Impact of Digital Video Analytics on Accuracy of Chemobehavioural Phenotyping in Aquatic Toxicology

[Abstract] Chemobehavioural phenotypic analysis using small aquatic model organisms is becoming an important toolbox in aquatic ecotoxicology and neuroactive drug discovery. The analysis of the organisms’ behavior is usually performed by combining digital video recording with animal tracking software. This software detects the organisms in the video frames, and reconstructs their movement trajectory using image processing algorithms. In this work we investigated the impact of video file characteristics, video optimization techniques and differences in animal tracking algorithms on the accuracy of quantitative neurobehavioural endpoints. We employed larval stages of a free-swimming euryhaline crustacean Artemia franciscana,commonly used for marine ecotoxicity testing, as a proxy modelto assess the effects of video analytics on quantitative behavioural parameters. We evaluated parameters such as data processing speed, tracking precision, capability to perform high-throughput batch processing of video files. Using a model toxicant the software algorithms were also finally benchmarked against one another. Our data indicates that variability in video file parameters; such as resolution, frame rate, file containers types, codecs and compression levels, can be a source of experimental biases in behavioural analysis. Similarly, the variability in data outputs between different tracking algorithms should be taken into account when designing standardized behavioral experiments and conducting chemobehavioural phenotyping

A review of 28 free animal tracking software: current features and limitations

This version of the article has been accepted for publication, after peer review and is subject to Springer Nature’s AM terms of use, but is not the Version of Record and does not reflect post-acceptance improvements, or any corrections. The Version of Record is available online at: http://dx.doi.org/10.1038/s41684-021-00811-1[Abstract]: Well-quantified laboratory studies can provide a fundamental understanding of animal behavior in ecology, ethology and ecotoxicology research. These types of studies require observation and tracking of each animal in well-controlled and defined arenas, often for long timescales. Thus, these experiments produce long time series and a vast amount of data that require the use of software applications to automate the analysis and reduce manual annotation. In this review, we examine 28 free software applications for animal tracking to guide researchers in selecting the software that might best suit a particular experiment. We also review the algorithms in the tracking pipeline of the applications, explain how specific techniques can fit different experiments, and finally, expose each approach’s weaknesses and strengths. Our in-depth review includes last update, type of platform, user-friendliness, off- or online video acquisition, calibration method, background subtraction and segmentation method, species, multiple arenas, multiple animals, identity preservation, manual identity correction, data analysis and extra features. We found, for example, that out of 28 programs, only 3 include a calibration algorithm to reduce image distortion and perspective problems that affect accuracy and can result in substantial errors when analyzing trajectories and extracting mobility or explored distance. In addition, only 4 programs can directly export in-depth tracking and analysis metrics, only 5 are suited for tracking multiple unmarked animals for more than a few seconds and only 11 have been updated in the period 2019–2021

Dielectrophoresis of micro/nano particles using curved microelectrodes

Dielectrophoresis, the induced motion of polarisable particles in non-homogenous electric field, has been proven as a versatile mechanism to transport, immobilise, sort and characterise micro/nano scale particle in microfluidic platforms. The performance of dielectrophoretic (DEP) systems depend on two parameters: the configuration of microelectrodes designed to produce the DEP force and the operating strategies devised to employ this force in such processes. This work summarises the unique features of curved microelectrodes for the DEP manipulation of target particles in microfluidic systems. The curved microelectrodes demonstrate exceptional capabilities including (i) creating strong electric fields over a large portion of their structure, (ii) minimising electro-thermal vortices and undesired disturbances at their tips, (iii) covering the entire width of the microchannel influencing all passing particles, and (iv) providing a large trapping area at their entrance region, as evidenced by extensive numerical and experimental analyses. These microelectrodes have been successfully applied for a variety of engineering and biomedical applications including (i) sorting and trapping model polystyrene particles based on their dimensions, (ii) patterning carbon nanotubes to trap low-conductive particles, (iii) sorting live and dead cells based on their dielectric properties, (iv) real-time analysis of drug-induced cell death, and (v) interfacing tumour cells with environmental scanning electron microscopy to study their morphological properties. The DEP systems based on curved microelectrodes have a great potential to be integrated with the future lab-on-a-chip systems.<br /

Miniaturized Embryo Array for Automated Trapping, Immobilization and Microperfusion of Zebrafish Embryos

Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP). The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale

An engineered sgsh Mutant Zebrafish recapitulates molecular and behavioural pathobiology of sanfilippo syndrome A/MPS IIIA

