Steps are presented towards the development of a new bioluminescence tomography (BLT) imaging system for in vivo small animal studies. A 2-mirror-based multi-view data collection scheme is investigated in conjunction with multi-spectral imaging, leading to the production of 3D volumetric maps of molecular source distributions in simulation and in physical phantom studies by way of a finite element model (FEM) based reconstruction method. A proof of concept is subsequently demonstrated showing a full work flow from data acquisition to 3D reconstruction. Results suggest that the multi-view mirror-based approach represents a strong improvement over standard single-view methods, with improvements of up to 58% in source localisation accuracy being observed for deep sources.© (2011) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.