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J. P. Greenberg, T. Duncker, J. R. Sparrow, T. Smith, F. C. Delori; Absolute Autofluorescence (AbsAF) Measurements: Method and Validation. Invest. Ophthalmol. Vis. Sci. 2010;51(13):6366.
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© ARVO (1962-2015); The Authors (2016-present)
To evaluate instrumentation for reproducible AbsAF lipofuscin measurements in vivo.
AbsAF measurement was done on 46 normal subjects (ages 4 to 57, 1 eye per subject) with clear crystalline lenses using the HRA2 scanning laser ophthalmoscope (Heidelberg Engineering Inc., Vista, CA) modified by insertion of a fluorescence reference chip in the retinal image plane to correct for variable laser power and individual acquisition sensitivity (Fig. 1). Corrections were also made for optical media density T-1om,488 T-1om,emi from normative data on lens transmission spectra [Pokorny J et al, Appl. Opt., 1987] and for refractive error with an appropriate scale factor SC (Eqn. 1). The emission efficiency of the reference fluorophore was assessed by fluorophotometry and found to be stable after exposure to 100 hours of blue light. Each original image was a mean of 9 raw images without histogram stretching. Median AbsAF values in the central 3000 micron region and in a temporal 1500 micron region were measured after 15 seconds of bleaching. Ex vivo animal lipofuscin measurements and their correlation to AbsAF values obtained from the scanning technique are in progress.
Median central and temporal AbsAF were highly correlated with age, ranging from 18 to 226 AbsAF units centrally and from 17 to 262 AbsAF units temporally over all normal subjects. Temporal AbsAF was always higher than any other region. Reproducibility was verified by 3 repeat scans of 17 eyes (for the central region, mean SD = 2.14 and range = 0.00 to 4.95, and for the temporal region, mean SD = 3.51 and range = 0.00 to 10.15). 31 subjects had both eyes imaged (mean SD between OD and OS central regions = 4.95 and range = 0.70 to 12.73).
Accurate, reproducible AbsAF measurement of in vivo lipofuscin levels can be achieved in humans with clear media. For retinal degenerations, this may enhance understanding of disease processes, serve as a more sensitive marker of disease progression, and allow for earlier diagnosis.
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