In vivo imaging of the human retina using a two-photon excited fluorescence ophthalmoscope

Noninvasive imaging of endogenous retinal fluorophores, including vitamin A derivatives, is vital to developing new treatments for retinal diseases. Here, we present a protocol for obtaining in vivo two-photon excited fluorescence images of the fundus in the human eye. We describe steps for laser characterization, sys-tem alignment, positioning human subjects, and data registration. We detail data processing and demonstrate analysis with example datasets. This technique al-lays safety concerns by allowing for the acquisition of informative images at low laser exposure. For complete details on the use and execution of this protocol, please refer to Boguslawski et al. (2022).1

Automated summary

This article presents a protocol for noninvasive imaging of endogenous retinal fluorophores in the human eye. It describes the steps for obtaining two-photon excited fluorescence images of the fundus, including laser characterization, system alignment, subject positioning, and data processing. The technique allows for informative images to be acquired at low laser exposure, addressing safety concerns.