Holding your breath with flow cytometry
Though no one knew it, a substantial increase in the growth of a particular harmful marine alga — Dinophysis acuminata — was threatening seafood lovers along the Gulf of Mexico. Consumption of the algae, through tainted shellfish, is not life-threatening, but it can lead to nausea, cramping, vomiting and diarrhea. The toxin produced by the algae remains whether or not the shellfish is cooked.
Fortunately, researchers detected a bloom of the algae in the waters of Port Aransas, Texas, using an automated underwater cell analyzer developed at the Woods Hole Oceanographic Institution in Massachusetts dubbed the “Imaging FlowCytobot.” Coastal managers subsequently closed shellfish beds in parts of Texas for a month, thus preventing human consumption of the algae.
Introducing the Imaging FlowCytobot into the waters off Texas was a joint effort by Robert J. Olson and Heidi M. Sosik, plankton biologists at Woods Hole who also developed the instrument, and biological oceanographer Lisa Campbell of Texas A&M University, College Station. The technology initially was developed to probe scientific questions that could not be answered using conventional methods: Olson and Sosik are interested in the reasons for which species bloom at particular times and places and in the impact they have on the rest of the ecosystem.
“Because the ocean is highly variable (in space and time), doing this kind of research has always been hampered by lack of technologies for making observations,” Sosik said. Conventional approaches involve collecting water samples from only a few places and at only a few time points, transporting them to the laboratory and conducting labor-intensive analysis using manual microscopic inspections, for example. “We got involved in transforming flow cytometry to fit this observational gap [high-resolution measurements at the organism level in the ocean environment].”
The Imaging FlowCytobot helps to address this gap, offering automation, submersibility and robustness in the demanding environment of the ocean. The instrument combines flow cytometry with video imaging of microscopic plankton to image organisms for identification and to measure chlorophyll fluorescence and scattered light from the organisms. A customized quartz flow cell with hydrodynamic focusing creates a sample stream of seawater, and single cells pass through a beam from a 635-nm diode laser made by Power Technology Inc. of Alexander, Ark. Cells containing chlorophyll emit fluorescence at 680 nm as they do so, and this fluorescence triggers a Hamamatsu xenon flashlamp strobe, which illuminates the flow cell with a 1-μs flash of light. A monochrome CCD camera made by Uniq Vision Inc. of Santa Clara, Calif., captures an image of the cell in question. A Zeiss microscope objective collects the images, as well as the scattered light and fluorescence from the cells.
An important enabling capability for the development of the instrument was the advent of cabled ocean observatory facilities such as the Martha’s Vineyard Coastal Observatory, which introduce high power and high-data bandwidth to the ocean. Without these facilities, Sosik said, the researchers could not have even imagined a demanding application like an automated submersible flow cytometer, with its large size, much higher than usual power needs and high data rates.
The Imaging FlowCytobot — an instrument that combines flow cytometry and video imaging to study microscopic plankton and its relationship to the surrounding ecosystem — allowed researchers to discover an especially abundant bloom of harmful algae off the coast of Texas. Shellfish beds in the area were closed temporarily to prevent human consumption of the algae through tainted shellfish. Here the researchers are deploying the instrument, which was designed especially to withstand the demands of the underwater environment.
The Imaging FlowCytobot transfers images and data to a shore-based laboratory, where software designed by the researchers automatically classifies the organisms by taxonomic group.
The investigators first deployed the Imaging FlowCytobot in the ocean — successfully, but perhaps too much so. “We were overwhelmed by data, especially millions of cell images,” Sosik said. “We had to develop novel data analysis approaches to transform the technology from a gee-whiz demonstration into a tool that was really useful for answering research questions about how plankton ecosystems function.”
In the fall of 2007, they deployed the streamlined system at the University of Texas Marine Sciences Institute laboratory in the Mission Bay Aransas National Estuarine Research Reserve. Originally, they were planning only to observe another toxic alga, Karenja brevis. Their discovery of the Dinophysis bloom was incidental to this initial goal, though ultimately very fortunate.
The system automatically classifies plankton by taxonomic group. Developers and Woods Hole biologists Robert J. Olson and Heidi M. Sosik, shown here examining plankton-filled water samples in Olson’s Woods Hole laboratory, are developing two additional prototypes — one to enable cell sorting based on image characteristics and the other to provide additional measurements.
Olson and Sosik continue to develop the technology. They have two lab-based prototypes that they hope will evolve into new submersible FlowCytobots. They are designing the first of these to enable physical cell sorting based on image characteristics, allowing them to choose a particular type of microscopic organism and physically separate it from the many other types of plankton present in the same seawater, and thus to study it in more detail.
They are designing the second prototype to provide additional measurements to accompany the cell images. These include fluorescence related to DNA content and pulse shapes for individual particles as they pass through the instrument’s laser beam, providing information about cell growth and morphology.
Contact: Heidi M. Sosik, Woods Hole Oceanographic Institution; e-mail: firstname.lastname@example.org.
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