Whole-Mosquito Imaging Offers a New Window into Malaria Parasites

When Benjamin Liffner, a parasite biologist at Adelaide University, learned how to perform expansion microscopy as a postdoctoral researcher at Indiana University, he immediately appreciated the method’s implications for his field.

“When you study parasites, good microscopy is a necessity because malaria parasites are one by 1.5 microns,” said Liffner. And as eukaryotic organisms, parasites contain several organelles densely packed in their small bodies, requiring specialized microscopy to visualize these crucial structures.

This had Liffner rethinking how he defined his research goals. “I had always really considered myself to be a parasite person first,” he said. “[But] I realized that I'm probably 50-50 parasite person and microscopy person.”

So, Liffner established his lab with the aim of utilizing specialized microscopy to better understand parasite biology. He and his team recently standardized a protocol that helped them peek into malaria parasites within whole mosquitoes using expansion microscopy, offering a way to study the microbes in their host’s anatomical context.

The road to imaging Plasmodium in whole mosquitoes was long. In 2023, Liffner and his colleagues had initially developed protocols to image parasites in cultured red blood cells, followed by a method to visualize the microbes in dissected salivary glands and midguts of mosquitoes in 2024.^1,2 Soon afterwards, Liffner and Joe van den Bergh, a member in Liffner's lab, sought to build on this. “We came up with this idea that said, ‘Well, rather than taking the salivary glands and the midgut out of the mosquito, why don't we just try and expand the whole mosquito,’” said Liffner.

The researchers went through a laborious process to optimize the quantity of reagents and incubation time periods to prepare the sample for expansion microscopy. After multiple iterations, the team figured out a way to crosslink the mosquito exoskeleton with a hydrogel, such that adding water to the sample would expand it physically for microscopy.

“Seeing the whole mosquito was cool, but we [were] interested in the parasites inside it, which are only a tiny, tiny fraction of the whole mosquito,” said Liffner, explaining that imaging a thicker sample proved to be challenging. When he shared his unsuccessful attempts at an expansion microscopy conference at Göttingen last year, Miguel Cardoso Mestre, a graduate student at Cambridge University, offered an alternative method that he and his colleagues had tested.³

This involved a setup with a water immersion objective lens and an imaging dish with the same refractive index as water, which helped them image at a greater depth within the sample. Convinced, Liffner tried the setup with his mosquito specimens. “That for us was really the game changer,” he said. The alternate protocol, coupled with staining for protein and DNA, helped Liffner and his team visualize parasites within a mosquito’s body with preserved internal anatomy.

While Liffner was excited to see the parasites within the mosquito, he admitted that he designed this project to tackle the technical challenge rather than to answer any specific biological question. But the moment he saw the images and realized that they offered a better view of the parasites than ever seen, he realized the immense potential of the tool.

Going forward, he hopes that people use this technique to answer interesting questions. “What I would most love to see about this is for people to use it for applications that I fundamentally couldn't have predicted,” said Liffner. “My hope is to be surprised by what people do with it.”