HCS was developed in late 1990s to apply high-throughput techniques to the study of intact cellular systems. Over the past decade, HCS has become a mainstream technology in the pharmaceutical industry and is employed at all stages of the drug development pipeline. Increasing public availability of large-scale screening resources, through initiatives such as the Molecular Libraries Program, has also spurred an interest in HCS among academic biomedical researchers. Although HCS offers the potential to capture a vast realm of biological complexity, in practice assays are often constrained by reliance on grossly simplified biological models such as immortalized cell lines and simple culture conditions. Proposals to address these limitations typically focus on transitioning HCS assays into more biologically relevant in vitro systems such as primary cells, stem cells, three-dimensional organotypic models, and mixed co-cultures. A more powerful approach would be applying HCS directly to in vivo models that more accurately mimic normal development, disease pathology, and systemic responses to perturbing agents. Here, we have here demonstrated such an approach.
With only minor modifications, our technology can generate high-resolution 3D reconstructions of any organ or tissue that has been labeled using a suitable chromogenic staining protocol. Of particular relevance for zebrafish researchers, our technology can easily be applied to larvae that have been stained by whole mount in situ hybridization (WISH) using antisense riboprobes to visualize gene expression patterns. This can allow virtually any organ, tissue, or underlying signaling pathway to be interrogated under normal and pathological conditions using HIP. When adapting OPT to new stains, it is essential to use a compatible clearing agent that optimizes the optical properties of the fixed tissue in such a way that light scattering is minimized and transmission in the visible spectra is maximized. In the case of Alcian blue, optical clearing is achieved using a 1% trypsin solution followed by equilibration in PBST. In the case of WISH, optical clearing may require imaging stained larvae in a 2:1 benzyl benzoate:benzyl alcohol (BB:BA) solution, which is compatible with most substrates used for colorimetric detection of alkaline phosphatase activity and allows for almost complete clearing of fixed and yolk-containing tissue.
By coupling high-throughput 3D tomography with automated image analysis, our platform can generate quantitative morphological metrics to create standardized phenomic databases. Phenomic databases produced by HIP can be readily compared between independent laboratories and across multiple studies. This approach allows the full diversity of morphological phenotypes in a given genetic or chemical screen to be documented and subsequently analyzed using data mining and bioinformatics tools. A major strength of HIP is that it allows for unbiased screens in which mechanistic relationships can be determined without a priori
knowledge. Using our platform, we are able to cluster chemicals based on their unique hyperdimensional phenotypic signatures and reveal biologically significant correlations that agree with known mechanisms of action in mammals. Importantly, at no point during this study did we need to introduce metrics that would specifically distinguish between different classes of chemicals. Automated quantitative whole-animal phenotyping technologies such as HIP will be particularly important for large-scale initiatives like the Zebrafish Phenome Project, which aims to systematically characterize loss-of-function mutations in every protein-coding gene30
. In addition, our technology will allow in vivo
characterization of the diversity of large chemical libraries and natural-product libraries with hard-to-predict targets.