Yushi Huang

Fishing on chips: Up-and-coming technological advances in analysis of zebrafish and Xenopus embryos

Biotests performed on small vertebrate model organisms provide significant investigative advantages as compared with bioassays that employ cell lines, isolated primary cells, or tissue samples. The main advantage offered by whole‐organism approaches is that the effects under study occur in the context of intact physiological milieu, with all its intercellular and multisystem interactions. The gap between the high‐throughput cell‐based in vitro assays and low‐throughput, disproportionally expensive and ethically controversial mammal in vivo tests can be closed by small model organisms such as zebrafish or Xenopus. The optical transparency of their tissues, the ease of genetic manipulation and straightforward husbandry, explain the growing popularity of these model organisms. Nevertheless, despite the potential for miniaturization, automation and subsequent increase in throughput of experimental setups, the manipulation, dispensing and analysis of living fish and frog embryos remain labor‐intensive. Recently, a new generation of miniaturized chip‐based devices have been developed for zebrafish and Xenopus embryo on‐chip culture and experimentation. In this work, we review the critical developments in the field of Lab‐on‐a‐Chip devices designed to alleviate the limits of traditional platforms for studies on zebrafish and clawed frog embryo and larvae.

A Millifluidic System for Analysis of Daphnia magna Locomotory Responses to Water-born Toxicants

Aquatic toxicity testing in environmental monitoring and chemical risk assessment is critical to assess water quality for human use as well as predict impact of pollutants on ecosystems. In recent years, studies have increasingly focused on the relevance of sub-lethal effects of environmental contaminants. Sub-lethal toxicity endpoints such as behavioural responses are highly integrative and have distinct benefits for assessing water quality because they occur rapidly and thus can be used to sense the presence of toxicants. Our work describes a Lab-on-a-Chip system for the automated analysis of freshwater cladoceran Daphnia magna locomotory responses to water-born toxicants. The design combines a Lab-on-a-Chip system for Daphnia sp. culture under perfusion with time-resolved videomicroscopy and software tracking locomotory activity of multiple specimens. The application of the system to analyse the swimming behaviour of water fleas exposed to different concentrations of water-born toxicants demonstrated that Lab-on-a-Chip devices can become important research tools for behavioural ecotoxicology and water quality biomonitoring.

Unsuitable use of DMSO for assessing behavioral endpoints in aquatic model species

Dimethyl sulfoxide (DMSO) is a universally used aprotic solvent with the ability to permeate biological membranes and thus is commonly used to achieve appropriate biological availability of hydrophobic toxicants. While DMSO as a carrier medium has a reportedly low toxicity and is routinely employed in ecotoxicology, very little is known about its effect on dynamic behavioral parameters. This study presents a comparative analysis of the lethal and behavioral effects of exposures to DMSO concentrations of 0.1-10% on several test species such as: neonates of the freshwater crustacean Daphnia magna, nauplii of the marine crustacean Artemia franciscana, the marine crustacean Allorchestes compressa, embryos and larvae of the freshwater fish Danio rerio. The results demonstrated that DMSO did not cause statistically significant mortality even at concentrations close to 1% but induced clear and significant behavioral abnormalities in response to sublethal concentrations on all test species. These included hypoactivity syndrome in A. franciscana, A. compressa, D. magna and zebrafish larvae while a slight time-dependent hyperactivity response was observed in zebrafish embryos. For the majority of test species, behavioral changes such as moving distance, acceleration and burst movement were often observed during the first hours of exposure. These results indicate that caution should be exercised when using DMSO as a carrier solvent in experiments assessing behavioral endpoints.

Enabling rapid behavioral ecotoxicity studies using an integrated lab-on-a-chip systems

