Expanding Insights: Harnessing Expansion Microscopy for Super-Resolution Analysis of HIV-1-Cell Interactions

Expansion microscopy has recently emerged as an alternative technique for achieving high-resolution imaging of biological structures. Improvements in resolution are achieved by physically expanding samples through embedding in a swellable hydrogel before microscopy. However, expansion microscopy has been rarely used in the field of virology. Here, we evaluate and characterize the ultrastructure expansion microscopy (U-ExM) protocol, which facilitates approximately four-fold sample expansion, enabling the visualization of different post-entry stages of the HIV-1 life cycle, focusing on nuclear events. Our findings demonstrate that U-ExM provides robust sample expansion and preservation across different cell types, including cell-culture-adapted and primary CD4+ T-cells as well as monocyte-derived macrophages, which are known HIV-1 reservoirs. Notably, cellular targets such as nuclear bodies and the chromatin landscape remain well preserved after expansion, allowing for detailed investigation of HIV-1-cell interactions at high resolution. Our data indicate that morphologically distinct HIV-1 capsid assemblies can be differentiated within the nuclei of infected cells and that U-ExM enables detection of targets that are masked in commonly used immunofluorescence protocols. In conclusion, we advocate for U-ExM as a valuable new tool for studying virus-host interactions with enhanced spatial resolution.