A Practical Guide to Ultrastructural Expansion Microscopy (U-ExM) in Trypanosoma brucei

Expansion Microscopy (ExM) is a powerful method that enables super-resolution fluorescence microscopy, beyond the diffraction limit of light, with conventional fluorescence microscopes. Briefly, macromolecules of the cell are "anchored" using acrylamide:formaldehyde, incorporated into an acrylamide/sodium cross-linked polymer, and then uniformly expanded by osmotic pressure. The molecule of interest is detected by specific labeling either prior to or after expansion. Here, we focus on the detection of proteins by Ultrastructural Expansion Microscopy (U-ExM), which achieves a resolution of 70-50 nm. In this variant, the labeling is done after the expansion; compared with pre-expansion labeling, this increases the resolution and also reduces molecular crowding and thus allows staining even in organisms with cell walls, cuticles, and dense extracellular matrices. We have used U-ExM to study the nuclear pore complex and cytoskeleton components of Trypanosoma brucei. Expansion microscopy has been readily adapted to Kinetoplastea species and should be also adaptable to virtually any Euglenozoa.