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1 Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology (MIT)
In animals, most of the complex physiological response mechanisms triggered by genetic mutations and biologically active molecules cannot be predicted using cell culture models, while animal level studies are progressing slowly. In this study, we used a micro-resolution high-throughput optical projection tomography system combined with an efficient large-particle flow sorting system ( Union Biometrica ) to achieve an overall multi-dimensional scan of vertebrates . With unprecedented speed and detail, the system automatically captures hundreds of unique morphological features and complex phenotypes in translucent zebrafish larvae at three dimensional levels. By aggregating and quantifying multiple phenotypic features, we are able to simultaneously detect and classify subtle changes in multiple biological processes. We refer to this method as an in vivo multidimensional phenotypic study . To validate the enormous role of multidimensional phenotypic research methods in vivo, we used 200 independent morphometric parameters to analyze the effects of dozens of teratogenic agents (which can cause abnormal cartilage formation) and identify them with known mechanisms of teratogens. Similarities and differences.
High-throughput screening for morphological changes caused by many factors is essential for modern biological research and drug screening. In the current study, effective drugs obtained at high-throughput screening ( HTS ) at the cytological level were not satisfactory in further animal-level studies and clinical studies. Even the most complex in vitro models do not fully reveal the complexity and composition of organ formation. In order to overcome the shortcomings of in vitro models in drug screening and other applications, more and more chemical drugs use zebrafish (Danio rerio) individuals for screening research. Zebrafish larvae are currently used in vertebrate models for drug screening. Large-scale genetic level screening for zebrafish has been underway for decades, but because the results read are often not quantified or limited to a limited number of parameter records, a more complete solution is needed.
To hundreds of phenotypic changes recorded quantitative analysis, and high content screening (the HCS), we apply a high throughput of optical projection tomography system (high-throughput optical projection tomography system , OPT), the system combines efficient The large particle flow sorting system ( Union Biometrica ), combined with special image processing algorithms, is compatible with a wide range of microscopes to achieve microscopic resolution of 3D organs or complex morphological parameters. This technology helps us quickly obtain information on hundreds of unique morphological features and complex phenotypes from a large number of animal samples. 
A New Method for High-throughput Study of Multidimensional Phenotypes of Vertebrates
Nature Communications 4, February 12, 2013, Article number:1467, doi:10.1038/ncomms2475
Carlos Pardo-Martin, Amin Allalou, Jaime Medina, Peter M. Eimon, Carolina Wählby Mehmet Fatih Yanik
2 Division of Health Sciences and Technology, MIT,
3 School of Engineering and Applied Sciences, Harvard University
4 Centre for Image Analysis, Science for Life Laboratory, Uppsala University, Sweden.
5 Imaging Platform, Broad Institute of Massachusetts Institute of Technology and Harvard
6 Department of Biological Engineering, MIT
Keywords :
Large particle flow sorting system, vertebrate, zebrafish (zebrafish , Danio rerio) , in vivo multidimensional phenotypic study, teratogen, high-throughput optical projection tomography system, high-throughput screening ( HTS ), high content Screening ( HCS ), OPT system, 3D reconstruction
Summary:
background:
results and analysis:
1. Union Biometrica BioSorter ® Flow Cytometry (New High-Throughput Sorting OPT System) : Group of Jet Systems and Microscopic Systems. BioSorte ® does not require pre-treatment of zebrafish samples, avoiding the most time-consuming steps in conventional OPT technology .
2. Image correction process: Obtain accurate tomographic reconstruction images through multi-step image software correction.
3. Three-dimensional reconstruction and evaluation of craniofacial malformations : Each zebrafish larvae recorded up to 320 frames of images and visualized the complex phenotype of zebrafish larvae by three-dimensional reconstruction techniques . The chromatogram of the image shows the craniofacial skeletal changes of the 5dpf zebrafish larvae treated with three different drugs. The three drugs are: retinoic acid, cyclopamine, and flusilazole.
4. Drug screening research example: effects of different concentrations of cyclopamine on the growth of three cartilage
5. In vivo multi-dimensional phenotypic analysis: In order to fully quantify the phenotypic variation of zebrafish larvae , the researchers calculated 202 independent indicators for reconstruction of 3D phenotype maps. These parameters include length, 3D orientation, surface area, and various bone volumes.
6. Array distribution of phenotypic changes in zebrafish larvae : Each row represents the effect of a drug on zebrafish larvae . The test results are expressed in color, red indicates that the phenotype of larvae is decreased relative to untreated larvae, and green indicates increased expression.
in conclusion:
Traditional drug high content screening ( HSS ) technology is limited to simplified biological models, such as cell lines that are wirelessly proliferated, and traditional techniques face numerous problems when performing HCS studies using primary cells, in vivo animal models, and the like. This study used a micro-resolution high-throughput optical projection tomography system combined with an efficient large particle flow sorting system ( Union Biometrica ) to achieve an overall multi-dimensional scan of vertebrates . The present technology can provide 3D reconstruction images of vertebrate organs and tissues. For the study of zebrafish larvae , the present technology can provide high-content screening ( HCS ) for individual levels of animals by providing researchers with various phenotypic characteristics of individual samples . Provides a viable technology platform.
Related introduction:
This is the only fully automated high-throughput sorting system on the market that is capable of sorting 1-1500um bioparticles. His tubing is large in diameter and can be used to sort large volumes of biological particles, so it can be applied to small model organisms such as zebrafish, nematodes, fruit flies, etc. It can also be applied to large volume cells: embryonic stem cells. Islet cells, hepatocytes, cell clusters, etc., can also be used for high-throughput drug screening of pharmaceutical compounds. The technical core of this instrument is the airflow sorting device, which sorts the particles by airflow. This method is very gentle, ensuring the activity and integrity of the collected biological organisms or sensitive substances.
Literature for zebrafish research by the Union Biometrica flow sorting system:
1.Presenting VAST BioImagerTM: A new modular, expandable platform to automate the orientation of 2-7 day old zebrafish larvae for imaging
2.Development and Validation of an Automated High-Throughput System for Zebrafish In Vivo Screenings
3.Fully automated cellular-resolution vertebrate screening platform with parallel animal processing
4.Fully automated cellular-resolution vertebrate screening platform with parallel animal processing
5.A High-Throughput Screen for Tuberculosis Progression
6.Zebrafish high throughput screening using robotic injection technology
7.High-throughput in vivo vertebrate screening.
8.Zebrafish High-Throughput Screening of Innate Immune Responses.
9.A High-Throughput Assay To Measure Whole Body Metabolic Rate Using Zebrafish Larvae.
10.A Whole-Animal Microplate Assay for Metabolic Rate Using Zebrafish
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