Simplifying In Vivo Retinal Imaging with Correction-Free Two-Photon Microscopy
Bar-Noam AS, Farah N, Shoham S. Correction-free remotely scanned two-photon in vivo mouse retinal imaging. Light Sci Appl 2016;5(1):e16007. doi: 10.1038/lsa.2016.7.
Background: Non-invasive fluorescence imaging of the mouse retina is essential for studying retinal structure and function in health and disease. While two-photon microscopy provides optical sectioning and reduced phototoxicity, its in vivo use in mice has been limited by the need for adaptive optics to correct ocular aberrations. Overcoming these limitations would allow simpler and more accessible retinal imaging.
Hypothesis: This study hypothesized that model-based optical design combined with remote focusing could enable high-resolution, correction-free two-photon imaging of the mouse retina through the intact pupil.
Methods: The authors developed a two-photon imaging setup that used a tunable infrared laser and an electronically tunable lens combined with offset optics to scan axially without corneal contact. Optical performance was modeled analytically and with ray-tracing software. Mouse retinal imaging was performed using fluorescein and genetically encoded calcium indicators. Image acquisition was conducted with ScanImage, and image analysis was performed using standard image processing tools.
Results: The optical model accurately predicted in vivo focal shifts and resolution. The system generated high-quality, optically sectioned three-dimensional angiograms and cellular-resolution images of GCaMP-expressing retinal cells. Functional calcium imaging revealed robust, repeatable light-evoked responses in retinal ganglion cells without apparent retinal damage across repeated sessions.
Conclusions: This study demonstrated that a simple, adaptive-optics–free two-photon imaging system can achieve correction-free, cellular-resolution in vivo retinal imaging and functional calcium recordings, providing a practical and accessible baseline for future multimodal retinal imaging applications.
