The quantitative imaging of trace elements in biological samples can bring information on their assimilation pathways and help understanding their toxicity or essentiality. This study presents the development of two quantitative LA-ICP MS methodologies and their comparison for Se, Hg, Cu, Zn and Mo imaging in rainbow trout. The first method is based on spiked polymer films (dextran) allowing internal standardization with Ge and Te. The second method relies on the matrix-matched standards (MMS) methodology, with homogenized and spiked trout muscle and calf liver. For Se, Cu, Zn and Mo, linear regressions with correlation coefficient above 0.994 were obtained by LA-ICP MS analysis for both methods. For Hg, only MMS calibration provided linear regression as polymer films exhibited Hg unstable signals during LA-ICP MS analysis. Element concentrations spiked in muscle and liver MMS were also confirmed by ICP MS and used to validate the polymer films method as values obtained for Se, Hg, Mo and Cu were in good agreement. While LODs of polymer film method were in the range of 0.06–0.6 µg.g−1 for Cu, Se, Mo, and 5 µg.g−1 for Zn, values were higher for MMS method (from 0.06 (Mo) to 2.2 (Cu) µg.g−1, and 30 of µg.g−1 for Zn). Particular attention has been paid to sample embedding and water was found to minimize internal standard response bias. Both methods were then applied to the quantitative mapping of these five elements in rainbow trout fry supplemented or not with organic forms of Se and Hg through parental and direct feeding. Calibrations were analysed before and after sample imaging evidencing a lower bias between the two quantifications with polymer films calibration than with MMS calibration. For the first time, elements concentrations were determined at 60 × 60 µm spatial resolution in specific tissues or organs of entire fry. Depending on the element, polymer films quantification was in agreement with trout muscle (Cu, Se) or with calf liver MMS quantification (Zn).