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Rowena Schultz, Lydia Sauer, Christine A Curcio, Daniel Meller, Martin Hammer; How are fluorescence lifetimes of chorioretinal tissue in human donor eyes affected by fixation?. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3207.
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Fluorescence lifetime imaging ophthalmoscopy (FLIO) is an emerging imaging modality for in vivo identification and characterization of endogenous retinal and sub-retinal fluorophores. In vivo full characterization of fluorophores is difficult; therefore, ex vivo analysis of donor tissues is also done for comparison. Our purpose was to investigate the influence of post mortem fixation on RPE autofluorescence (AF) lifetimes in comparison with fresh frozen eyes, all subject to cryo-sectioning.
From each of 2 pairs of donor eyes (<6 hr after death), the left eye was frozen without prior fixation, and the right eye was preserved in 4% paraformaldehyde. Macula and periphery of both eyes in each pair were cryo-sectioned together at 12µm thickness. Fluorescence decay images of cryo-sections from non-fixed and fixed eyes were recorded in two spectral channels (ch1: 498-560nm; ch2: 560-720nm). Decays were approximated by a series of three exponentials, resulting in three lifetimes (τ1-τ3). Their amplitude-weighted mean (τm) per channel and pixel was utilized as the main parameter for statistical analysis. We used FLIO (Heidelberg-Engineering, Heidelberg, Germany) with additional optics turning the ophthalmoscope into a microscope.
An AF signal was found in the RPE layer and intermittently in the choroid of fixed and non-fixed eyes. RPE lifetimes of fixed eyes were longer than those of non-fixed cryo-sections but not significantly (ch1: 426.3ps (interquartile range 139.3 ps) vs. 576.5 ps (153 ps), p = 0.42 (Wilcoxon test on paired samples), ch2: 355.2 ps (47.1 ps) vs. 391.2 ps (110.8 ps), p=0.78). Subgroup analysis of macular and peripheral RPE revealed a significant lifetime difference only for the periphery in ch2 (p=0.015) but not for the macular samples and generally not in ch1.
Fixation seems not to alter RPE fluorescence lifetimes substantially. However, ex-vivo lifetimes were generally longer than that found for fundus AF in vivo1. Further study will focus on differences among the chorioretinal layers of the same histological sections of normal and AMD eyes, each will be subject to these same fixation and processing methods.1. Schmidt J, Peters S, Sauer L, et al. Fundus autofluorescence lifetimes are increased in non-proliferative diabetic retinopathy. Acta Ophthalmol 2016.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
Infrared (IR) reflection image and pseudo-colored images of AF lifetimes for Ch1 and Ch2.
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