In Vivo Functional Imaging of Retinal Neurons Using Red and Green Fluorescent Calcium Indicators
Adaptive optics retinal imaging of fluorescent calcium indicators is a minimally invasive method used to study retinal physiology over extended periods of time. It has potential for discovering novel retinal circuits, tracking retinal function in animal models of retinal disease, and assessing vision restoration therapy. We previously demonstrated functional adaptive optics imaging of retinal neurons in the living eye using green fluorescent calcium indicators; however, the use of green fluorescent indicators presents challenges that stem from the fact that they are excited by short-wavelength light. Using red fluorescent calcium indicators such as jRGECO1a, which is excited with longer-wavelength light (~560 nm), makes imaging approximately five times safer than using short-wavelength light (~500 nm) used to excite green fluorescent calcium indicators such as GCaMP6s. Red fluorescent indicators also provide alternative wavelength imaging regimes to overcome cross talk with the sensitivities of intrinsic photoreceptors and blue light-activated channelrhodopsins. Here we evaluate jRGECO1a for in vivo functional adaptive optics imaging of retinal neurons using single-photon excitation in mice. We find that jRGECO1a provides similar fidelity as the established green indicator GCaMP6s.
KeywordsAdaptive optics jRGECO1a Calcium indicator Retinal imaging Ganglion cells
We thank Sophia Zhao for making the jRGECO1a virus, Jie Zhang for designing and constructing the visual stimulus apparatus, and Keith Parkins for programing data acquisition software. This work was supported by grants from the National Eye Institute of the National Institutes of Health, EY001319 and EY021166; an Unrestricted Grant to the University of Rochester Department of Ophthalmology from Research to Prevent Blindness, New York, New York; as well as a Beckman-Argyros Award from the Arnold and Mabel Beckman Foundation. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. The Genetically-Encoded Neuronal Indicator and Effector (GENIE) Project and the Janelia Farm Research Campus of the Howard Hughes Medical Institute have generously allowed these GECI materials to be distributed with the understanding that requesting investigators need to acknowledge the GENIE Program and the Janelia Farm Research Campus in any publication in which the material was used, specifically Vivek Jayaraman, PhD; Rex A. Kerr, PhD; Douglas S. Kim, PhD; Loren L. Looger, PhD; and Karel Svoboda, PhD from the GENIE Project, Janelia Farm Research Campus, Howard Hughes Medical Institute.
- Dana H, Sun Y, Hasseman JP et al (2014) Improved red protein indicators for in vivo calcium imaging. In: Proceedings of the Society for Neuroscience, Washington DCGoogle Scholar
- Dubra A, Harvey Z (2010) Registration of 2D images from fast scanning ophthalmic instruments. In: Biomedical image registration lecture notes in computer science. Springer, BerlinGoogle Scholar
- Zhang J, Sabarinathan R, Bubel T et al (2016) Spectral dependence of light exposure on retinal pigment epithelium (RPE) disruption in living primate retina. Invest Ophthalmol Vis Sci 57:2220–2220Google Scholar