Semi-automated MicroED system unveils multiple polymorphs in fish-derived guanine crystals

Certain fish display silvery or blue colour, not through pigments, but through specialized guanine crystals that exist within iridophore cells in their skin. Within these cells, guanine (C₅H₅N₅O) crystals are precisely arranged in stratified layers that reflect incident light, resulting in structural colour. This phenomenon has garnered growing scientific attention due to its potential applications in advanced materials. However, the minute size of guanine crystals significantly hinders their structural characterization. Guanine crystals exist in multiple polymorphic forms. Previous powder X-ray diffraction (PXRD) studies have revealed that fish-derived guanine primarily adopts the anhydrous α- and/or β-polymorphs. More recently, MicroED analyses have identified the β-polymorph in salmon. Nonetheless, the crystal forms in other fish species remain insufficiently characterized. Due to their nanoscale dimensions and sensitivity to electron irradiation, reliable structural determination requires data collection from a large number of crystals. Here we established a semi-automated MicroED data collection and processing system. Initial validation was performed using synthetic guanine crystallized under acidic and basic conditions. Subsequently, guanine crystals were analyzed from three fish species: Pacific saury, Pacific cutlassfish and blue damselfish. Our findings revealed the presence of both α- and β-polymorphs in all three species, corroborating previous PXRD reports and demonstrating the efficacy of our system for the structural investigation of submicrometer-scale biogenic crystals.