Mucopolysaccharidosis IIIA (MPS IIIA, Sanfilippo syndrome type A), a paediatric neurological lysosomal storage disease, is caused by impaired function of the enzyme N‐sulfoglucosamine sulfohydrolase (SGSH) resulting in impaired catabolism of heparan sulfate glycosaminoglycan (HS GAG) and its accumulation in tissues. MPS IIIA represents a significant proportion of childhood dementias. This condition generally leads to patient death in the teenage years, yet no effective therapy exists for MPS IIIA and a complete understanding of the mechanisms of MPS IIIA pathogenesis is lacking. Here, we employ targeted CRISPR/Cas9 mutagenesis to generate a model of MPS IIIA in the zebrafish, a model organism with strong genetic tractability and amenity for highthroughput screening. The sgshΔex5−6 zebrafish mutant exhibits a complete absence of Sgsh enzymatic activity, leading to progressive accumulation of HS degradation products with age. sgshΔex5−6 zebrafish faithfully recapitulate diverse CNS‐specific features of MPS IIIA, including neuronal lysosomal overabundance, complex behavioural phenotypes, and profound, lifelong neuroinflammation. We further demonstrate that neuroinflammation in sgshΔex5−6 zebrafish is largely dependent on interleukin‐1β and can be attenuated via the pharmacological inhibition of Caspase‐1, which partially rescues behavioural abnormalities in sgshΔex5−6 mutant larvae in a context‐dependent manner. We expect the sgshΔex5−6 zebrafish mutant to be a valuable resource in gaining a better understanding of MPS IIIA pathobiology towards the development of timely and effective therapeutic interventions.publishedVersionPeer reviewe

3D-Printed Chips: Compatibility of Additive Manufacturing Photopolymeric Substrata with Biological Applications

Additive manufacturing (AM) is ideal for building adaptable, structurally complex, three-dimensional, monolithic lab-on-chip (LOC) devices from only a computer design file. Consequently, it has potential to advance micro- to milllifluidic LOC design, prototyping, and production and further its application in areas of biomedical and biological research. However, its application in these areas has been hampered due to material biocompatibility concerns. In this review, we summarise commonly used AM techniques: vat polymerisation and material jetting. We discuss factors influencing material biocompatibility as well as methods to mitigate material toxicity and thus promote its application in these research fields

Microfluidics: Emerging prospects for anti-cancer drug screening

Cancer constitutes a heterogenic cellular system with a high level of spatio-temporal complexity. Recent discoveries by systems biologists have provided emerging evidence that cellular responses to anti-cancer modalities are stochastic in nature. To uncover the intricacies of cell-to-cell variability and its relevance to cancer therapy, new analytical screening technologies are needed. The last decade has brought forth spectacular innovations in the field of cytometry and single cell cytomics, opening new avenues for systems oncology and high-throughput real-time drug screening routines. The up-and-coming microfluidic Lab-on-a-Chip (LOC) technology and micro-total analysis systems (μTAS) are arguably the most promising platforms to address the inherent complexity of cellular systems with massive experimental parallelization and 4D analysis on a single cell level. The vast miniaturization of LOC systems and multiplexing enables innovative strategies to reduce drug screening expenditures while increasing throughput and content of information from a given sample. Small cell numbers and operational reagent volumes are sufficient for microfluidic analyzers and, as such, they enable next generation high-throughput and high-content screening of anti-cancer drugs on patient-derived specimens. Herein we highlight the selected advancements in this emerging field of bioengineering, and provide a snapshot of developments with relevance to anti-cancer drug screening routines

Ecotoxicology Goes on a Chip: Embracing Miniaturized Bioanalysis in Aquatic Risk Assessment

Biological and environmental sciences are, more than ever, becoming highly dependent on technological and multidisciplinary approaches that warrant advanced analytical capabilities. Microfluidic lab-on-a-chip technologies are perhaps one the most groundbreaking offshoots of bioengineering, enabling design of an entirely new generation of bioanalytical instrumentation. They represent a unique approach to combine microscale engineering and physics with specific biological questions, providing technological advances that allow for fundamentally new capabilities in the spatiotemporal analysis of molecules, cells, tissues, and even small metazoan organisms. While these miniaturized analytical technologies experience an explosive growth worldwide, with a substantial promise of a direct impact on biosciences, it seems that lab-on-a-chip systems have so far escaped the attention of aquatic ecotoxicologists. In this Critical Review, potential applications of the currently existing and emerging chip-based technologies for aquatic ecotoxicology and water quality monitoring are highlighted. We also offer suggestions on how aquatic ecotoxicology can benefit from adoption of microfluidic lab-on-a-chip devices for accelerated bioanalysis.The authors acknowledge grants sponsored by the Australian Research Council DECRA DE130101046 (D.W.), Vice-Chancellor’s Senior Research Fellowship, RMIT University, Australia (D.W.), and Australian Government, Department of Defence (D.W.).Peer reviewe