High-resolution fluorescence lifetime and infrared spectroscopic characterization of pathogenic retinal pigment epithelium cell organelles (HiResi4RPE)

• Applicants: Dr. Martin Hammer; Professor Dr. Rainer Heintzmann; Dr. Daniela Täuber
• Subject Area: Ophthalmology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 542825796

Within HiResi4RPE we plan to apply two newly developed high resolution imaging methods to the investigation of retinal pigment epithelium (RPE) organelles for enhancing the understanding of the pathogenesis of age-related macular degeneration (AMD) and its clinical diagnosis & treatment. AMD is a disease of the outer retina and the RPE. Fluorescence Lifetime Imaging Ophthalmoscopy (FLIO) is used for non-invasive clinical imaging. The measured fluorescence originates from auto-fluorescent granules (melanin, lipofuscin, and melano-lipofuscin) in the RPE. The distribution of autofluorescent granules as well as the respective fluorescence lifetimes are different in RPE of AMD and control eyes. So far, the variation in fluorescence lifetimes has not been investigated on a sub-cellular level. We plan to use a combination of image scanning microscopy (ISM) with fluorescence lifetime imaging microscopy (FLIM) to assess the distribution of fluorescence lifetimes on the scale of individual granules in the RPE layer. We further suggest to complement this approach by the exploration of chemical changes using high-resolution mid-IR photoinduced force microscopy (PiF-IR). Our proposed research project consists of the following four work packages: WP1: ISM-FLIM will be applied to histological RPE sections of human AMD and control donor eyes. This will enable the characterization of changes in the fluorescence lifetime on a single granule level. WP2: An established transwell-culture model of porcine RPE cells will be fed native and photo-oxidized photoreceptor outer segments, inducing AMD-like cell damage. The effect of this treatment on cell integrity, survival, and function will be tested by measuring the trans-epithelial electrical resistance, ELISA, immune-histochemistry, and Western blot. WP3: Cell cultures from WP2 will be subject to high-resolution fluorescence lifetime imaging using ISM-FLIM. Furthermore, lipofuscin-, melanolipofuscin-, and melanin granules will be isolated and separated by a sucrose gradient. Single granules will be investigated using ISM-FLIM. WP4: Granule fractions, isolated in WP3, as well as in histologic sections, will be investigated by PiF-IR and FTIR imaging for chemical characterization of the granule surfaces and bodies, respectively. The obtained mid IR-spectra will be compared with that of pristine RPE fluorophores. The combined analysis of complementary high-resolution ISM-FLIM and PiF-IR image data will allow to identify the influence of single fluorophores on fluorescence lifetime changes in AMD-like pathology. This will, firstly, enhance the understanding of molecular mechanisms in AMD, and secondly, support the understanding of clinical FLIO imaging and, thus, strengthen its application in AMD diagnostics.

• DFG Programme: Research Grants