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Images collected by intravital microscopy suffer from motion artifacts which necessitates the use of image registration techniques to reconstruct three-dimensional or time series data. In this paper, we describe a multi-resolution/multi-level approach to non-rigid image registration using B-splines with a coarse-to-fine strategy. A control lattice with a coarse grid spacing is used first to account for slowly changing deformations. Next, a control lattice with a finer grid spacing is used to account for more localized and quickly changing deformations. We demonstrate this method on a sequence of images acquired from the lung of a living rat.