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

Understanding the biological impacts of plastic pollution
requires
an effective methodology to detect unlabeled microplastics in environmental
samples. Detecting unlabeled microplastics in an organism generally
requires a digestion protocol, which results in the loss of spatial
information on the distribution of microplastic within the organism
and could lead to the disappearance of the smaller plastics. Fluorescence
microscopy allows visualization of ingested microplastics but many
labeling strategies are nonspecific and label biomass, thus limiting
our ability to distinguish internalized plastics. While prelabeled
plastics can be used to avoid nonspecific labeling, this approach
precludes the detection of environmental microplastics in organisms.
Also, using prelabeled microplastics can affect the viability of the
organism and impact plastic uptake. Thus, a method was developed that
employs nonspecific labeling with a tissue-clearing technique. Briefly,
unlabeled microplastics are stained with a fluorescent dye after ingestion by the organism. The tissue-clearing technique
then removes tissue-bound dye while rendering the structurally intact
organism transparent. The internalized plastics remain stained and
can be visualized in the cleared tissue with fluorescence microscopy.
The technique is demonstrated using polystyrene beads in living aquatic
organismsTigriopus californicusandDaphnia magnaand by spiking a model vertebrate (Cephalochordata) with different microplastics.