Small size, optical transparency of complex organs, and ease of culture make the zebrafish (Danio Rerio
) larva an ideal organism for large-scale in vivo
genetic and chemical studies of many processes that cannot be replicated in vitro
. Zebrafish models of several human diseases have been developed1-5
. Lead compounds discovered by screening chemical compound libraries for efficacy in zebrafish disease models have been useful for pharmaceutical discovery due to the high level of conservation of drug activity between mammals and zebrafish1-5
Visualizing most zebrafish organs requires manipulating and properly orienting the animals. Even with confocal or two-photon microscopy, optical access is often impeded by pigmentation, by intervening organs such as eyes and heart, or by the highly autofluorescent yolk sac. Current methods to address these challenges involve treatment with the toxic chemical phenylthiourea to suppress pigmentation6
, manually transferring animals from multiwell plates or reservoirs, and manually orienting them in viscous media such as agar. These processes are too slow and unreliable for high-throughput screens To improve the throughput and complexity of zebrafish screens, we developed the vertebrate automated screening technology (VAST) that automatically manipulates and images animals on the fly, eliminating the need for any manual handling ( and Online Methods).
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 (more ...)
Each cycle of VAST includes the following steps: loading, detection, positioning, orienting, focusing, imaging, laser manipulation, and dispensing. Such an automated system makes both genetic and pharmaceutical whole-organism screens possible7
(Supplementary Fig. 1
). During loading, the system extracts larvae either from a multiwell plate or a reservoir. A high-speed photodetection system composed of a photodiode and two LEDs discerns the entry of larvae into the loading tube. The photodiode senses transmitted light from one LED and scattered light from the other LED. By simultaneously monitoring both the transmission and scattering signals, the system discriminates larva from air bubbles and debris with 100% reliability (n
= 1000). After loading and photodetection, the larva moves from the larger loading tube into a capillary8,9
positioned within the field-of-view (FOV) of an optical imaging and manipulation system. The capillary has a refractive index similar to water, allowing the use of high numerical-aperture (NA) water-immersion objectives that require short working distances. Using a fast camera and an automated image processing algorithm, the larva is coarsely positioned by the syringe pump. Next, a 3-axis stage automatically moves the capillary assembly to precisely position the larva’s head at the center of the FOV. The larva is then rotationally oriented by a pair of stepper motors. Thus, larvae can be arbitrarily positioned and oriented. In addition, larvae can be automatically re-oriented, thus allowing visualization of organs from multiple angles. At the end of the cycle, animals can be dispensed back into either individual wells or larger containers by executing the loading process in reverse. See Online Methods and Supplementary Fig. 2
Larvae are imaged through two objective lenses: an upright, high-resolution water-immersion objective and an inverted air objective. This allows both wide-field fluorescence imaging and high-resolution confocal microscopy. Imaging most phenotypes requires larvae to be oriented at specific angles. For instance, the midline crossing of the Mauthner motor neuron axons that project into spinal cord is only visible when directly observed from the hindbrain (, 0°). At less favorable orientations, the structure is obscured. We performed a small-scale test screen on a similar phenotype – the midline crossing of retinal axon projections to the optic tectum ( and Supplementary Fig. 3
). Screening for retinal axon misguidance mutants had previously led to the discovery of astray
mutation in robo2
. Homozygous astray
(ast/ast) zebrafish fail to exhibit proper midline crossing, while the projections are normal in heterozygous (ast/+) zebrafish. Using our system, we were able to distinguish wild type larvae from robo2
with a sensitivity of 100% and specificity of 98.8% for a 96-well plate with 83 randomly seeded mutants. The 1.20% false negative error in identification of ast/ast animals was due to the rare cases of mutants with strong phenotypic similarity to wild type. The system could thus be used for large-scale chemical screens for small molecules that rescue such misguidance.
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 (more ...)
In conjunction with sample positioning and orientation, VAST also allows in vivo
optical manipulations such as localized activation of fluorescent reporters and ion channels, uncaging of compounds, and femtosecond laser microsurgery12
with subcellular precision. We illustrate the use of the system to study neuronal regeneration13
following injury by laser microsurgery14
(). The lateral-neuron axon fiber bundle projecting along the trunk of a larva is visible when the larva is laterally oriented. The bundle is axotomized by focusing near-infrared femtosecond laser pulses on it. This process is semi-automated15
: The user selects a cell body by clicking on a graphical user interface. An algorithm measures the user-specified distance from the cell body to the point of axotomy along the axon and the position stage automatically moves the selected axonal region to the focal spot of the laser for axotomy. The regenerating axonal fibers are shown at 18 and 24 hours post-axotomy in . The laser pulses are delivered with high precision (1.7 μm)12
, and the subsequent response of the tissue to the laser (i.e. immediate retraction of nerve fibers) show some variability, leading to a distribution of cut sizes (8.1 ± 5.5 μm, n
= 30, ±s.d.) (). 100% of the animals recovered from surgery within 30 minutes with no apparent morphological abnormality, and 100% of the animals survived 24 hours post surgery (n
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 (more ...)
A complete cycle of loading, positioning, cellular-resolution imaging, and dispensing an animal takes less than 16 seconds (). Axotomy requires an additional 2 seconds. Screening an entire multiwell plate took 31.85 minutes with an average of 19.9 seconds per well, which includes the additional interval for retracting, moving and inserting the loading apparatus, and sealing the wells. Animals can be loaded and imaged multiple times for time-lapse assays. Performed manually, assays of similar complexity require about 10 minutes per animal, and the assays are error-prone.
Average duration of screening steps
Zebrafish larvae are delicate and particularly susceptible to injury by tearing at sharp edges or by deformation. The most significant potential for damaging larvae occurs as they enter the loading tube at the high aspiration rates needed to achieve the desired throughput. To lessen this risk, the flow is started at a low initial rate and increased at an acceleration of 42 μl·s−2 until a larva is detected by the high-speed photodetector. The maximum flow rate is limited to 330 μl·s−1. At this maximum rate, no injury occurs while larvae are traveling within the tubing. After a larva is detected by the photodetector, the control software decreases the aspiration rate to 83 μl·s−1 to allow automated recognition by the camera.
We assessed the health of 450 larvae screened at 2 dpf at three different initial aspiration rates (). The assessment was based on both functional and morphological criteria. At all flow rates, heartbeat and touch response matched those of controls. Tearing of yolk was never observed (n = 450). At the highest initial flow rate of 330 μl·s−1, 2.0% of the animals exhibited morphological abnormality. With a slightly slower initial aspiration rate (increasing the screening time by approximately 1 second), all health criteria matched those of controls (). Post-manipulation developmental delay was measured by monitoring the time of appearance of the swimming bladder. There was no significant difference (One way ANOVA, P = 0.94, 3 independent experiments with n = 50 for each group) between the development of larvae that were manipulated by the system and control animals, even at the highest flow rates (). Among the different larval age groups tested (i.e. 2-7 dpf, n > 100 per age), no difference in health assessment was observed.
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).
In summary, we demonstrate a high-throughput vertebrate screening platform with cellular-resolution imaging and manipulation capabilities that should permit large-scale in vivo study of complex processes such as organ development, neural degeneration and regeneration, stem cell proliferation, cardiovascular, immune, endocrine, and nervous system functions, pathogenesis, cancer, and tissue specificity and toxicity of drugs.