A novel prototype instrument for mutli-spectral imaging applications is described. The device is based on an acousto-optic tuneable filter (AOTF) that can be easily attached to many standard imaging systems (e.g. endoscope or fluorescence microscope). The instrument developed offers significant advantages over typical fixed-filter based systems in terms of flexibility, performance and diagnostic potential. The selected AOTF was designed to have a large acceptance aperture suitable for imaging applications. Any filtering centre-wavelength in the visible range (450 to 700nm) can be rapidly selected by either random access or by continuous tuning thus providing a versatile performance. The prototype instrument has been demonstrated for in-vivo applications where it was attached to the eyepiece of a commercial endoscope allowing simultaneous white light and fluorescence endoscopy. Autofluorescence of endogenous protoporphyrin IX (PpIX), a biomarker of diseased tissues undergoing an inflammatory response, was mapped in vivo on a rat model. The AOTF device was also coupled to the viewing port of a commercial fluorescence microscope thus realising a powerful fluorescence imaging spectrometer capable of detecting and mapping fluorescent biomolecules in vitro.© (2005) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.