Quantitative Measurements of Autofluorescence with the Scanning Laser Ophthalmoscope

PURPOSE. To evaluate the feasibility and reliability of a standardized approach for quantitative measurements of fundus autofluorescence (AF) in images obtained with a confocal scanning laser ophthalmoscope (cSLO). METHODS. AF images (30 degrees) were acquired in 34 normal subjects (age range, 20-55 years) with two different cSLOs (488-nm excitation) equipped with an internal fluorescent reference to account for variable laser power and detector sensitivity. The gray levels (GLs) of each image were calibrated to the reference, the zero GL, and the magnification, to give quantified autofluorescence (qAF). Images from subjects and fixed patterns were used to test detector linearity with respect to fluorescence intensity, the stability of qAF with change in detector gain, field uniformity, effect of refractive error, and repeatability. RESULTS. qAF was independent of detector gain and laser power over clinically relevant ranges, provided that detector gain was adjusted to maintain exposures within the linear detection range (GL < 175). Field uniformity was better than 5% in a central 20 degrees-diameter circle but decreased more peripherally. The theoretical inverse square magnification correction was experimentally verified. Photoreceptor bleaching for at least 20 seconds was performed. Repeatability (95% confidence interval) for same day and different-day retests of qAF was +/- 6% to +/- 14%. Agreement (95% confidence interval) between the two instruments was <11%. CONCLUSIONS. Quantitative AF imaging appears feasible. It may enhance understanding of retinal degeneration, serve as a diagnostic aid and as a sensitive marker of disease progression, and provide a tool to monitor the effects of therapeutic interventions. (Invest Ophthalmol Vis Sci. 2011; 52: 9379-9390) DOI: 10.1167/iovs.11-8319