Behavioral ecotoxicity tests are gaining an increasing recognition in environmental toxicology. Behavior of sensitive bioindicator species can change rapidly in response to an acute exposure to contaminants and thus has a much higher sensitivity as compared to conventional LC50 mortality tests. Furthermore, behavioral endpoints seems to be very good candidates to develop early-warning biomonitoring systems needed for rapid chemical risk assessment. Behavioral tests are non-invasive, fast, do not harm indicator organisms (behavioural changes are very rapid) and are thus fully compatible with 3R (Replacement – Reduction – Refinement) principle encouraging alternatives to conventional animal testing. These characteristics are essential when designing improved ecotoxicity tests for chemical risk assessment. In this work, we present a pilot development of miniaturized Lab-on-a-Chip (LOC) devices for studying toxin avoidance behaviors of small aquatic crustaceans. As an investigative tool, LOCs represent a new direction that may miniaturize and revolutionize behavioral ecotoxicology. Specifically our innovative microfluidic prototype: (i) enables convening “caging” of specimens for real-time videomicroscopy; (ii) eliminates the evaporative water loss thus providing an opportunity for long-term behavioral studies; (iii) exploits laminar fluid flow under low Reynolds numbers to generate discrete domains and gradients enabling for the first time toxin avoidance studies on small aquatic crustaceans; (iv) integrates off-the-chip mechatronic interfaces and video analysis algorithms for single animal movement analysis. We provide evidence that by merging innovative bioelectronic and biomicrofluidic technologies we can deploy inexpensive and reliable systems for culture, electronic tracking and complex computational analysis of behavior of bioindicator organisms.

Automation of daphtoxkit-F biotest using a microfluidic lab-on-a-chip technology

An increased rigor in water quality monitoring is not only a legal requirement, but is also critical to ensure timely chemical hazard emergency responses and protection of human and animal health. Bioindication is a method that applies very sensitive living organisms to detect environmental changes using their natural responses. Although bioindicators do not deliver information on an exact type or intensity of toxicants present in water samples, they do provide an overall snapshot and early-warning information about presence of harmful and dangerous parameters. Despite the advantages of biotests performed on sentinel organisms, their wider application is limited by the nonexistence of high-throughput laboratory automation systems. As a result majority of biotests used in ecotoxicology require time-consuming and laborious manual procedures. In this work, we present development of a miniaturized Lab-on-a-Chip (LOC) platform for automation and enhancement of acute ecotoxicity test based on immobilization of a freshwater crustacean Daphnia magna (Daphtoxkit-FTM). Daphnids’ immobilization in response to sudden changes in environment parameters is fast, unambiguous, and easy to record optically. We also for the first time demonstrate that LOC system enables studies of sub-lethal ecotoxic effects using behavioral responses of Daphnia magna as sentinels of water pollution. The system working principle incorporated a high definition (HD) time-resolved video data analysis to dynamically assess impact of the reference toxicant on swimming behavior of D. magna. Our system design combined: (i) microfluidic device for caging of Daphnia sp.; (ii) mechatronic interface for fluidic actuation; (iii) video data acquisition; and (iv) algorithms for animal movement tracking and analysis.

Integrated microfluidic technology for sub-lethal and behavioral marine ecotoxicity biotests

Changes in behavioral traits exhibited by small aquatic invertebrates are increasingly postulated as ethically acceptable and more sensitive endpoints for detection of water-born ecotoxicity than conventional mortality assays. Despite importance of such behavioral biotests, their implementation is profoundly limited by the lack of appropriate biocompatible automation, integrated optoelectronic sensors, and the associated electronics and analysis algorithms. This work outlines development of a proof-of-concept miniaturized Lab-on-a-Chip (LOC) platform for rapid water toxicity tests based on changes in swimming patterns exhibited by Artemia franciscana (Artoxkit M™) nauplii. In contrast to conventionally performed end-point analysis based on counting numbers of dead/immobile specimens we performed a time-resolved video data analysis to dynamically assess impact of a reference toxicant on swimming pattern of A. franciscana. Our system design combined: (i) innovative microfluidic device keeping free swimming Artemia sp. nauplii under continuous microperfusion as a mean of toxin delivery; (ii) mechatronic interface for user-friendly fluidic actuation of the chip; and (iii) miniaturized video acquisition for movement analysis of test specimens. The system was capable of performing fully programmable time-lapse and video-microscopy of multiple samples for rapid ecotoxicity analysis. It enabled development of a user-friendly and inexpensive test protocol to dynamically detect sub-lethal behavioral end-points such as changes in speed of movement or distance traveled by each animal.

