High-throughput in vivo vertebrate screening.

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.

Sub-wavelength nanofluidics in photonic crystal sensors.

We introduce a novel sensor scheme combining nano-photonics and nano-fluidics on a single platform through the use of free-standing photonic crystals. By harnessing nano-scale openings, we theoretically and experimentally demonstrate that both fluidics and light can be manipulated at sub-wavelength scales. Compared to the conventional fluidic channels, we actively steer the convective flow through the nanohole openings for effective delivery of the analytes to the sensor surface. We apply our method to detect refractive index changes in aqueous solutions. Bulk measurements indicate that active delivery of the convective flow results in better sensitivities. The sensitivity of the sensor reaches 510 nm/RIU for resonance located around 850 nm with a line-width of approximately 10 nm in solution. Experimental results are matched very well with numerical simulations. We also show that cross-polarization measurements can be employed to further improve the detection limit by increasing the signal-to-noise ratio.

Organ-targeted high-throughput in vivo biologics screen identifies materials for RNA delivery.

Therapies based on biologics involving delivery of proteins, DNA, and RNA are currently among the most promising approaches. However, although large combinatorial libraries of biologics and delivery vehicles can be readily synthesized, there are currently no means to rapidly characterize them in vivo using animal models. Here, we demonstrate high-throughput in vivo screening of biologics and delivery vehicles by automated delivery into target tissues of small vertebrates with developed organs. Individual zebrafish larvae are automatically oriented and immobilized within hydrogel droplets in an array format using a microfluidic system, and delivery vehicles are automatically microinjected to target organs with high repeatability and precision. We screened a library of lipid-like delivery vehicles for their ability to facilitate the expression of protein-encoding RNAs in the central nervous system. We discovered delivery vehicles that are effective in both larval zebrafish and rats. Our results showed that the in vivo zebrafish model can be significantly more predictive of both false positives and false negatives in mammals than in vitro mammalian cell culture assays. Our screening results also suggest certain structure-activity relationships, which can potentially be applied to design novel delivery vehicles.

Fully automated cellular-resolution vertebrate screening platform with parallel animal processing.

The zebrafish larva is an optically-transparent vertebrate model with complex organs that is widely used to study genetics, developmental biology, and to model various human diseases. In this article, we present a set of novel technologies that significantly increase the throughput and capabilities of our previously described vertebrate automated screening technology (VAST). We developed a robust multi-thread system that can simultaneously process multiple animals. System throughput is limited only by the image acquisition speed rather than by the fluidic or mechanical processes. We developed image recognition algorithms that fully automate manipulation of animals, including orienting and positioning regions of interest within the microscope's field of view. We also identified the optimal capillary materials for high-resolution, distortion-free, low-background imaging of zebrafish larvae.

Large-scale plasmonic microarrays for label-free high-throughput screening.

Microarrays allowing simultaneous analysis of thousands of parameters can significantly accelerate screening of large libraries of pharmaceutical compounds and biomolecular interactions. For large-scale studies on diverse biomedical samples, reliable, label-free, and high-content microarrays are needed. In this work, using large-area plasmonic nanohole arrays, we demonstrate for the first time a large-scale label-free microarray technology with over one million sensors on a single microscope slide. A dual-color filter imaging method is introduced to dramatically increase the accuracy, reliability, and signal-to-noise ratio of the sensors in a highly multiplexed manner. We used our technology to quantitatively measure protein-protein interactions. Our platform, which is highly compatible with the current microarray scanning systems can enable a powerful screening technology and facilitate diagnosis and treatment of diseases.

Spatial control of cellular adhesion using photo-crosslinked micropatterned polyelectrolyte multilayer films.

Cellular patterning on biomaterial surfaces is important in fundamental studies of cell-cell and cell-substrate interactions, and in biomedical applications such as tissue engineering, cell-based biosensors, and diagnostic devices. In this study, we combined the layer-by-layer polyelectrolyte multilayer deposition and photolithographic technique to create an easy and versatile technique for cell patterning. Poly(acrylic acid) (PAA) conjugated with 4-azidoaniline was interwoven in PAA/polyacrylamide (PAM) multilayer films. After UV irradiation through a photo mask, the UV-exposed areas were crosslinked and the unexposed areas were rinsed away by alkaline water, resulting in micropatterns. Cell patterns were formed when the cell adhesion was limited to the base substrate, but not on the multilayer films. The stability of cell patterns could be modulated by simply modification of the surface chemistry of base substrate and PEM films with conjugation of bioactive macromolecules. This technique can be also applied to other PEM systems with proper rinsing protocol, and many types of substrates. Cell co-culture systems can be also achieved by this technique.