Fig. 1.

Multi-thread Vertebrate Automated Screening Technology (VAST). The platform consists of three subsystems that operate simultaneously: loading, imaging, and unloading. Larvae are automatically loaded to the platform from individual wells of a mesh-filter multiwell plate positioned by a motorized x y stage. The mesh-filter insert allows easy transfer of larvae into the system. In order to avoid a drop in the fluid level within multiwell plate (while larva is being aspirated by the loading nozzle), a circulator is set nearby the loading nozzle (inset A) that consists of a fluid source and an aspirator where the tip of the aspirator is slightly elevated with respect to the tip of the fluid source. A zebrafish discriminator with a brightfield and a fluorescence photodetection system (inset B) discriminates the passage of fluorescent larvae from non-fluorescent ones, air bubbles and debris. Two step motors hold a capillary immersed in a water bath along its axis of rotation; this assembly is mounted on a three-axis position stage (not shown) and held between an upright microscope and an inverted microscope. A multifocal confocal head with a cooled electron-multiplying charge-coupled device (EM-CCD) camera and a second large-area charge-coupled device (CCD) are used for high-speed confocal and wide-field fluorescence imaging, respectively. A high-speed CCD camera connected to the inverted microscope allows rapid bright-field imaging for positioning and orienting the larvae.