Expansion microscopy helps chart the planktonic universe

Plankton are the invisible engines of life on Earth, producing much of the planet’s oxygen and forming the foundation of the oceanic food chain. They are also incredibly diverse, with tens of thousands of species described so far, and many more waiting to be discovered. Among them, protists,  tiny, single-celled organisms, stand out for their extraordinary diversity and evolutionary significance, yet for decades, scientists could study them only through genomic data, as reliable imaging methods were lacking. 

During the COVID-19 pandemic, EMBL Group Leader Gautam Dey received a Zoom call from his collaborator Omaya Dudin, then leading a group at EPFL. Dudin had just adapted a new technique to visualise the internal architecture of Ichthyosporea — a marine protist closely related to animals and fungi — overcoming the long-standing barrier of its impermeable cell walls. The new technique called expansion microscopy, first developed by scientists at MIT, USA, and further optimised into ultrastructure expansion microscopy (U-ExM) for exploring sub-cellular ultrastructure by Paul Guichard and Virginie Hamel at the University of Geneva, had made the cell wall permeable, and the protist's inner structures could now be clearly visualised and studied.

Determined to study more marine organisms with this method, Dudin, Dey, Guichard and Hamel started a cooperation that, three years later, has succeeded in generating almost encyclopedic knowledge of hundreds of protist species and is on its path to creating a planetary atlas of plankton.

The EMBL-led Traversing European Coastlines (TREC) expedition was a great opportunity for the researchers to dive deeper into the internal structures of different marine microbes. Recently published in Cell, their collaboration has yielded unprecedented and in-depth insights into the cellular architecture of over 200 plankton species, in particular eukaryotes – organisms that have a cell nucleus enclosed by a membrane. This was the first step in PlanExM, a TREC plug-in project which aims to reveal the world of planktonic ultrastructural diversity with expansion microscopy.

Unveiling cellular secrets with ultrastructure expansion microscopy

At Roscoff, France, one of the TREC expedition's first prominent sampling stops, the Station Biologique hosts one of the most complete culture collections of marine microorganisms in Europe. The team asked Ian Probert, the facility's manager, how many samples they might receive to run an expansion microscopy pilot – expecting about 20 species – only to find the doors of the full facility, with over 200 species, being opened to them.

"We spent three days and nights just fixing those samples. This was a treasure trove we could not let go of," said co-first author Felix Mikus, who completed his PhD from the Dey Group and is now a postdoc in Dudin’s current lab at the University of Geneva. 

Expansion microscopy, a relatively young technique only turning 10 years old this year, works by physically 'expanding' biological samples. The sample – which can contain single-celled organisms, cells, or tissues – is first embedded in a clear gel. Then, the gel is allowed to expand by absorbing water. Marvellously, many of the cell's internal structures remain intact during the process and expand more or less proportionally, allowing scientists to 'magnify' the sample four or even 16 times without needing to resort to lenses. 

“When combined with regular light microscopy methods, expansion microscopy allows scientists to bypass the standard wavelength barriers which limit how small a structure can be resolved using light microscopy,” said Guichard and Hamel. 

Using the Roscoff samples, as well as a second culture collection from Bilbao, Spain, the researchers went on to perform one of the most extensive investigations to date of diversity of the cytoskeleton – the filamentous network that forms the underlying structure of eukaryotic cells. In particular, researchers looked at microtubules – hollow, tubular filaments that help the cell maintain its structure, divide, and move, and centrins – a family of proteins found in the structures that organise microtubules inside cells. 

“We were able to map features of microtubule and centrin organisation across many different eukaryotic groups,” said Hiral Shah, EIPOD Postdoctoral Fellow in EMBL's Dey and Schwab groups and co-first author of the study. “The scale of the study, with many species characterised in each group, opens up the possibility to make evolutionary predictions. For instance, dinoflagellates, one of the most diverse groups found in oceans across the planet, are well-represented in our study. We were able to map the presence of tubulin and centrin structures associated with the cell cortex or the flagella in these species.”

“U-ExM is transforming how we explore protist ultrastructure,”  said Armando Rubio Ramos, co-first author of the study and Postdoctoral Fellow at Hamel & Guichard’s research group at the University of Geneva. “By combining this technique with high-throughput imaging and comparative analyses, we can begin to decode how cellular architecture has diversified across evolution. It’s a bridge between molecular data and the physical organisation of life at the microscopic scale.” 

According to the researchers, this analysis not only helps us understand the fundamental principles that underlie the organisation of a eukaryotic cell, it also offers clues to the evolutionary history of the cytoskeleton architecture in these species. Moreover, it demonstrates the potential of expansion microscopy as a powerful tool for studying complex samples, including those collected directly from marine ecosystems. 

“Our adventures with expansion microscopy are only beginning,” said Dey. “This is perhaps the first high-resolution microscopy technique that has the potential to match the scale and ambition of large biodiversity genomics projects, enabling us in the near future to associate new multiomics data with cellular physiology at scale across the tree of life.”  

With Thomas Richards from Oxford University joining the team, Dey and Dudin were also successful in obtaining a prestigious Moore Foundation Grant worth CHF 2 million to continue this project. 

“The next step is to selectively look deeper into certain species within this broad collection to answer specific questions, such as understanding how mitosis and multicellularity evolved and the phenotypic diversity that underlie major evolutionary transitions,” Dudin said.