Application of expansion microscopy on developing Arabidopsis seeds

Expansion microscopy (ExM) improves image resolution of specimens without requirements of sophisticated techniques or equipment. Probes or proteins are anchored onto an acrylamide gel matrix which is then expanded with osmotic pressure. As the physical distance between two signal points increases, previously confounded signals can be resolved while their relative spatial locations are retained. ExM has been successfully applied to several animal tissues, but its application to plant tissues was only recently demonstrated. Here we provide a detailed ExM protocol for plant tissues using fluorescent immunostaining of developing Arabidopsis thaliana (Arabidopsis) seeds as an example. This modified ExM protocol enables expansion of ovule/seed samples, and preserves the majority of fluorescent protein signals in the expanded samples. The fluorescent immunostaining observed using this protocol demonstrates the feasibility of detecting cellular events and subcellular structures in expanded plant samples. This ExM protocol variant for plants can serve as a guideline for applying ExM to various plant tissues and help increase the resolution of corresponding microscopy based studies.