High-throughput in vivo vertebrate screening

Abstract

We demonstrate a high-throughput platform for cellular-resolution in vivo chemical and genetic screens on zebrafish larvae. The system automatically loads zebrafish from reservoirs or multiwell plates, and positions and rotates them for high-speed confocal imaging and laser manipulation of both superficial and deep organs within 19 s without damage. We performed small-scale test screening of retinal axon guidance mutants and neuronal regeneration assays in combination with femtosecond laser microsurgery.

Figure 1

Schematic of zebrafish manipulation and imaging platform. Larvae are automatically loaded to the system from either reservoirs or multi-well plates. Reservoirs are connected to the system via fluidic valves and a bubble mixer prevents the larvae from settling. The multiwell plate sits on a motorized x-y stage, which positions individual wells below a larva-loading (red arrow) and a water-supply (blue arrow) tube, both held by a silicone rubber block. The block seals the well surfaces as a piston moves the tubes into the wells. A photodetection system including two LEDs and one high-speed photodiode (PD) discriminates the passage of a larva from air bubbles and debris. Two stepping motors hold a capillary along its axis of rotation; this assembly is mounted on a 3-axis position stage (not shown) and held between an upright microscope for confocal imaging and an inverted microscope for bright-field imaging. A multifocal confocal head with a cooled electron-multiplying (EM) CCD camera and a second large-area CCD connected to the upright port are used for for high-speed confocal and wide-field fluorescence imaging. A high-speed CCD camera connected to the inverted port allows rapid bright-field detection and positioning of larvae. A femtosecond laser beam used for microsurgery is directed to the upper beam path by a dichroic filter and focused on the sample through the objective.

Figure 2

Orientation, imaging, and screening of zebrafish larvae. (a) Schematic (left) showing the midline crossing of Mauthner axons. Right panels show confocal images of EGFP-expressing Mauthner cells at 0° and 15°. Magnified versions of marked insets are shown in each case. Scale bar, 150 μm (50 μm for insets). (b) Widefield fluorescence images illustrating the phenotypes distinguished in a small-scale test screen. Images show GFP-labeled axons of retinal ganglion neurons projecting to the optic tectum in larvae of the indicated genotypes. Images are representative of the phenotypes seen in mutant fish (see Supplementary Figure 3 for more examples). White arrows point to the misguided projections. Scale bar, 150 μm.

Figure 3

Laser microsurgery and neuronal regeneration. (a) Wide-field fluorescence images of GFP-expressing lateral-neuron axon fibres in a 3dpf larva are shown at the indicated times pre- and post- axotomy. (b) The distribution of laser cut sizes is shown (n = 30). Scale bar, 75 μm.

Figure 4

Quantitative assessment of animal health. (a) Survival and abnormality of larvae as a function of initial flow rate (n = 150 for each rate). (b) Appearance time of swimming bladder in screened and control fish (n = 50).