Coimbra Health School 1 article published in JoVE Biochemistry Analysis of Fluorescent-Stained Lipid Droplets with 3D Reconstruction for Hepatic Steatosis Assessment Karina Garcia1,2,3,4, André Alves1,2,3, Teresa M. Ribeiro-Rodrigues1,2,3, Flávio Reis1,2,3, Sofia Viana1,2,3,4 1Institute of Pharmacology & Experimental Therapeutics & Coimbra Institute for Clinical and Biomedical Research (iCBR), Faculty of Medicine, University of Coimbra, 2Center for Innovative Biomedicine and Biotechnology (CIBB), University of Coimbra, 3Clinical Academic Center of Coimbra (CACC), 4Polytechnic Institute of Coimbra, Coimbra Health School Herein, we demonstrate an optimized BODIPY 493/503 fluorescence-based protocol for lipid droplet characterization in liver tissue. Through the use of orthogonal projections and 3D reconstructions, the fluorophore allows for successful discrimination between microvesicular and macrovesicular steatosis and may represent a complementary approach to the classical histological protocols for hepatic steatosis assessment.