Image subtraction has been an extremely useful tool for capturing subtle changes in pixel intensity with extremely high
temporal resolution, and has been used for decades in the astronomy and metal corrosion fields. However, to date, image
subtraction has not been used as a mainstream technique for investigating morphological changes in cells, tissues, or
whole organisms. We introduce a user-friendly differential imaging technique for monitoring real time (~msec) changes
in morphology within the micrometer to millimeter spatial scale. The technique is demonstrated by measuring
morphological changes morphology for biomedical (bone stress), agricultural (crop root elongation), and environmental
(zooplankton ecotoxicology) applications. Subtle changes in growth that would typically only be observed by highly
skilled experts are easily resolved via image subtraction and the use of convolution kernels. When coupled with
techniques characterizing real time biochemical transport (e.g., respiration, ion/substrate transport), physiology can be
directly quantified with a high temporal and spatial resolution. Because of the ease of use, this technique can be readily
applied to any field of science concerned with bridging the gap between form and function.
The detection of harmful chemicals and biological agents in real time is a critical need for protecting water quality. We
studied the real-time effects of five environmental contaminants with differing modes of action (atrazine,
pentachlorophenol, cadmium chloride, malathion, and potassium cyanide) on respiratory oxygen consumption in 2-day
post-fertilization fathead minnow (Pimephales promelas) eggs. Our objective was to assess the sensitivity of fathead
minnow eggs using the self-referencing micro-optrode technique to detect instantaneous changes in oxygen consumption
after brief exposures to low concentrations of contaminants. Oxygen consumption data indicated that the technique is
indeed sensitive enough to reliably detect physiological alterations induced by all contaminants. After 2 h of exposure,
we identified significant increases in oxygen consumption upon exposure to pentachlorophenol (100 and 1000 μg/L),
cadmium chloride (0.0002 and 0.002 μg/L), and atrazine (150 μg/L). In contrast, we observed a significant decrease in
oxygen flux after exposures to potassium cyanide (5.2, 22, and 44 μg/L) and atrazine (1500 μg/L). No effects were
detected after exposures to malathion (200 and 340 μg/L). We have also tested the sensitivity of Daphnia magna
embryos as another animal model for real-time environmental biomonitoring. Our results are so far encouraging and
support further development of this technology as a physiologically coupled biomonitoring tool for the detection of
environmental toxicants.
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