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H Heier, O Brinchmann-Hansen; Reliable measurements from fundus photographs in the presence of focusing errors.. Invest. Ophthalmol. Vis. Sci. 1989;30(4):674-677.
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When measuring the width of the blood column and the superimposed central light reflex of retinal vessels from fundus photographs, we find that the measurements are affected by focusing error, unless certain steps are taken to reduce its effect. Thus, we recommend using a scanning microdensitometer to extract the density profile of the blood column from the negative, and convert the density profile to an intensity profile by means of the characteristic curve of the film. In this paper we model the imaging process on a computer, and find that the half value width of the intensity profile is relatively insensitive to focus error. However, the maximum intensity of the central light reflex is still sensitive to focus error. Both these results are in agreement with experimental studies.
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