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Near infrared autofluorescence imaging of retinal pigmented epithelial cells using 663 nm excitation



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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Fig. 1: AOSLO and NIRAF data from a healthy volunteer.
Fig. 2: Infrared autofluorescence (IRAF) and near-infrared autofluorescence (NIRAF) imaging of the right eye of a patient with degenerative maculopathy (~6° deg. eccentricity toward temporal region).
Fig. 3: Signal-to-noise ratio and Michelson contrast were similar for each excitation wavelength.


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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.

Author information




EAR and JAS conceived the experiments, KVV and EAR conducted the experiment(s), EAR, KKD and KVV analyzed the results. VCS conducted the clinical imaging. MZ developed the custom electronics and control software. KVV wrote the paper, EAR edited the paper, and all other co-authors carefully reviewed the paper for scientific content.


The Edward N. & Della L. Thome Memorial Foundation to J-AS; Departmental startup funds from the University of Pittsburgh to EAR; NIH CORE Grant P30 (EY08098); the Eye and Ear Foundation of Pittsburgh; NVIDIA GPU Grant Program and from an unrestricted grant from Research to Prevent Blindness, New York, N.Y., USA.

Corresponding author

Correspondence to Kari V. Vienola.

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Competing interests

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.

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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).

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