Trypanosoma brucei is a single cell parasite that causes Human African Trypanosomiasis (HAT), which is also known as African sleeping sickness. When Justin Wiedeman, a PhD student in Dr. Kojo Mensa-Wilmot’s lab, started investigating protein kinase TbCK1.2, which is an enzyme found near the flagellum of Trypanosoma brucei, he found that he needed a tool for visualizing the membranes of this parasite, and none of the membrane probes on the market quite did the job. Wiedeman turned to mCLING, a dye that has been developed to track the membranes of neurons using super-resolution microscopy, to see if he could adapt the technology to T. brucei membranes.
“Researchers have been forced to use crude methods to outline trypanosomes in fluorescence microscopy,” said Wiedeman, “We are the first group to solve this general problem in super-resolution microscopy of T. brucei. mCLING labels the flagellum and plasma membrane vividly, sometimes providing details of cell structure that rivals images obtained with scanning electron microscopy.”
Other current leading methods limit images to a two-dimensional representation of the cell and prohibit the ability to determine cell periphery, making it difficult to determine the orientation of organelles within the cell.
Using a combination of standard-resolution and super-resolution fluorescence microscopy, he was able to confirm mCLING labels the plasma and flagellar membranes of T. brucei. He discovered mCLING could be used to track endocytosis (the process of importing molecules into the cell) in real time. Using the Zeiss ELYRA S1 super-resolution microscopy, mCLING allowed for a 3D reconstruction of the parasite. This is the first time such an image has been reported, and will allow for the study of trypanosomes in finer detail than ever before.