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Review
. 2021 Sep 15;148(18):dev199369.
doi: 10.1242/dev.199369. Epub 2021 Oct 1.

Tissue clearing and 3D imaging in developmental biology

Alba Vieites-Prado  1 , Nicolas Renier  1
Affiliations
Review

Tissue clearing and 3D imaging in developmental biology

Alba Vieites-Prado et al. Development. .

Abstract

Tissue clearing increases the transparency of late developmental stages and enables deep imaging in fixed organisms. Successful implementation of these methodologies requires a good grasp of sample processing, imaging and the possibilities offered by image analysis. In this Primer, we highlight how tissue clearing can revolutionize the histological analysis of developmental processes and we advise on how to implement effective clearing protocols, imaging strategies and analysis methods for developmental biology.

Keywords: 3D imaging; Light-sheet microscopy; Tissue clearing.

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Conflict of interest statement

Competing interests The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Genealogy of tissue-clearing methods applied to developmental biology. The tree illustrates the first publication of each method. Only methods compatible with embryology are listed. Aqueous-based methods (blue); hydrogel crosslink-based methods (green); organic-solvent-based methods (purple). Arrows indicate a derived method. See Table 1 for more details.
Fig. 2.
Fig. 2.
Common modules used in tissue-clearing methods. The six modules of tissue-clearing protocols are indicated, with examples of chemicals used in the different protocols. *, reduction or instability of the genetically-encoded protein (XFP) fluorescence signal; **, complete loss of XFP fluorescence; aqueous-based methods (blue); hydrogel crosslink-based methods (green); organic-solvent-based methods (purple).
Fig. 3.
Fig. 3.
Optimization of light-sheet microscopy for embryology: maximizing the resolution. (A) Schematic of light-sheet illumination and light collection (example of the ultramicroscope). (B) Importance of the numerical aperture (NA) for the light-sheet generation and its effects on the homogeneity of the optical plane and the size of the field of view. Examples are given with vascular labeling: at low axial resolution, the vessels appear continuous, whereas at high resolution they are shown with their cross-sections as points. (C) Types of illumination. Ultramicroscopes incorporate multiple angles or dual-side illumination to reduce the shadows and improve the illumination width in large samples. The MesoSPIM uses dual-sided illumination and horizontal scanning to speed up the system efficiently. Finally, the Zeiss systems use a pivoted illumination system to reduce the shadows and incorporate a mechanical arm to rotate the sample.
Fig. 4.
Fig. 4.
Possible optimization of tissue-clearing protocols in early and late embryos. Standard protocols should be followed by default, but in some situations, optimizations are needed to reveal a weak signal or difficult staining. This figure provides some suggestions for possible modifications to enhance the signal of a few complex organs.
Fig. 5.
Fig. 5.
Examples of applications of tissue clearing to developmental biology. (A-D) Examples of image analysis. (A) Imaris surface segmentation of a GW8 human embryo, showing the urogenital system. This type of semi-automated segmentation allows researchers to highlight structures of interest, even in complex or noisy data (Belle et al., 2017). (B) Automated axon segmentation of cortico-fugal projections of the barrel cortex with TrailMap, annotated to the mouse Allen Brain Atlas template (https://allensdk.readthedocs.io/en/latest/reference_space.html) with the regional color code of the Allen Brain Atlas. The brain reference template is shown in gray for orientation. (C) 3D view and sagittal projection (300 µm thickness) of a P2 mouse brain stained for the vasculature [CD31/podocalyxin (blue); Sm22 (pink)] with iDISCO+. (D) Coronal slice of the vascular graph from C obtained through an automated segmentation using TubeMap (ClearMap2). Blood vessels were automatically segmented from the 3D scan obtained in C, and embedded into a graph representation, with vessels coded as edges and bifurcations as nodes. The image shows a 3D coronal slice through the reconstructed graph, which reveals the orientations and densities of vessels across different brain regions. Images in B-D kindly provided by Grace Houser and Elisa de Launoit (Paris Brain Institute, France; unpublished). (E-I) Applications of tissue clearing to evo-devo studies. (E) Short-tailed fruit bat (Carollia perspicillata) in developmental stage 19, stained with Alcian Blue and cleared with BABB, imaged with a bright-field stereomicroscope. Image kindly provided by Idoia Quintana-Urzainqui, Paola Bertucci, Peter Warth, Chi-Kuo Hu and Richard Behringer (University of Texas MD Anderson Cancer Center, TX, USA; unpublished). (F) 3D view of an African house snake embryo (8 days post oviposition) stained for Robo3 (red) and βIII-Tub (green) with 3DISCO and imaged with a light-sheet ultramicroscope. Image kindly provided by Alain Chédotal (The Vision Institute, France), produced as described in Friocourt et al. (2019). (G) Longfin inshore squid and Hawaiian bobtail squid stained for acetylated tubulin with the DEEPClear protocol. Adapted from Pende et al. (2020). (H) Fruit fly (Drosophila melanogaster) pupa expressing GFP in sensory neurons, cleared with FlyClear (Pende et al., 2018). Images (G,H) kindly provided by Marco Pende (Vienna University of Technology, Austria). (I) 3D light-sheet image of the brain of a spotted gar injected in the eyes with two cholera toxin β tracers (Vigouroux et al., 2021). Scale bars: 400 µm.
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