Hyperspectral scanning laser optical tomography is developed to provide spectrally resolved volume data sets with high spectral resolution for large mesoscopic samples. It can be used to resolve largely overlapping fluorophores, as demonstrated by the 3D fluorescence hyperspectral reconstruction of a dual‐labelled mouse thymus gland sample and to distinguish between signals from autofluorescence of diseased and normal tissue without prior knowledge. Further details can be found in the article by Lingling Chen, Guiye Li, Li Tang, et al. ( e201800221 ).