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Polarized light imaging (PLI) is a new method which quantifies and visualizes nerve fiber direction. In this study, the educational value of PLI sections of the human brainstem were compared to histological sections stained with Luxol fast blue (LFB) using e-learning modules. Mental Rotations Test (MRT) was used to assess the spatial ability. Pre-intervention, post-intervention, and long-term (1 week) anatomical tests were provided to assess the baseline knowledge and retention. One-on-one electronic interviews after the last test were carried out to understand the students' perceptions of the intervention. Thirty-eight medical students, (19 female and 19 males, mean age 21.5 ± SD 2.4; median age: 21.0 years) participated with a mean MRT score of 13.2 ± 5.2 points and a mean pre-intervention knowledge test score of 49.9 ± 11.8%. A significant improvement in both, post-intervention and long-term test scores occurred after learning with either PLI or LFB e-learning module on brainstem anatomy (both P < 0.001). No difference was observed between groups in post-intervention test scores and long-term test scores (P = 0.913 and P = 0.403, respectively). A higher MRT-score was significantly correlated with a higher post-intervention test score (r k  = 0.321; P < 0.05, respectively), but there was not a significant association between the MRT- and the long-term scores (r k = -0.078; P = 0.509). Interviews (n = 10) revealed three major topics: Learning (brainstem) anatomy by use of e-learning modules; The "need" of technological background information when studying brainstem sections; and Mnemonics when studying brainstem anatomy. Future studies should assess the cognitive burden of cross-sectional learning methods with PLI and/or LFB sections and their effects on knowledge retention., (© 2021 The Authors. Anatomical Sciences Education published by Wiley Periodicals LLC on behalf of American Association for Anatomy.)