Use of multi-photon laser-scanning microscopy to describe the distribution of xenobiotic chemicals in fish early life stages

To better understand the mechanisms by which persistent bioaccumulative toxicants (PBTs) produce toxicity during fish early life stages (ELS), dose-response relationships need to be understood in relation to the dynamic distribution of chemicals in sensitive tissues. In this study, a multi-photon laser scanning microscope (MPLSM) was used to determine the multi-photon excitation spectra of several polyaromatic hydrocarbons (PAHs) and to describe chemical distribution among tissues during fish ELS. The multi-photon excitation spectra revealed intense fluorescent signal from the model fluorophore, pentamethyl-difluoro-boro-indacene (BODIPY®), less signal from benzo[a]pyrene and fluoranthene, and no detectable signal from pyrene. The imaging method was tested by exposing newly fertilized medaka (Oryzias latipes) eggs to BODIPY® or fluoranthene for 6h, followed by transfer to clean media. Embryos and larvae were then imaged through 5 days post-hatch. The two test chemicals partitioned similarly throughout development and differences in fluorescence intensity among tissues were evident to a depth of several hundred microns. Initially, the most intense signal was observed in the oil droplet within the yolk, while a moderate signal was seen in the portion of the yolk containing the yolk-platelets. As embryonic development progressed, the liver biliary system, gall bladder, and intestinal tract accumulated strong fluorescent signal. After hatch, once the gastrointestinal tract was completely developed, most of the fluorescent signal was cleared. The MPLSM is a useful tool to describe the tissue distribution of fluorescent PBTs during fish ELS.