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Purpose: To demonstrate the application of high-resolution spectral-domain optical coherence tomography (SD-OCT) for three-dimensional (3D) retinal imaging of small animals and quantitative retinal information extraction using 3D segmentation of the OCT images., Methods: A high-resolution SD-OCT system was built for in vivo imaging of rodent retina. OCT fundus images similar to those acquired with a scanning laser ophthalmoscope (SLO) were constructed from the measured OCT data, which provided precise spatial registration of the OCT cross-sectional images on the fundus. A 3D segmentation algorithm was developed for calculation of the retinal thickness map. OCT images were compared by histologic examination., Results: High-quality OCT images of the retinas of mice (B6/SJLF2 for normal retina, rhodopsin-deficient Rho(-/-) for photoreceptor degeneration, and LH(BETA)T(AG) for retinoblastoma) and rat (Wistar) were acquired. The OCT images compared well with histology. Not only was a 3D image of the tumor in a retinoblastoma mouse model successfully imaged in vivo but the tumor volume was extracted from the 3D image. Retinal thickness maps were calculated that enabled successful quantitative comparison of the retinal thickness distribution between the normal (202.3 +/- 9.3 microm) and the degenerative (102.7 +/- 12.6 microm) mouse retina., Conclusions: High-resolution spectral-domain OCT provides unprecedented high-quality 2D and 3D in vivo visualization of retinal structures of mouse and rat models of retinal diseases. With the capability of 3D quantitative information extraction and precise spatial registration, the OCT system made possible longitudinal study of ocular diseases that has been impossible to conduct.