Microfuidic Devices and Open Access Tool for Localized Microinjection and Heart Monitoring of Drosophila Melanogaster

This thesis aims to address the current research gaps associated with the use of Drosophila larvae as an in-vivo model for cardiac toxicity and cardiac gene screening. In objective 1, we have developed a hybrid multi-tasking microfluidic platform that enables desired orientation, reversible immobilization, and localized microinjection of intact Drosophila larvae for recording heart activities upon injection of controlled dosages of different chemicals. In objective 2. we have developed software for in-vivo quantification of essential heartbeat parameters on intact Drosophila larvae. Several image segmentation and signal processing algorithms were developed to detect the heart, extract the heartbeat signal, and quantify heart rate and arrhythmicity index automatically, while other heartbeat parameters were quantified semi-automatically using the M-mode. In objective 3a, we demonstrated the application of our microfluidic device and heartbeat quantification software for investigating the effect of different chemicals (e.g., serotonin and heavy metals) on Drosophila larval heart function. Also, we applied our technology to genetically modified Drosophila larvae to investigate the effect of metal responsive transcription factor (MTF-1) against heavy metals cardiac toxicity (objective 3b)

Microfuidic Devices and Open Access Tool for Localized Microinjection and Heart Monitoring of Drosophila Melanogaster

This thesis aims to address the current research gaps associated with the use of Drosophila larvae as an in-vivo model for cardiac toxicity and cardiac gene screening. In objective 1, we have developed a hybrid multi-tasking microfluidic platform that enables desired orientation, reversible immobilization, and localized microinjection of intact Drosophila larvae for recording heart activities upon injection of controlled dosages of different chemicals. In objective 2. we have developed software for in-vivo quantification of essential heartbeat parameters on intact Drosophila larvae. Several image segmentation and signal processing algorithms were developed to detect the heart, extract the heartbeat signal, and quantify heart rate and arrhythmicity index automatically, while other heartbeat parameters were quantified semi-automatically using the M-mode. In objective 3a, we demonstrated the application of our microfluidic device and heartbeat quantification software for investigating the effect of different chemicals (e.g., serotonin and heavy metals) on Drosophila larval heart function. Also, we applied our technology to genetically modified Drosophila larvae to investigate the effect of metal responsive transcription factor (MTF-1) against heavy metals cardiac toxicity (objective 3b)

An Automated Device to Increase Screening Throughput of Zebrafish Larvae

The use of the zebrafish as an animal model alternative to mammalian species has spawned research advancements in several medical fields. Since the zebrafish shares a high degree of sequence and functional homology with mammals, studies using this organism can provide in-depth insight into host response to disease and provide a platform for testing a range of treatment options. The optical transparency of zebrafish at early stages of development permits easy assessment of the effects of treatments, occurrence of tumors and other abnormal growth, disease progression, and immune response, to name only a few. These characteristics make it ideal for studying human diseases such as the Influenza A Virus (IAV). Recent research reveals that zebrafish produce sialic acids that are identical to that of humans. IAV possesses receptors that bind directly to sialic acid receptors, permitting infection. The conventional method of IAV transmission is by aerosol, and since the zebrafish does not accommodate this mode of entry, the virus is injected into the specimen under study. Zebrafish larvae are fragile and susceptible to deformation, therefore handling can be challenging. The larvae typically require manipulation during preparatory procedures prior to assessment, a process that can be time consuming and stressful for the organism. In this thesis, I describe the development of a device designed to eliminate the problems associated with manipulating zebrafish larvae by automatically conducting specimen from a reservoir directly into an entrapment dock, where it will be immobilized for injection and rapidly removed post-injection. This will help to significantly reduce the handling time of large sample sets, thereby increasing the screening throughput. Zebrafish have fast growth rateshence preparatory procedures for analysis like injection should be as quick and efficient as possible. This will reduce the likelihood that a large group of fish will transition to different stages of development prior to analysis. The device employs a system to conduct 48-72 hour old zebrafish through a liquid medium (egg water) using a syringe pump. The complete system consists of three main subsystems, namely the pump, optical detection and entrapment components. A 3D printed housing encloses the electrical components of the entire system. The device works by aspirating individual fish through a tube via a pressure gradient created with a syringe pump. Each cycle of the device involves the following steps: (1) loading, (2) sensing, (3) trapping, (4) injection, and (5) flushing. During loading, a single larva is extracted from the reservoir and conducted through a tube past the optical detection subsystem. At the sensing stage, the optical detection subsystem composed of a photodiode and a laser, senses transmitted light from the laser and discerns the entry of larva from air bubbles and debris with precision. Upon larva recognition, the specimen is then conducted to the entrapment dock (step 3) where it will be immobilized for injection (step 4). The final step (5) involves conducting the larva out of the entrapment dock and subsequently out of entire system for further analysis. This device will primarily serve IAV researchers who intend to introduce vaccines, pathogens and other experimental materials into many individual zebrafish larvae

