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. Two-photon microscopy: correction-free imaging of the mouse retina A correction-free, non-invasive technique for in vivo two-photon imaging of the mouse retina has been developed. Two-photon imaging offers many advantages for in vivo visualization of the retinas of small mammals, but has never been realized until now. Modeling and experiments by researchers at the Technion – Israel Institute of Technology revealed that focusing on the retina is strongly constrained by commonly used imaging lenses and the optics of the mouse eye. The researchers developed a two-photon imaging system capable of imaging the retina and whose focal plane is remotely scanned using an electronically tunable lens. They obtained high-quality, optically sectioned fundus images of fluorescent microstructures down to the cellular level, and performed the first functional two-photon calcium imaging of a live mouse retina. Their system has significant advantages over conventional wide-field imaging systems.