Alternating-Luminophore Electrochemiluminescence Microscopy Reveals Developmental Stages of Brine Shrimp

Brine shrimp (Artemia sp.) are model organisms used in ecotoxicity studies and other important biological testing. These mesoscale (l × w × h = 100s - 1000s μm) organisms mature rapidly and are ideal candidates for whole-organism imaging due to their ease of culture, definable anatomy, and well-documented development. We employ electrochemiluminescence (ECL) microscopy to examine both the chemical environment and morphology of these developing lower-level crustaceans on an electrified interface. We survey the tris-(2,2'-bipyridyl)-ruthenium-(II) ([Ru-(bpy)₃]²⁺) and luminol luminophores, highlighting important trade-offs across developmental stages. In the luminol system, positive ECL emission occurs from the cyst (egg) despite no addition of coreactant, thus pointing toward the potential location of reactive oxygen species in the local chemical environment near the cyst. We find that the kinetic regime (lower potentials) of the [Ru-(bpy)₃]²⁺ to be confined to the electrode surface and the Artemia. In contrast, in the mass-transfer-limited regime (higher potentials), shadow ECL dominates as Artemia acts as a blocking entity. Furthermore, we sum ECL micrographs and analyze the resulting composite images to evaluate trade-offs between temporal resolution and ECL intensity. Overall, this work establishes a foundation for mesoscale imaging of multicellular organisms and highlights the challenges and opportunities associated with ECL microscopy and luminophore choice.