Cars as a Method for the Detection of Toxic Pollutants in MIBA: The Case of Phthalates on Danio renio’s Larva

Living organisms (eggs and larvae of Danio rerio) have been incubated in an innovative microincubator MIBA with various concentrations of Di-N-Butyl Phthalate (DBP) derivatives for different times. Images were acquired with a macromultiphoton or CARS microscope and probe DBP (control) and its derivative N3-aDBP were detectable thanks to the signal emitted by the alkyne and a signal possible thanks to the auto-fluorescence of the aromatic ring. Using fluorescence imaging and CARS microscopy methods, we investigated the value of Raman-CARS that could vibrate unnatural groups (N3 or C≡C) previously grafted onto the DBP molecule in zebrafish. Contrary to other products, N3-aDBP probe has been detected continuingly by macro-fluorescence in larva but CARS signal was only detected in FLIM mode (Fluorescence Lifetime Imaging Microscopy). To conclude, CARS microscopy can be used to image toxic pollutants even by integrating photo-activatable probes. In our case, photo-physical reactions following the irradiation of the probe lead to new photoproducts that are not identified and difficult to characterize in fluorescence and specific CARS signal.