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

• Published in: STAR protocols Vol. 4; no. 2; p. 102225
• Main Authors: Bogusławski, Jakub, Tomczewski, Sławomir, Dąbrowski, Michał, Komar, Katarzyna, Milkiewicz, Jadwiga, Palczewska, Grażyna, Palczewski, Krzysztof, Wojtkowski, Maciej
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 16.06.2023; Elsevier
• Subjects: Bioinformatics; Biotechnology and Bioengineering; Clinical Protocol; Health Sciences; Molecular/Chemical Probes; Neuroscience; Physics; Protocol; Neuroscience; Clinical Protocol; Molecular/Chemical Probes; Biotechnology and Bioengineering; Bioinformatics; Physics; Health Sciences
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2023.102225

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, system alignment, positioning human subjects, and data registration. We detail data processing and demonstrate analysis with example datasets. This technique allays 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 Bogusławski et al. (2022).1 [Display omitted] •Protocol for in vivo imaging of the human retina with two-photon excited fluorescence•Noninvasively shows the fluorophores’ distribution in the human retina•The protocol is supplemented with data processing software and an exemplary dataset•Potential for monitoring eye diseases in the earliest stages before structural damage Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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, system alignment, positioning human subjects and data registration. We detail data processing and demonstrate analysis with example datasets. This technique allays safety concerns by allowing for the acquisition of informative images at low laser exposure.