Retinal tissue preparation for high-resolution live imaging of photoreceptors expressing multiple transgenes

Live imaging has become the favorite method in recent years to study the protein transport, localization and dynamics in live cells. Protein transport is extremely essential for proper function of photoreceptors. Aberration in the proper transport of proteins gives rise to the loss of photoreceptor and blindness. On the other hand, the ease of generation of transgenic Xenopus laevis tadpoles and the advantage of high resolution live confocal imaging provide new insight into understanding protein dynamics in photoreceptors. There are several steps for quantifying and visualizing fluorescently tagged proteins in photoreceptors starting with assembly of plasmids, generation of transgenic tadpoles, preparation of retinal tissues, imaging the transgenic photoreceptors and finally analyzing the recorded data. The focus of this manuscript is to describe how to prepare retinal tissues suited for live cell imaging and provide our readers with a tutorial video. We also give a summary of steps leading to a successful experiment that might be designed for imaging the ultrastructures of photoreceptors, the expression of two or more different fluorescently tagged proteins, their localization, distribution, or protein dynamics within photoreceptors. • Retinal tissue live imaging demonstrates the ultrastructures of photoreceptors. • High resolution live confocal imaging provides new insight into understanding the pathophysiology of photoreceptors.