Microfluidics for Investigation of Electric-Induced Behaviors of Zebrafish Larvae

Zebrafish has emerged as a model organism for studying the genetic, neuronal and behavioral bases of diseases and for drug screening. Being a vertebrate, they are phylogenetically closer to humans than invertebrates, possess complex organs and the overall organization of their brain shows structural similarities with human. They are small at larval stages, optically transparent and easy to culture. In addition, zebrafish models of human diseases and genetic mutants are widely available. These characteristics make this vertebrate model an ideal organism for neurodegeneration study and drug screening from the molecule to whole organism level. Despite these attractive features, the conventional zebrafish screening methods used for movement-based behavioral tests are mostly time-consuming, uncontrollable, qualitative, low-throughput and inaccurate. Zebrafish larvae behavioral response to various stimulations including optical and chemical stimuli, have been already investigated. However, zebrafish sensory-motor responses to electrical signals, a controllable stimulus which its potential in inducing locomotion response was proven in research done before, have not been broadly studied. Examples of research questions remaining to be answered are if zebrafish electric induced response is sensitive to different electric current intensities, voltage drops, multiple electrical stimulation, and the electric field direction. The involvement of different pathways and genes in this response and its potential for utilization in disease studies and chemical screening, and drug discovery can also be investigated. This research aims to enhance our understanding of zebrafish electric-induced response via presenting novel microfluidic devices that address the challenges associated with monitoring the behavioral activities of zebrafish larvae in response to various electrical signals. In Objective 1 of the thesis, we designed a microfluidic device to deliver electrical stimuli to the awake and partially immobilized zebrafish larvae, screen and study their phenotypic behavioral responses and analyze the outputs. Behavioral response was characterized in terms of response duration and tail beat frequency. A multi-phenotypic microfluidic device was also developed to study the effect of electric stimulation on the heartrate. In Objective 2, attention was given to investigate the effect of electric current, voltage, and field direction on the zebrafish larvae’s response to find an optimized setting which can induce a traceable response in zebrafish. Using different habituation-dishabituation strategies, we also investigated if the zebrafish larvae show adaptation towards repeated exposures to electric stimuli. In Objective 3, we developed a quadruple-fish device to enhance the behavioral throughput of our microfluidic platform and showed the technique's effectiveness for larger sample size and faster behavioral assay. In Objective 4, our quadruple-fish device was employed to investigate the involvement of dopaminergic neurons in electric-induced movement response of zebrafish larvae. Lastly, since we could monitor the electric-induced behavioral responses of zebrafish larvae, in Objective 5, the applicability of our proposed technique in chemical toxicity and gene screening assays was investigated. This study is expected to introduce a microfluidic platform for on-demand and phenotypic behavioral screening of zebrafish larvae with applications in chemical screening and drug discovery

Hybrid Microfluidic Devices For On-Demand Manipulation and Screening of Neurons and Organs of Small Model Organisms

Caenorhabditis elegans and Drosophila melanogaster are widely used model organisms for neurological and cardiac studies due to their simple neuronal and cardiac systems, genome similarity to humans, and ease of maintenance in laboratories. However, their 50m-1mm sizes and continuous mobility impede their precise spatiotemporal manipulation, thereby, reducing the throughput of biological assays. By integrating glass capillaries into microfluidic devices and using 3D-printed fixtures for precise control, we have developed hybrid lab-on-a-chip devices to facilitate the processes of animal manipulation and stimuli control, using modules for single-organism selection, orientation, imaging and chemical stimulation. These microdevices enabled us to manipulate organisms individually and to orient them at any desired direction for imaging purposes. The applications of these hybrid microdevices were demonstrated in the optical and fluorescent imaging of C. elegans cells as well as cardiac screening of Drosophila larvae. This technique can be applied in fundamental biology, toxicology, and drug discovery

