Pre-gelation staining expansion microscopy (PS-ExM) for visualization of the Plasmodium liver stage

Abstract

Fluorescence and light microscopy are important tools in the history of natural science. However, the resolution of microscopes is limited by the diffraction of light. One possible method to circumvent this physical restriction is the recently developed expansion microscopy (ExM). By physically expanding the sample in a swellable hydrogel, biomolecules are separated in space allowing to image molecules that are otherwise difficult to study due to their size or complexity. Furthermore, the improved resolution is useful to study protein localization and interactions. However, the original ultrastructure ExM (U-ExM) protocol is very time-consuming, and some epitopes are lost during the process. In this study, we developed a shortened pre-gelation staining ExM (PS-ExM) protocol and tested it to investigate the Plasmodium liver stage. The protocol presented in this study allows expanding pre-stained samples, which results in shorter incubation times, better preservation of some epitopes, and the advantage that non-expanded controls can be performed alongside using the same staining protocol. The protocol applicability was accessed throughout the Plasmodium liver stage showing isotropic five-fold expansion. Furthermore, we used PS-ExM to visualize the association of lysosomes to the parasitophorous vacuole membrane (PVM) as an example of visualizing host-pathogen interaction. We are convinced that this new tool will be helpful for a deeper understanding of the biology of the Plasmodium liver stage.