Microfluidic Chips for In Vivo Imaging of Cellular Responses to Neural Injury in Drosophila Larvae

With powerful genetics and a translucent cuticle, the Drosophila larva is an ideal model system for live imaging studies of neuronal cell biology and function. Here, we present an easy-to-use approach for high resolution live imaging in Drosophila using microfluidic chips. Two different designs allow for non-invasive and chemical-free immobilization of 3rd instar larvae over short (up to 1 hour) and long (up to 10 hours) time periods. We utilized these ‘larva chips’ to characterize several sub-cellular responses to axotomy which occur over a range of time scales in intact, unanaesthetized animals. These include waves of calcium which are induced within seconds of axotomy, and the intracellular transport of vesicles whose rate and flux within axons changes dramatically within 3 hours of axotomy. Axonal transport halts throughout the entire distal stump, but increases in the proximal stump. These responses precede the degeneration of the distal stump and regenerative sprouting of the proximal stump, which is initiated after a 7 hour period of dormancy and is associated with a dramatic increase in F-actin dynamics. In addition to allowing for the study of axonal regeneration in vivo, the larva chips can be utilized for a wide variety of in vivo imaging applications in Drosophila

RECEIVED 1 APRIL; ACCEPTED 9 JULY; PUBLISHED ONLINE

The nematode C. elegans is an excellent model organism for studying behavior at the neuronal level. Because of the organism's small size, it is challenging to deliver stimuli to C. elegans and monitor neuronal activity in a controlled environment. To address this problem, we developed two microfluidic chips, the 'behavior' chip and the 'olfactory' chip for imaging of neuronal and behavioral responses in C. elegans. We used the behavior chip to correlate the activity of AVA command interneurons with the worm locomotion pattern. We used the olfactory chip to record responses from ASH sensory neurons exposed to high-osmotic-strength stimulus. Observation of neuronal responses in these devices revealed previously unknown properties of AVA and ASH neurons. The use of these chips can be extended to correlate the activity of sensory neurons, interneurons and motor neurons with the worm's behavior. How neural circuits process information to generate behavior is a fundamental question in neuroscience. To address this question, one should observe an animal in a well-controlled environment, in which a specific behavior can be generated and corresponding neuronal activity monitored. Ideally such an environment should not disturb normal neuronal function and should be able to reveal the specific neuronal circuit under study. C. elegans, with its optically accessible, stereotyped and compact nervous system, has drawn great scientific attention because of its diverse repertoire of behavioral outputs and its genetic conservation with vertebrates. Initial efforts to measure activity in the C. elegans nervous system relied on electrophysiological recordings from single neurons in dissected worms 1 . The recent development of genetically encoded fluorescent calcium indicators 2 has spawned an increasing interest in optical imaging approaches that permit the tracking of calcium transients in individual neurons in vivo in intact worms 3 . Although transgenic worms that express neuron-specific indicators can now routinely be generated, the present methods for confining and stimulating the worm during imaging are not ideal. The typical experimental setup involves application of glue onto specific segments of the worm to achieve permanent immobilization on a hydrated agar pad. Fluid-filled pipettes, temperature-controlled plates and sharp electrodes have been used in the past to deliver chemical, thermal and mechanical stimuli, respectively 4,5 . Whether the organic glue is toxic to the worm and how it influences neuronal activity are difficult to determine. Moreover, the delivery of chemical stimuli to the glued worm cannot be precisely controlled or separated from mechanical stimuli associated with fluid flow. More concerns arise when the circuit controlling locomotion is under study. The glue immobilizes the worm, not allowing muscles and stretch-receptor neurons, if any, to contract and relax normally. This mechanically restricted microenvironment might affect the function of the proprioceptive sensory neurons as well as motor neurons. Most importantly, the glue setup does not permit most behaviors to be generated, visualized, quantified or correlated to neuronal activity in real time. A system with two objectives 6 has been a welcome step toward simultaneous neuronal-behavior analysis, as has been a new system for tracking thermosensory neurons (albeit at low optical resolution) in freely moving worms 7 . Recent advances in microfabrication technology permit the construction of well-controllable microenvironments with applications ranging from cell analysis to tissue engineering RESULTS The behavior chip The first microfluidic device, the behavior chi

Probing the physiology of ASH neuron in Caenorhabditis elegans using electric current stimulation

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/98694/1/ApplPhysLett_99_053702.pd

A Portable, Optical Scanning System for Large Field of View, High Resolution Imaging of Biological Specimens

Large field-of-view (FOV), high resolution imaging of biological specimens isa challenging task, requiring sophisticated and bulky optical systems. Such systems cannot be used for diagnosing or monitoring a disease at the point-of-care. To address this need, we developeda portable, optical system that can image—with a 2.88 μm resolution—large areas (6 mm × 40 mm) from various biological samples by performing scanning in one direction. This is achieved through the use of a microfabricated, mini-lens array. We demonstrated that our system can detect single cells from a smear blood test and thus validating our vision for its use at the point-of-care

