Book excerpt: Expansion microscopy is a laboratory technique that enables nanoscale imaging of biological samples with conventional light microscopes. While expansion microscopy has traditionally been applied to specimens consisting of tissue and adherent cell culture, it has not been optimized for specimens consisting of cells in suspension. In this work, a straightforward expansion microscopy protocol was developed for suspension cells. This protocol was validated across multiple cell types including in vitro and in vivo disease models, and multiple expansion microscopy versions encompassing different methods of sample fixation, anchoring, and gelation. Suspension cells imaged after conducting the protocol exhibited increased resolution compared to images of the initial raw sample, as well as a high rate of sample retention at a variety of initial concentrations. These findings suggest the potential for the wide use of expansion microscopy to study suspension cells, which provide a versatile and scalable system for investigating cellular processes and developing therapeutic treatments. The protocol created in this work can be directly used in the future to interrogate suspension cells at nanoscale resolution to identify underlying molecular and morphological mechanisms.