Expansion Microscopy of Mouse Photoreceptor Cilia

The small size of ciliary structures that underlies photoreceptor function and inherited ciliopathies requires imaging techniques adapted to visualizing them at the highest possible resolution. In addition to powerful super-resolution imaging modalities, emerging approaches to sample preparation, including expansion microscopy (ExM), can provide a robust route to imaging specific molecules at the nanoscale level in the retina. We describe a protocol for applying ExM to whole retinas in order to achieve nanoscale fluorescence imaging of ciliary markers, including tubulin, CEP290, centrin, and CEP164. The results are consistent with those from other super-resolution fluorescence techniques and reveal new insights into their arrangements with respect to the subcompartments of photoreceptor cilia. This technique is complimentary to other imaging modalities used in retinal imaging, and can be carried out in virtually any laboratory, without the need for expensive specialized equipment.