AbstractBackground To study the mapping from the retina to the brain, typically a small region of the retina is injected with a dye, which then propagates to the retina’s target structures. To determine the location of the injection, usually the retina is dissected out of the eye, flattened and then imaged, causing tears and stretching of the retina. The location of the injection is then estimated from the image of the flattened retina. Here we propose a new method that avoids dissection of the retina.ResultsWe have developed IntactEye, a software package that uses two orthogonal images of the intact retina to locate focal injections of a dye. The two images are taken while the retina is still inside the eye. This bypasses the dissection step, avoiding unnecessary damage to the retina, and speeds up data acquisition. By using the native spherical coordinates of the eye, we avoid distortions caused by interpreting a curved structure in a flat coordinate system. Our method compares well to the projection method and to the Retistruct package, which both use the flattened retina as a starting point. We have tested the method also on synthetic data, where the injection location is known. Our method has been designed for analysing mouse retinas, where there are no visible landmarks for discerning retinal orientation, but can also be applied to retinas from other species.Conclusions IntactEye allows the user to precisely specify the location and size of a retinal injection from two orthogonal images taken of the eye. We are solving the abstract problem of locating a point on a spherical object from two orthogonal images, which might have applications outside the field of neuroscience.