Complete infiltrating brain tumor margin resection continually eludes neurosurgeons due to inherent limitations of current margin localization techniques. A need exists for an objective, on-site, real-time imaging system which can accurately localize brain tumor margins and therefore be used as a basis for image-guided surgery. Optical biopsy methods are a proven means for successful brain tissue discrimination, indicating promise for spectral imaging to fill such a need. Before testing spectral imaging for surgical guidance, various spectral imaging modalities must be systematically compared to determine the modality most conducive to the clinical setting. A liquid crystal tunable filter spectral imaging system was characterized for field of view, spatial and spectral resolution, and ability to retain spectral features acquired from a clinical single-pixel spectroscopy system. For a 35-mm diameter field of view, the system possessed a spatial resolution of 50 μm in both image dimensions and a spectral resolution which monotonically increased from 10 to 30 nm over the tuning range of the filter. Differences between imaging and single-pixel spectra for location and FWHM of fluorescence peaks from two fluorescent dye targets were summarily less than 3 nm. However, two remediable artifacts were introduced to imaging system spectra during spectral sensitivity correction.© (2003) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.