Changing the Flow of Ocean Science

As Hurricane Emily bore down on Bermuda in 1987, Researcher David Phinney and Bigelow Laboratory co-founder Clarice Yentsch were there as part of a research mission that was revolutionizing microbial oceanography.

The storm rocked the island, toppled trees, and knocked out the power, bringing the team’s research to a halt. When the skies cleared, the scientists emerged to survey the destruction left in the hurricane’s wake. Phinney, like all good Mainers, was well-versed with a chainsaw and promptly went to work alongside colleagues and locals, clearing fallen trees to begin the recovery.

This kind of scrappy enthusiasm is the same that defined the team’s endeavor to advance what would become one of Bigelow Laboratory’s biggest contributions to ocean science – a technique called flow cytometry. Today, the technique is used around the world to study aquatic environments. It has been instrumental in pivotal breakthroughs, from advances in genomics technologies to the discovery of new species.

Flow cytometry is a way of quickly counting and characterizing large quantities of microscopic particles, such as cells, viruses, bacteria, and phytoplankton. It works by suspending particles in a fluid where they are forced to flow, single file, in a stream. The particles are then passed in front of laser beams where they exhibit different characteristics based on their size and fluorescence. Using this information, researchers can determine the size, shape, and basic identity of the particles, which can then be used to sort particles based on these parameters.

One of the major benefits of flow cytometry is its speed. Traditional microscope techniques can be used to count and characterize cells, but may take weeks or months. Flow cytometers can count and isolate tens to hundreds of thousands of cells per second.

“In graduate school, I spent the whole summer in a dark room counting bacteria in samples of seawater under a microscope, and just about everything I did that summer, a flow cytometer could do in a day,” said Deborah Bronk, Bigelow Laboratory President and CEO. “It has revolutionized the study of phytoplankton and bacteria.”

The technique has allowed scientists to study particles and cells in greater numbers and smaller sizes than previously possible, enabling studies that tackle harder questions. It even led to the discovery of the most abundant phytoplankton, and possibly the most abundant photosynthetic organism, on Earth: Prochlorococcus.

The introduction of flow cytometry to aquatic science began at Bigelow Laboratory in the early 1980s. Yentsch was speaking about the difficulties of processing cells with a microscope to a colleague who had just gotten back from a biomedical conference. They started to wonder if flow cytometers, which were being used to count blood and cancer cells, might offer a solution. With the help of her Bigelow Laboratory colleagues, including Phinney, she began to pursue funding to test her hypotheses.

Others weren’t as quick to see the potential of the technology, and her first attempts to secure grant support were unsuccessful. Eventually she realized that it was not because the attempts were too grand, but because they were too humble.

“We were asking for the minimum amount and needed to think bigger,” she said. “We were advised that if we could demonstrate the possible usefulness of this tool in the ocean sciences, that agencies would not want to fund the Volkswagen – they would want to fund the Mercedes.”

Yentsch saw enormous opportunities for flow cytometry in the study of the ocean, which teems with microscopic life that forms the foundation of global ocean health. So, she and Phinney started working in Bermuda to prove the tool could work. The ocean around the island is home to some of the world’s smallest aquatic microbes, which she thought would be a perfect demonstration of the technology’s potential.

“We were trying to test it in the most difficult situation,” she said. “Had we not been so enthusiastic, we would have given up. But we kept doing more experiments. We kept going and getting more and more interesting results.”

By analyzing seawater samples using flow cytometry, the team completed work in a few months that might have otherwise taken a year. As the results came in, so did the funding. The National Science Foundation, the Office of Naval Research, and the Maine Department of Marine Resources soon stepped up to support the work, and Bigelow Laboratory became the first laboratory in the world to have a flow cytometer dedicated for use in aquatic research and the ocean science community.

Clarice and her team cultivated a whole new field of oceanographic research. The process wasn’t easy or immediate, but the team’s enthusiasm built the groundwork for the new tool.

“Our efforts really built a sense of community,” Yentsch said. “We were not just advancing our own work, but showing others how it could serve them and helping them build expertise.”

Bigelow Laboratory hosted workshops and steadily fostered a collaborative group of scientists that developed the techniques and protocols for how flow cytometry would be used in ocean science. Many of them later became senior research scientists at the lab or landed prominent positions, such as Richard Spinrad, who was recently nominated by President Joe Biden to lead the National Oceanic and Atmospheric Administration.

“Through our workshops and people testing flow cytometry on their own research, it brought a tremendous amount of attention to Bigelow,” Phinney said. “Clarice really brought the community together around this tool with her enthusiasm about what it could do.”

Almost 40 years after its introduction to ocean science, flow cytometry is now a staple of microbial oceanography and used at most oceanographic research institutes. It has also been instrumental to another breakthrough pioneered at Bigelow Laboratory – single-celled genomics, which allows scientists to analyze the genetic information contained in individual cells.

“Flow cytometry has completely changed our ability to quickly obtain cell estimates of what populations are out there, whether it be at the surface of the ocean, ocean depths, or deep in a mineshaft,” said Senior Research Scientist Nicole Poulton, the director of Bigelow Laboratory’s Center for Aquatic Cytometry. “This has enabled us to advance our understanding of the different microbes that are out there and how they are related to one another, which has revolutionized our understanding of the tree of life.”

Poulton has been the director of the Center since 2014. She took over the position from Michael Sieracki, who was director after Yentsch. Along with the role, she has taken up the mantle of growing a research community around flow cytometry. She regularly runs technical workshops to share her expertise, and she was elected as a council member to the International Society for the Advancement of Cytometry in 2020, as the only representative for aquatic and environmental cytometry.

From studying phytoplankton to microbial ecology, Poulton also conducts her own research using flow cytometry. One current project aims to better understand and detect microplastics with colleagues at the University of Minnesota Duluth.

Plastic is the most prevalent type of marine debris found in the ocean and the Great Lakes, and microplastics are particularly challenging to address – and to study. Scientists do not yet know the full impact of these particles, but they make their way through food webs and into the digestive systems of aquatic animals and the people that eat them.

Much like the phytoplankton and bacteria cells that originally inspired Yentsch to begin using flow cytometry, studying microplastics is currently arduous and prone to errors. Poulton believes that flow cytometry can speed up and improve this process.

“We can use flow cytometry to isolate even the smallest microplastics, so we can understand what type of plastics are most likely to end up in the aquatic food chain,” Poulton said. “By understanding how microplastics are distributed, we can begin to locate where they are likely going to be a problem.”

Poulton is also developing new techniques and instrumentation to address new challenges in ocean science. In collaboration with Senior Research Scientists Ramunas Stepanauskas, the Director of the Single Cell Genomics Center, she is currently working on the development of a new flow cytometer that uses both cell imaging and multiple lasers to improve the detection capabilities for isolating individual cells.

This advancement may again launch a new era of groundbreaking research.

“When a new tool is developed, you realize you can go back and ask research questions again, in a different way,” Yentsch said. “Suddenly you're able to look at the world, the ocean, or single cells in a totally new way, and everyone’s research leaps forward.”