Automatic multiple zebrafish larvae tracking in unconstrained microscopic video conditions

The accurate tracking of zebrafish larvae movement is fundamental to research in many biomedical, pharmaceutical, and behavioral science applications. However, the locomotive characteristics of zebrafish larvae are significantly different from adult zebrafish, where existing adult zebrafish tracking systems cannot reliably track zebrafish larvae. Further, the far smaller size differentiation between larvae and the container render the detection of water impurities inevitable, which further affects the tracking of zebrafish larvae or require very strict video imaging conditions that typically result in unreliable tracking results for realistic experimental conditions. This paper investigates the adaptation of advanced computer vision segmentation techniques and multiple object tracking algorithms to develop an accurate, efficient and reliable multiple zebrafish larvae tracking system. The proposed system has been tested on a set of single and multiple adult and larvae zebrafish videos in a wide variety of (complex) video conditions, including shadowing, labels, water bubbles and background artifacts. Compared with existing state-of-the-art and commercial multiple organism tracking systems, the proposed system improves the tracking accuracy by up to 31.57% in unconstrained video imaging conditions. To facilitate the evaluation on zebrafish segmentation and tracking research, a dataset with annotated ground truth is also presented. The software is also publicly accessible.

Miniaturized video-microscopy system for near real-time water quality biomonitoring using microfluidic chip-based devices

Biomonitoring studies apply biological responses of sensitive biomonitor organisms to rapidly detect adverse environmental changes such as presence of physic-chemical stressors and toxins. Behavioral responses such as changes in swimming patterns of small aquatic invertebrates are emerging as sensitive endpoints to monitor aquatic pollution. Although behavioral responses do not deliver information on an exact type or the intensity of toxicants present in water samples, they could provide orders of magnitude higher sensitivity than lethal endpoints such as mortality. Despite the advantages of behavioral biotests performed on sentinel organisms, their wider application in real-time and near realtime biomonitoring of water quality is limited by the lack of dedicated and automated video-microscopy systems. Current behavioral analysis systems rely mostly on static test conditions and manual procedures that are time-consuming and labor intensive. Tracking and precise quantification of locomotory activities of multiple small aquatic organisms requires high-resolution optical data recording. This is often problematic due to small size of fast moving animals and limitations of culture vessels that are not specially designed for video data recording. In this work, we capitalized on recent advances in miniaturized CMOS cameras, high resolution optics and biomicrofluidic technologies to develop near real-time water quality sensing using locomotory activities of small marine invertebrates. We present proof-of-concept integration of high-resolution time-resolved video recording system and high-throughput miniaturized perfusion biomicrofluidic platform for optical tracking of nauplii of marine crustacean Artemia franciscana. Preliminary data demonstrate that Artemia sp. exhibits rapid alterations of swimming patterns in response to toxicant exposure. The combination of video-microscopy and biomicrofluidic platform facilitated straightforward recording of fast moving objects. We envisage that prospectively such system can be scaled up to perform high-throughput water quality sensing in a robotic biomonitoring facility.

Testing organic toxicants on biomicrofluidic devices: Why polymeric substrata can lead you into trouble

Advances in microfabrication technologies and manufacturing over last decade, allowed for inexpensive prototyping of microfluidic chip-based devices for biomedical studies in biocompatible and optically transparent elastomeric polymers such as poly(dimethylsiloxane) (PDMS) and thermoplastics such as poly(methyl methacrylate) (PMMA). More resently, advanced additive manufacturing technologies such as stereolithography (SLA), capable of reproducing feature sizes less than 50 μm, pave a way towards a new generation of microfabrication techniques. The latter promise new methods to enable accelerated design, validation and optimisation of optical-grade biomicrofluidic Lab-on-a-Chip (LOC) devices. The main limitation, however, of virtually all polymers that are used to both manufacture LOC devices as well as to provide fluidic interconnects is their significant hydrophobicity. Conventionally the hydrophobic properties were postulated to impede wetting and priming of the polymeric chip-based devices and tubing interconnects. Such issues were often solved with plasma treatment or ethanol priming to help wet the polymeric substrata and also reduce the nucleation and persistence of air bubbles. In this work, we present evidence that use of certain hydrophobic polymers is a significant impediment in performing ecotoxicity tests of organic chemicals on biomicrofluidic devices. We report on electrostatic interaction between polymers and toxicants that lead to non-covalent adsorption and rapid depletion of chemicals from the tested media. This introduces a significant bioanalytical bias irrespectively of the fact that microfluidic tests are preformed under continuous perfusion.