RESUMEN
Systems biology methods, such as transcriptomics and metabolomics, require large numbers of small model organisms, such as zebrafish embryos. Manual separation of mutant embryos from wild-type embryos is a tedious and time-consuming task that is prone to errors, especially if there are variable phenotypes of a mutant. Here we describe a zebrafish embryo sorting system with two cameras and image processing based on template-matching algorithms. In order to evaluate the system, zebrafish rx3 mutants that lack eyes due to a patterning defect in brain development were separated from their wild-type siblings. These mutants show glucocorticoid deficiency due to pituitary defects and serve as a model for human secondary adrenal insufficiencies. We show that the variable phenotypes of the mutant embryos can be safely distinguished from phenotypic wild-type zebrafish embryos and sorted from one petri dish into another petri dish or into a 96-well microtiter plate. On average, classification of a zebrafish embryo takes approximately 1 s, with a sensitivity and specificity of 87% to 95%, respectively. Other morphological phenotypes may be classified and sorted using similar techniques.
Asunto(s)
Animales de Laboratorio/clasificación , Embrión no Mamífero , Mutación , Fenotipo , Pez Cebra/clasificación , Animales , Procesamiento de Imagen Asistido por Computador , Imagen Óptica , Sensibilidad y EspecificidadRESUMEN
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.
Asunto(s)
Evaluación Preclínica de Medicamentos/métodos , Embrión no Mamífero/efectos de los fármacos , Embrión no Mamífero/efectos de la radiación , Procesamiento de Imagen Asistido por Computador/métodos , Pruebas de Toxicidad/métodos , Animales , Humanos , Pez CebraRESUMEN
OBJECTIVE: A variety of medical robots have been developed in recent years. MRI, including MR angiography and morphological imaging, with its excellent soft-tissue contrast is attractive for the development of interventional MRI-guided therapies and operations. This paper presents a telerobotic device for use in CT- and/or MR-guided radiological interventions. A robotic device for precise needle insertion during MR-guided therapy of spinal diseases will be briefly described. MATERIALS AND METHODS: Actuation of robots in an MRI environment is difficult due to the presence of strong magnetic fields. Therefore, the robot was constructed of nonmagnetic materials. The system frame was built from polyether ether ketone (PEEK) and fiber-reinforced epoxy, and actuated using ultrasonic and pneumatic motors. Completely MR-compatible sensors were developed for positioning control. RESULTS: Accuracy evaluation procedures and phantom tests were performed, with the required accuracy of approximately 1 mm being achieved and no significant artifacts being caused by the robotic device during MR image acquisition.