFP)×Tg(kdrl:rasCherry) zebrafish (3.5 dpf) visualized with (A,C) SPIM or (B,D) SPIM (red/green) combined with optical tomography (gray). The dotted lines in the sagittal sections indicate the position of the transverse section and vice versa. (E-F′) Lateral (E) and dorsal (F) views of the sample created with weighted intensity projection. The boxed regions in E,F are enlarged in E′,F′ to illustrate the fine details in the data. Scale bars: 100 µm.
Mentions: To assess the value of the multimodal imaging, we recorded and superimposed optical tomography and SPIM volumes of a Tg(neurog1:GFP)×Tg(kdrl:rasCherry) embryo expressing green fluorescent protein (GFP) in neuronal cells and mCherry in endothelial cells. The fluorescence detected with SPIM was sparse and difficult to assign to any anatomical region (Fig. 2A,C). Only the additional optical tomography information placed the fluorescence data in the proper anatomical context within the zebrafish in all of the reconstructed sections (Fig. 2B,D; supplementary material Fig. S3) and in the entire volume (Fig. 2E,F; supplementary material Movie 4). Registration of the two modalities was straightforward, as both datasets were acquired using the same detector in short succession (supplementary material Fig. S4). Only the relative z-position of the reconstructed volume had to be determined by registration (supplementary material Fig. S5). We conclude that the multimodal system provides a comprehensive visualization of the specimen, making it well suited for in vivo localization analysis of gene expression in the entire zebrafish.Fig. 2.

Bottom Line: Fluorescently labeled structures can be spectrally isolated and imaged at high resolution in living embryos by light sheet microscopy.We found that the bright-field contrast of unstained specimens in a selective plane illumination microscopy (SPIM) setup can be exploited for in vivo tomographic reconstructions of the three-dimensional anatomy of zebrafish, without causing phototoxicity.We report multimodal imaging of entire zebrafish embryos over several hours of development, as well as segmentation, tracking and automatic registration of individual organs.

View Article: PubMed Central - PubMed

Affiliation: Max Planck Institute of Molecular Cell Biology and Genetics, Dresden 01307, Germany Politecnico di Milano, Dipartimento di Fisica, Milano 20133, Italy.

Show MeSH
Related in: MedlinePlus