An improved interstitial diffuse optical tomography (iDOT) system has been developed to characterize the optical properties of prostate gland during the photodynamic therapy (PDT). Multiple cylindrical light diffusers with different lengths (instead of point sources used in an earlier version) and isotropic detectors are introduced interstitially in the prostate gland in-vivo. During the data acquisition, linear sources and detectors are stepping into prostate sequentially controlled by a motorized system. A computerized multi-channel attenuator system is developed to automatically control the power strength of each linear source and on times to speed up data acquisition. Three dimensional optical properties are obtained by solving the inverse problem of steady-state diffusion equation based on an adjoint model with Moore- Penrose scheme. The convergence, accuracy and the speed of the algorithms are tested in mathematical phantoms and in prostate simulating phantoms with known optical properties. For comparison, the optical properties of tissue simulating phantoms are also reconstructed using iDOT with multiple isotropic point sources. Data acquisition time in iDOT using linear sources is at least 10 times faster than using the point sources with the total data acquisition time to be less than 1 minutes. Reconstruction results showed both algorithms can successfully recover the optical properties. Reconstruction using linear sources/detectors acquisition mode is 20 times faster than the point sources/detectors method (30 minutes vs. 4 hours on a 3.4 GHz Pentium PC with 4 GB memory). We have demonstrated that linear-source/detector acquisition mode out-performs the point-source mode, and is more practical to be implemented in the clinical settings.© (2008) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.