Small molecule high-throughput screenings are essential for the fields of drug discovery and toxicology. Such screenings performed on whole animals are more physiologically relevant leading to more predictive results. However, due to challenges in automation, high costs and absence of miniaturized solutions for animal-based experiments, high throughput screenings based on animal models are still in its infancy. Here a platform for miniaturized high throughput whole-organism screenings is presented. The new platform is based on patterns of hydrophilic spots separated by superhydrophobic borders. The difference in wettability of spots and borders generates the effect of discontinuous dewetting and allows for formation of arrays of microdroplets that incorporate single fish embryos. Due to the flat border-less nature of the platform, the system is compatible with single-step collection of embryos and pipetting-free parallel addition of chemical libraries using the "sandwiching method." The system is miniaturized and allows for incubation of embryos in volumes as low as 5 µL. Finally, the platform realizes surface tension based immobilization of single embryos inside of each microcompartment and permits high-throughput microscopic analysis directly on the platform. Thus, this method combines the advantages of microarrays, such as highthroughput and simplicity, with the power of in vivo experiments. Fish-Microarrays
Over the last years, the zebrafish (Danio rerio) has become a key model organism in genetic and chemical screenings. A growing number of experiments and an expanding interest in zebrafish research makes it increasingly essential to automatize the distribution of embryos and larvae into standard microtiter plates or other sample holders for screening, often according to phenotypical features. Until now, such sorting processes have been carried out by manually handling the larvae and manual feature detection. Here, a prototype platform for image acquisition together with a classification software is presented. Zebrafish embryos and larvae and their features such as pigmentation are detected automatically from the image. Zebrafish of four different phenotypes can be classified through pattern recognition at 72 hours post fertilization (hpf), allowing the software to classify an embryo into two distinct phenotypic classes: wild-type versus variant. The zebrafish phenotypes are classified with an accuracy of 79-99% without any user interaction. A description of the prototype platform and of the algorithms for image processing and pattern recognition is presented.
Quantifying cardiac functions in model organisms like embryonic zebrafish is of high importance in small molecule screens for new therapeutic compounds. One relevant cardiac parameter is the fractional shortening (FS). A method for semi-automatic quantification of FS in video recordings of zebrafish embryo hearts is presented. The software provides automated visual information about the end-systolic and end-diastolic stages of the heart by displaying corresponding colored lines into a Motion-mode display. After manually marking the ventricle diameters in frames of end-systolic and end-diastolic stages, the FS is calculated. The software was evaluated by comparing the results of the determination of FS with results obtained from another established method. Correlations of 0.96 < r < 0.99 between the two methods were found indicating that the new software provides comparable results for the determination of the FS.
The zebrafish (Danio rerio) is a well-established vertebrate model organism. Its embryos are used extensively in biology and medicine to perform chemical screens to identify drug candidates or to evaluate teratogenicity and embryotoxicity of substances. Behavioral readouts are increasingly used to assess the effects of compounds on the nervous system. Early stage zebrafish show characteristic behavioral features at stages between 30 and 42 hours post fertilization (hpf) when exposed to a short and bright light flash. This so-called Photomotor Response (PMR) is a reaction of the nervous system of the fish and can be used as a marker in screenings for neuroactive chemicals. To probe a broad and diverse chemical space, many different substances have to be tested and repeated observations are necessary to warrant statistical significance of the results. Although PMR-based chemical screens must use a large number of specimens, there is no sophisticated, automated high-throughput platform available which ensures minimal human intervention. Here we report a PMR platform that was developed by combining an improved automatic sample handling with a remotely controllable microscope setup and an image analysis pipeline. Using infrared illumination during automatic sample preparation, we were able to eliminate excess amounts of visible light that could potentially alter the response results. A remotely controlled microscope setup allows us to screen entire 96-well microtiter plates without human presence that could disturb the embryos. The development of custom video analysis software, including single egg detection, enables us to detect variance among treated specimens and extract easy to interpret numerical values representing the PMR motion. By testing several neuroactive compounds we validated the workflow that can be used to analyze more than one thousand zebrafish eggs on a single 96-well plate.
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