Using a Novel Multiplexed Algal Cytological Imaging (MACI) Assay and Machine Learning as a Way to Characterize Complex Phenotypes in Plant-Type Organisms

High-throughput phenotypic profiling assays, popular for their ability to characterize alternations in single-cell morphological feature data, have been useful in recent years for predicting cellular targets and mechanisms of action (MoAs) for different chemicals and novel drugs. However, this approach has not been extensively used in environmental toxicology due to the lack of studies and established methods for performing this kind of assay in environmentally relevant species. Here, we developed a multiplexed algal cytological imaging (MACI) assay, based on the subcellular structures of the unicellular microalgae, Raphidocelis subcapitata, a toxicology and ecological model species. Several different herbicides and antibiotics with unique MoAs were exposed to R. subcapitata cells, and MACI was used to characterize cellular impacts by measuring subtle changes in their morphological features, including metrics of area, shape, quantity, fluorescence intensity, and granularity of individual subcellular components. This study demonstrates that MACI offers a quick and effective framework for characterizing complex phenotypic responses to environmental chemicals that can be used for determining their MoAs and identifying their cellular targets in plant-type organisms.