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D Schweitzer, M Hammer, R Anders, T Doebbecke; Evaluation of Time-resolved Autofluorescence Images of the Human Fundus . Invest. Ophthalmol. Vis. Sci. 2002;43(13):2538.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To analyze dynamic fluorescence data obtained from living human fundus and to determine effect of provocation tests ( breathing of 100% oxygen, bleaching of photoreceptors) on fluorescence lifetime. Methods: Images of time-resolved fluorescence are obtained by a modified laser scanner ophthalmoscope, equipped with a pulse laser (λ=458nm, 200 ps FWHM, repetition rate 77.3 MHz) and photo-multiplier in time-correlated single photon counting technique. The photon number of single pixels was enlarged by binning of 2x2 up to 32x 32 pixels. Decay behavior of collected photons was approximated by mono-, bi-, and tri-exponential model functions. In provocation tests, time-resolved fluorescence images were taken before, during 6 min breathing of 100% oxygen and 15 min after breathing normal air. Furthermore, changes in time-resolved autofluorescence were investigated between dark adapted and bleached eye. Results: Bi-exponential approximation is best suited for interpretation of time-resolved fundus fluorescence, resulting in resolution of 200x200m2. Presentation of lifetime-distribution in regions of interest allows determination of influences of cut off wavelength and of provocation tests. In papillo-macular bundle (PMB) maxima of occurrence in lifetime distribution were found at 0.5, 1.2, 2.9, and 3.8ns (λcut off= 515nm). In contrast, broad maxima were found in papilla at 0.5, 3, and 5.5ns. Examining a limited emission range (λcut off=550nm), maxima are shifted from 1.2 to 1ns and from 2.9 to 2.4ns in PMB. Clear changes in lifetime distribution after breathing of 100% oxygen can be determined in papilla for fluorophores with decay times around 1.3 and 3.4ns. Whereas change at 1.3ns is returned after 15min breathing normal air, change at 3.4ns consists also after this time. In PMB of dark adapted eye, frequency of lifetime is reduced at 1.2 and 3ns, but increased at 4.4ns. Conclusions: Marking selected lifetimes in graph of bi-exponentially determined lifetime distribution, corresponding locations in fundus image can be visualized. Changes in lifetime after oxygen breathing mean that other fluorophores than lipofuscin in AMD are detectable which can be utilized also in glaucoma research. CR: Non. Supported by German TMWFK A 309-00015
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