Multiphoton Excitation in Retinal Imaging and Functional Measurements

In the retina, two-photon (2P) excitation induces fluorescence emission both from endogenous fluorophores, including vitamin A metabolites that sustain vision, and from exogenous dyes or fluorescent proteins expressed selectively in individual retinal cells. The simultaneous absorption of two near infrared photons generates 2P excited fluorescence and results in high signal-to-noise ratio images deep in the tissue, enabling acquisition of 3D volumes over the entire thickness of the retina in an intact eye. Advancements in noninvasive 2P-based techniques offer detailed characterization of retinal structure and function at subcellular levels. This capability is especially valuable for measuring transfection efficiency of newly developing gene-editing approaches, including clustered regularly interspaced short palindromic repeats-CRISPR-associated nuclease 9 (CRISPR-Cas9) systems and viral-vector-mediated therapies, designed to treat inherited eye diseases. Furthermore, the simultaneous absorption of two photons by visual pigments can directly initiate phototransduction cascades, providing unique insights into precise detection of photoreceptor sensitivity and unexplored mechanisms of visual perception. Two-photon processes enable real-time study of biochemical transformations in living retinal tissue, advancing the development of novel treatments and facilitating assessment of therapeutic interventions.