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Measuring whole body fluorescence and movement in freely swimming transgenic zebrafish for measuring stress from exposure to environmental stressors


OBJECTIVE: The objective of this project is to develop a turnkey system of software and hardware necessary to measure whole-body fluorescence in freely swimming zebrafish under stress from environmental contaminants or stressors such as Hg, Pb or temperature. DESCRIPTION: Military operations at installations or when deployed produce environmental contaminants. Some are the result of military test and training activities (e.g., heavy metals) and others result from routine base activities (e.g., chemical spills). Assessing the impacts of environmental contamination is difficult especially at very low concentrations over long time periods. Relating exposure to changes in behavior and the connections to animal fitness and populations are even more difficult to establish. Facility managers would benefit from tools that relate exposure to changes in stress and behavior in animals under low concentrations of environmental contaminants. The offeror will develop a turnkey system of hardware (cameras, lighting, test domains) and software to quantify the whole-body fluorescence and movement (x,y,z, time) in freely swimming transgenic zebrafish. The system will record the spatial position (e.g. via video) and the fluorescence intensity (measured as pixel intensity) of a freely swimming fish at a high frequency (minimum of 1 measurement/sec). The test domain can be of various sizes with a minimum size being a typical 10 gallon aquarium. Larger sizes are also of interest. The system must be able to track one fish or multiple fish (e.g. 10 fish simultaneously). The system will then be able to post-process the measured fish track and pixel intensity values to produce metrics of behavior (e.g. swim speed and angle), provide animations of the track, and to colorize pixel intensity estimates according to which protein (e.g., mCherry or GFP) is being illuminated. A function graphical user interface that facilitates camera calibration, accuracy estimation, and experiment implementation should be included. PHASE I: The offeror will design architecture and integration methodology for the integrated hardware and software. The design should incorporate the ability to record spactial position with the ability to post-process the measured values and provide visual output, all within a function graphical user interface. The desired Phase I product is a report that describes the design architecture and integration methodology needed to develop a hardware and software system that meets the requirments laid out in the topic description. PHASE II: Phase II Year 1 shall produce a working hardware and software system alpha version that will highlight the expected hardware performance tradeoffs, data storage and operation via the graphic user interface. Phase II Year 2shall produce a beta version of the system, fully capable of measuring movement and fluorescence in a single freely swimming fish. The working system shall record all relevant data and produce output files that can be used for further analysis. The beta version software platform should have an intuitive user interface with flexible application to a range of problems (e.g., different flourescent proteins, different stressors, different lightning requirements) and different sized test domains (e.g. 10 gallon up to a larger at yet to be determined size). Particular attention should be applied to making sure that measurments are repeatable and have a manageable level of noise. The offerer will provide a report that documents the the design utilized for a turnkey system to measure whole-body fluorescence in freely swimming zebrafish under stress from environmental contaminants or stressors such as Hg, Pb or temperature; and the results of a functionality demonstration. PHASE III: This program could be expanded to include a wider range of fluorescent proteins and settings and to handle motile fish simultaneously. This type of program may have great value from a toxicology and behavior study perspective, and potentially as a method to monitor the integrity of municipal and agricultural water supplies. A full experimental demonstration of the ability to measure movement and whole body fluorescence should be part of this phase. This technology has potential commercial applications in the areas of water and waste management. REFERENCES: Blackburn, Jessica S, Sali Liu, Aubrey R Raimondi, Myron S Ignatius, Christopher D Salthouse et al. 2011. High-throughput imaging of adult fluorescent zebrafish with an LED fluorescence macroscope. Nature Protocols 6 (2) (February): 22941. Brandon W. Kusik, Michael J. Carvan III, Ava J. Udvadia, 2008. Detection of Environmental Oxidative Stress using EPRE reporter zebrafish. Marine Biotechnology 10: 750-757. Grover, Dhruv, Junsheng Yang, Simon Tavare, and John Tower. 2008."Simultaneous Tracking of Fly Movement and Gene Expression Using GFP."BMC Biotechnology 8 (January): 93. doi:10.1186/1472-6750-8-93. Grover, Dhruv, John Tower, and Simon Tavare. 2008."O Fly, Where Art Thou?"Journal of the Royal Society, Interface / the Royal Society 5 (27) (October 6): 118191. doi:10.1098/rsif.2007.1333. Grover, Dhruv, Daniel Ford, Christopher Brown, Nicholas Hoe, Aysen Erdem, Simon Tavare, and John Tower. 2009."Hydrogen Peroxide Stimulates Activity and Alters Behavior in Drosophila Melanogaster."PloS One 4 (10) (January): e7580.
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