We present an optical system for small animal imaging that can combine various in vivo imaging modalities, including fluorescence (intensity and lifetime), spectral, and trans-illumination imaging. This system consists of light-tight box with ultrafast pulsed or cw laser light excitation, motorized translational and rotational stages, a telecentric lens for detection, and a cooled CCD camera that can be coupled to an ultrafast time-gated intensifier. All components are modular, making possible laser excitation at various wavelengths and pulse lengths, and signal detection in a variety of ways (multimode). Results of drug nanoconjugate carrier delivery studies in mice are presented. Conventional and spectrally-resolved fluorescence images reveal details of in vivo drug nanoconjugate carrier accumulation within the tumor region and several organs in real time. By multi-spectral image analysis of ex vivo specimens from the same mice, we were able to evaluate the extent and topology of drug nanoconjugate carrier distribution into specific organs and the tumor itself.© (2007) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.