A13-081 TITLE: Measuring whole body fluorescence and movement in freely swimming transgenic zebrafish for measuring stress from exposure to environmental stressors
TECHNOLOGY AREAS: Materials/Processes
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 is 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 under laboratory conditions. The fluorescent markers that are used mark genes that transcribe specific proteins. The genes and the proteins that are marked are generally conserved across all fish and perhaps most vertebrates, including humans. In fact, zebrafish cellular and genetic functions are often a model for studying human physiology. Mapping zebrafish protein transcription to other species and to humans is commonly done as the molecular mechanisms are conserved at the cellular level.
The system will consist of a test domain (tank) monitored by multiple video cameras. Appropriate illumination suitable to record behavior and excite the fluorescent protein of interest is required. The cameras 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). Pixel intensity will be extracted from image files of the fish and may be standardized between 0 and 1. Distance between the video camera and the test domain will depend on camera resolution and domain size at a minimum. For a given test the cameras, once positioned, will not be moved. The test domain can be of various sizes with a minimum size being a typical 10 gallon aquarium with limited turbidity. Larger sizes are also of interest depending on experimental needs and may require multiple cameras. The system must be able to track one fish or multiple fish (e.g. >200 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 spatial position with the ability to post-process the measured values and provide visual output, all within a function graphical user interface. The approach to design used should pay particular attention to how measurement noise will be managed with emphasis on system lighting design. 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 requirements 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 2 shall 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 fluorescent proteins, different stressors, different lighting requirements) and different sized test domains (e.g. 10 gallon up to a larger as yet to be determined size). Particular attention should be applied to making sure that measurements are repeatable and have a manageable level of noise. The offerer will provide a report that documents 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 DUAL USE APPLICATIONS: 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.