Fundus autofluorescence (AF) using adaptive optics scanning laser ophthalmoscopy (AOSLO) enables morphometric analysis of individual retinal pigmented epithelial (RPE) cells. However, only a few excitation wavelengths in the visible and near-infrared have been evaluated. Visible light excitation (<600 nm) presents additional safety hazards and is uncomfortable for patients. Near-infrared excitation (>700 nm) overcomes those problems but introduces others, including decreased AF signal and cone signatures that obscure RPE structure. Here we investigated the use of an intermediate wavelength, 663 nm, for excitation and compared it to 795 nm.
Subjects were imaged using AOSLO equipped with a detection channel to collect AF emission between 814 and 850 nm. Two light sources (663 and 795 nm) were used to excite the retinal fluorophores. We recorded 90 s videos and registered them with custom software to integrate AF images for analysis.
We imaged healthy eyes and an eye with pattern dystrophy. Similar AF microstructures were detected with each excitation source, despite ~4 times lower excitation power with 663 nm. The signal-to-noise values showed no meaningful difference between 663 nm and 795 nm excitation and a similar trend was observed for image contrast between the two excitation wavelengths.
Lower light levels can be used with shorter wavelength excitation to achieve comparable images of the microstructure of the RPE as have been obtained using higher light levels at longer wavelengths. Further experiments are needed to fully characterize AF across spectrum and determine the optimal excitation and emission bandwidths that balance efficiency, patient comfort, and efficacy.
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Delori FC. Spectrophotometer for noninvasive measurement of intrinsic fluorescence and reflectance of the ocular fundus. Appl Opt. 1994;33:7439–52.
Delori FC, Dorey CK, Staurenghi G, Arend O, Goger DG, Weiter JJ. In vivo fluorescence of the ocular fundus exhibits retinal pigment epithelium lipofuscin characteristics. Investig Ophthalmol Vis Sci. 1995;36:718–29.
von Rückmann A, Fitzke FW, Bird AC. Fundus autofluorescence in age-related macular disease imaged with a laser scanning ophthalmoscope. Investig Ophthalmol Vis Sci. 1997;38:478–86.
Rossi EA, Rangel-Fonseca P, Parkins K, Fischer W, Latchney LR, Folwell MA, et al. In vivo imaging of retinal pigment epithelium cells in age related macular degeneration. Biomed Opt Express. 2013;4:2527–39.
Keilhauer CN, Delori FC. Near-infrared autofluorescence imaging of the fundus: visualization of ocular melanin. Investig Ophthalmol Vis Sci. 2006;47:3556–64.
Morgan JIW, Dubra A, Wolfe R, Merigan WH, Williams DR. In Vivo Autofluorescence Imaging of the Human and Macaque Retinal Pigment Epithelial Cell Mosaic. Investig Ophthalmol Vis Sci. 2009;50:1350–9.
Granger CE, Yang Q, Song H, Saito K, Nozato K, Latchney LR, et al. Human Retinal Pigment Epithelium: In Vivo Cell Morphometry, Multispectral Autofluorescence, and Relationship to Cone Mosaic. Investig Ophthalmol Vis Sci. 2018;59:5705–16.
Liu Z, Tam J, Saeedi O, Hammer DX. Trans-retinal cellular imaging with multimodal adaptive optics. Biomed Opt Express. 2018;9:4246–62.
Liang J, Williams DR, Miller DT. Supernormal vision and high-resolution retinal imaging through adaptive optics. J Opt Soc Am A 1997;14:2884–92.
Roorda A, Romero-Borja F, Donnelly WJ III, Queener H, Hebert TJ, Campbell MCW. Adaptive optics scanning laser ophthalmoscopy. Opt Express. 2002;10:405–12.
Rangel-Fonseca P, Gómez-Vieyra A, Malacara-Hernández D, Wilson MC, Williams DR, Rossi EA. Automated segmentation of retinal pigment epithelium cells in fluorescence adaptive optics images. J Opt Soc Am A 2013;30:2595–604.
Vienola KV, Zhang M, Snyder VC, Sahel J-A, Dansingani KK, Rossi EA. Microstructure of the retinal pigment epithelium near-infrared autofluorescence in healthy young eyes and in patients with AMD. Sci Rep. 2020;10:9561.
Grieve K, Gofas-Salas E, Ferguson RD, Sahel JA, Paques M, Rossi EA. In vivo near-infrared autofluorescence imaging of retinal pigment epithelial cells with 757 nm excitation. Biomed Opt Express. 2018;9:5946–61.
Liu T, Jung H, Liu J, Droettboom M, Tam J. Noninvasive near infrared autofluorescence imaging of retinal pigment epithelial cells in the human retina using adaptive optics. Biomed Opt Express. 2017;8:4348–60.
Taubitz T, Fang Y, Biesemeier A, Julien-Schraermeyer S, Schraermeyer U. Age, lipofuscin and melanin oxidation affect fundus near-infrared autofluorescence. EBioMedicine . 2019;48:592–604.
Rossi EA, Granger CE, Sharma R, Yang Q, Saito K, Schwarz C, et al. Imaging individual neurons in the retinal ganglion cell layer of the living eye. Proc Nat Acad Sci USA. 2017;114:586–91.
Arathorn DW, Yang Q, Vogel CR, Zhang Y, Tiruveedhula P, Roorda A. Retinally stabilized cone-targeted stimulus delivery. Opt Express. 2007;15:13731.
Zhang M, Gofas-Salas E, Leonard BT, Rui Y, Snyder VC, Reecher HM, et al. Strip-based digital image registration for distortion minimization and robust eye motion measurement from scanned ophthalmic imaging systems. Biomed Opt Express. 2021;12:2353–72.
Laser Institute of America. American National Standard for Safe Use of Lasers. ANSI Z1361-2014.
Marmor MF, Byers B. Pattern Dystrophy of the Pigment Epithelium. Am J Ophthalmol. 1977;84:32–44.
Masella BD, Williams DR, Fischer WS, Rossi EA, Hunter JJ. Long-Term Reduction in Infrared Autofluorescence Caused by Infrared Light Below the Maximum Permissible Exposure. Investig Ophthalmol Vis Sci. 2014;55:3929–38.
Lyon T. Hazard analysis technique for multiple wavelength lasers. Health Phys. 1985;49:221–6.
The authors would like to thank Austin Roorda for sharing his AOSLO software with us, Pavan Tiruveedhula for electronics fabrication and software guidance and support, Jeff Speakman and Andrew Holmes for manufacturing custom mechanical components, and finally Jie Zhang, for sharing his original AOSLO optical design with us.
Some aspects of this work include technologies that Dr EAR is an inventor on for patents that are owned by the University of Rochester (US Patent No.: US 10,123,697 and US 10,092,181). Dr KKD is a consultant for Biogen. No other authors have any competing interests related to this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Vienola, K.V., Zhang, M., Snyder, V.C. et al. Near infrared autofluorescence imaging of retinal pigmented epithelial cells using 663 nm excitation. Eye (2021). https://doi.org/10.1038/s41433-021-01754-0