Correction-free remotely scanned two-photon in vivo mouse retinal imaging

Non-invasive fluorescence retinal imaging in small animals is an important requirement for an array of translational vision applications.The in vivo two-photon imaging of the mouse retina may enable the long-term investigation of the structure and function of healthy and diseased retinal tissue.However,to date,this has only been possible using relatively complex adaptive-optics systems.Here,the optical modeling of the murine eye and of the imaging system is used to achieve correction-free two-photon microscopy through the pupil of a mouse eye to yield high-quality,optically sectioned fundus images.By remotely scanning the focus using an electronically tunable lens,high-resolution three-dimensional fluorescein angiograms and cellular-scale images are acquired,thus introducing a correction-free baseline performance level for two-photon in vivo retinal imaging.Moreover,the system enables functional calcium imaging of repeated retinal responses to light stimulation using the genetically encoded indicator,GCaMP6s.These results and the simplicity of the new add-on optics are an important step toward several structural,functional,and multimodal imaging applications that will benefit from the tight optical sectioning and the use of near-infrared light.