Technologies bringing young Zebrafish from a niche field to the limelight

Fundamental life science and pharmaceutical research are continually striving to provide physiologically relevant context for their biological studies. Zebrafish present an opportunity for high-content screening (HCS) to bring a true in vivo model system to screening studies. Zebrafish embryos and young larvae are an economical, human-relevant model organism that are amenable to both genetic engineering and modification, and direct inspection via microscopy. The use of these organisms entails unique challenges that new technologies are overcoming, including artificial intelligence (AI). In this perspective article, we describe the state-of-the-art in terms of automated sample handling, imaging, and data analysis with zebrafish during early developmental stages. We highlight advances in orienting the embryos, including the use of robots, microfluidics, and creative multi-well plate solutions. Analyzing the micrographs in a fast, reliable fashion that maintains the anatomical context of the fluorescently labeled cells is a crucial step. Existing software solutions range from AI-driven commercial solutions to bespoke analysis algorithms. Deep learning appears to be a critical tool that researchers are only beginning to apply, but already facilitates many automated steps in the experimental workflow. Currently, such work has permitted the cellular quantification of multiple cell types in vivo, including stem cell responses to stress and drugs, neuronal myelination and macrophage behavior during inflammation and infection. We evaluate pro and cons of proprietary versus open-source methodologies for combining technologies into fully automated workflows of zebrafish studies. Zebrafish are poised to charge into HCS with ever-greater presence, bringing a new level of physiological context

Automation of Technology for Cancer Research

Zebrafish embryos can be obtained for research purposes in large numbers at low cost and embryos develop externally in limited space, making them highly suitable for high-throughput cancer studies and drug screens. Non-invasive live imaging of various processes within the larvae is possible due to their transparency during development, and a multitude of available fluorescent transgenic reporter lines. To perform high-throughput studies, handling large amounts of embryos and larvae is required. With such high number of individuals, even minute tasks may become time-consuming and arduous. In this chapter, an overview is given of the developments in the automation of various steps of large scale zebrafish cancer research for discovering important cancer pathways and drugs for the treatment of human disease. The focus lies on various tools developed for cancer cell implantation, embryo handling and sorting, microfluidic systems for imaging and drug treatment, and image acquisition and analysis. Examples will be given of employment of these technologies within the fields of toxicology research and cancer research

Novel technologies for high-throughput and high-content studies on zebrafish larvae

The zebrafish larva is an ideal candidate for in vivo high-throughput screening: it is a small vertebrate, it is optically transparent, possesses complex organs, and is easy to culture. In addition, genetic mutants and models of human diseases are widely available. Despite these attractive features there are no tools capable of screening at sufficient throughput and resolution to fully exploit the zebrafish. Here, I present a collection of technologies that enable high-throughput studies on zebrafish larvae at cellular resolution.Engineering and Applied Science

High-throughput vertebrate total analysis/screening platform

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2012.Cataloged from PDF version of thesis.Includes bibliographical references.High-throughput screening (HTS) is seen as one of the most promising technologies to facilitate biomedical studies and pharmaceutical discoveries. Although large varieties of in vitro HTS technologies have opened great opportunities, the speed of improvement has been limited by lack of advanced tools for in vivo screening on whole complex organisms, such as vertebrates. To address this issue, a high-throughput platform as a vertebrate total analysis/screening system (V-TAS) is proposed. This platform consists of two independent parts: an automated imaging system and an automated microinjection system. These two systems are designed for general high-content high-throughput pharmaceutical and genetic screens on whole zebrafish larvae, and therefore, are well-modularized for adapting different situations. Furthermore, to demonstrate the capability of V-TAS, a screen of lipidoid library for biologics delivery on thousands of animals was conducted. Very limited damage to the larvae was shown during the screening. In the end, the author also validated the hits discovered by V-TAS can be applied to more advanced animal models such as rats, and be more predictable than cell-based assays.by Tsung-Yao Chang.Ph.D

Advanced Microfluidic Assays for Caenorhabditis elegans

The in vivo analysis of a model organism, such as the nematode Caenorhabditis elegans, enables fundamental biomedical studies, including development, genetics, and neurobiology. In recent years, microfluidics technology has emerged as an attractive and enabling tool for the study of the multicellular organism. Advances in the application of microfluidics to C. elegans assays facilitate the manipulation of nematodes in high-throughput format and allow for the precise spatial and temporal control of their environment. In this chapter, we aim to illustrate the current microfluidic approaches for the investigation of behavior and neurobiology in C. elegans and discuss the trends of future development