An Optofluidic Lens Array Microchip for High Resolution Stereo Microscopy

We report the development of an add-on, chip-based, optical module—termed the Microfluidic-based Oil-immersion Lenses (μOIL) chip—which transforms any stereo microscope into a high-resolution, large field of view imaging platform. The μOIL chip consists of an array of ball mini-lenses that are assembled onto a microfluidic silicon chip. The mini-lenses are made out of high refractive index material (sapphire) and they are half immersed in oil. Those two key features enable submicron resolution and a maximum numerical aperture of ~1.2. The μOIL chip is reusable and easy to operate as it can be placed directly on top of any biological sample. It improves the resolution of a stereo microscope by an order of magnitude without compromising the field of view; therefore, we believe it could become a versatile tool for use in various research studies and clinical applications

Business Model Selection for Community Energy Storage: A Multi Criteria Decision Making Approach

This paper explores business models for community energy storage (CES) and examines their potential and feasibility at the local level. By leveraging Multi Criteria Decision Making (MCDM) approaches and real-world case studies in Europe and India, it presents insights into CES deployment opportunities, challenges, and best practices. Different business models, including community energy cooperatives, utility–community partnerships, demand response, energy services, and market mechanisms, are analyzed. The proposed method combines the MCDM method PROMETHEE II with the fuzzy set theory to obtain a complete CES business model ranking, addressing project uncertainties. The analysis emphasizes CES’s role in balancing local renewable energy supply and demand, facilitating energy sharing, and achieving energy independence. Findings prioritize models like Community Cooperative, Energy Arbitrage, and Energy Arbitrage Peak Shaving for CES with renewables. Environmental benefits include reduced diesel use and greenhouse gas emissions. Efficient cooperatives are advocated to recover costs and enable competitive energy prices. The paper highlights the need for novel value propositions to boost the energy transition in local communities. This research contributes to the discourse on CES business models, fostering knowledge exchange and promoting effective strategies for sustainable energy systems

On-Demand Isolation and Manipulation of C. elegans by In Vitro Maskless Photopatterning

Caenorhabditis elegans (C. elegans) is a model organism for understanding aging and studying animal behavior. Microfluidic assay techniques have brought widespread advances in C. elegans research; however, traditional microfluidic assays such as those based on soft lithography require time-consuming design and fabrication cycles and offer limited flexibility in changing the geometric environment during experimentation. We present a technique for maskless photopatterning of a biocompatible hydrogel on an NGM (Agar) substrate, enabling dynamic manipulation of the C. elegans culture environment in vitro. Maskless photopatterning is performed using a projector-based microscope system largely built from off-the-shelf components. We demonstrate the capabilities of this technique by building micropillar arrays during C. elegans observation, by fabricating free-floating mechanisms that can be actuated by C. elegans motion, by using freehand drawing to isolate individual C. elegans in real time, and by patterning arrays of mazes for isolation and fitness testing of C. elegans populations. In vitro photopatterning enables rapid and flexible design of experiment geometry as well as real-time interaction between the researcher and the assay such as by sequential isolation of individual organisms. Future adoption of image analysis and machine learning techniques could be used to acquire large datasets and automatically adapt the assay geometry.National Institutes of Health (U.S.). Microfluidics in Biomedical Sciences Training Program (5T32-EB005582)United States. Air Force Office of Scientific Research. Young Investigator Program (FA9550-11-1-0089

Photovoltaics Enabling Sustainable Energy Communities: Technological Drivers and Emerging Markets

In this paper, we investigate the economic benefits of an energy community investing in small-scale photovoltaics (PVs) when local energy trading is operated amongst the community members. The motivation stems from the open research question on whether a community-operated local energy market can enhance the investment feasibility of behind-the-meter small-scale PVs installed by energy community members. Firstly, a review of the models, mechanisms and concepts required for framing the relevant concepts is conducted, while a clarification of nuances at important terms is attempted. Next, a tool for the investigation of the economic benefits of operating a local energy market in the context of an energy community is developed. We design the local energy market using state-of-the-art formulations, modified according to the requirements of the case study. The model is applied to an energy community that is currently under formation in a Greek municipality. From the various simulations that were conducted, a series of generalizable conclusions are extracted

On-Demand Isolation and Manipulation of C. elegans by In Vitro Maskless Photopatterning.

Caenorhabditis elegans (C. elegans) is a model organism for understanding aging and studying animal behavior. Microfluidic assay techniques have brought widespread advances in C. elegans research; however, traditional microfluidic assays such as those based on soft lithography require time-consuming design and fabrication cycles and offer limited flexibility in changing the geometric environment during experimentation. We present a technique for maskless photopatterning of a biocompatible hydrogel on an NGM (Agar) substrate, enabling dynamic manipulation of the C. elegans culture environment in vitro. Maskless photopatterning is performed using a projector-based microscope system largely built from off-the-shelf components. We demonstrate the capabilities of this technique by building micropillar arrays during C. elegans observation, by fabricating free-floating mechanisms that can be actuated by C. elegans motion, by using freehand drawing to isolate individual C. elegans in real time, and by patterning arrays of mazes for isolation and fitness testing of C. elegans populations. In vitro photopatterning enables rapid and flexible design of experiment geometry as well as real-time interaction between the researcher and the assay such as by sequential isolation of individual organisms. Future adoption of image analysis and machine learning techniques could be used to acquire large datasets and automatically adapt the assay geometry