6/24/2023 0 Comments Wings 3d stitchingToday, this acceleration is enabling studies that could have been considered impossible or too ambitious until recently, such as screening complex phenotypes and inter-organ connections or analyzing the development of intricate 3D structures, such as the vascular and neural networks, the digestive tract, lung or any other tubular system. Light-sheet microscopy offered a gain of speed several orders of magnitude over scanning microscopes and the capacity to image very large samples (over 1 cm) however, it required the development of better tissue-clearing protocols because high transparency is paramount for this imaging modality. In 2007, the combination of light-sheet microscopes (or selective plane illumination microscopes) with tissue clearing on large samples ( Dodt et al., 2007) started a race to develop effective tissue-clearing protocols. Aqueous-based methods (blue) hydrogel crosslink-based methods (green) organic-solvent-based methods (purple). Only methods compatible with embryology are listed. The tree illustrates the first publication of each method. Genealogy of tissue-clearing methods applied to developmental biology. These early applications of tissue clearing in embryology enabled a host of studies on the development of the peripheral nervous system or the study of apoptosis during early organogenesis (e.g. Murray's method was combined with bright-field imaging of colorimetric enzymatic reactions or confocal microscopy of fluorescent dyes in cleared embryos of early developmental stages. This method is the foundation of current organic solvent-based protocols ( Fig. 1). In the late 1980s, Andrew Murray modified the method to clarify Xenopus eggs, using benzyl-alcohol and benzyl-benzoate (BABB), also known as Murray's method ( Dent et al., 1989). Werner Spalteholz developed the first tissue-clearing protocol ( Spalteholz, 1911, 1914) using methyl-salicylate and benzyl-benzoate (simplified as MSBB) to turn animals and large organs transparent, enabling the observation of their skeleton and internal organs. ![]() bone) and lipids, then to bleach pigments, and finally to homogenize the refractive index of left-over compounds. The aim of these protocols is to first remove compounds with outlier refractive indices and less informative value, usually water, hydroxyapatite (i.e. Reducing the heterogeneity of the refractive indices in a sample is the key concept behind tissue-clearing technology. This heterogeneity scatters light that, in practice, makes the sample opaque ( Richardson and Lichtman, 2015). The complex composition of biological samples produces a heterogenous refractive index, from 1.33 (water), up to 1.66 (bones), with intermediate indices for proteins and lipids (usually 1.4 to 1.6) ( Susaki and Ueda, 2016). ![]() The refractive index, or index of refraction, of a substance indicates how much it delays light propagation in a given medium compared with in a vacuum. Tissue-clearing techniques aim to re-establish a straight light path through the tissues by partially removing the components that deviate (scatter) or absorb light. From the 19th century, the studies of fertilization, zygotic activation, cleavage, gastrulation, neurulation and organogenesis have been performed in a host of model species drawing from the large pool of transparent embryos found in Protostomia, echinoderms, amphibians, teleosts and birds.Īlthough the early stages of embryogenesis can be amenable to direct observation, as the embryos develop the lipid content, the secretion of fibrous proteins in the extracellular matrix and accumulation of pigment perturb the propagation of light, turning the specimen opaque and, therefore, hampering the study of middle- to late-developmental stages in intact samples. It enables the visualization and manipulation of cells in vivo, preserving their natural interactions in intact organisms. Natural transparency is one of the most desired features of model organisms for developmental studies.
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