Tissue Clearing To Localize Microplastics via Three-Dimensional Imaging of Whole Organisms

Understanding the biological impacts of plastic pollutionrequiresan effective methodology to detect unlabeled microplastics in environmentalsamples. Detecting unlabeled microplastics in an organism generallyrequires a digestion protocol, which results in the loss of spatialinformation on the distribution of microplastic within the organismand could lead to the disappearance of the smaller plastics. Fluorescencemicroscopy allows visualization of ingested microplastics but manylabeling strategies are nonspecific and label biomass, thus limitingour ability to distinguish internalized plastics. While prelabeledplastics can be used to avoid nonspecific labeling, this approachprecludes the detection of environmental microplastics in organisms.Also, using prelabeled microplastics can affect the viability of theorganism and impact plastic uptake. Thus, a method was developed thatemploys nonspecific labeling with a tissue-clearing technique. Briefly,unlabeled microplastics are stained with a fluorescent dye after ingestion by the organism. The tissue-clearing techniquethen removes tissue-bound dye while rendering the structurally intactorganism transparent. The internalized plastics remain stained andcan be visualized in the cleared tissue with fluorescence microscopy.The technique is demonstrated using polystyrene beads in living aquaticorganismsTigriopus californicusandDaphnia magnaand by spiking a model vertebrate (Cephalochordata) with different microplastics. The method presented combines a tissue-clearingtechniqueand light sheet microscopy to image and localize microplastics thathave been dyed after internalization.