June 2020
Volume 61, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2020
Near infrared (NIR) imaging of the human retinal pigment epithelium (RPE) using super-resolution structured illumination microscopy (SIM)
Author Affiliations & Notes
  • Thomas Ach
    Dept of Ophthalmology, University Hospital Wuerzburg, Wuerzburg, Germany
  • Ioana-Sandra Tarau
    Dept of Ophthalmology, University Hospital Wuerzburg, Wuerzburg, Germany
  • Jost Hillenkamp
    Dept of Ophthalmology, University Hospital Wuerzburg, Wuerzburg, Germany
  • Sebastian Unger
    Leibniz Institute for Photonic Technologies, Jena, Germany
  • Christian Karras
    Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller University Jena, Jena, Germany
  • Christine Curcio
    Department of Ophthalmology, University of Alabama at Birmingham, Birmingham, Alabama, United States
  • Rainer Heintzmann
    Leibniz Institute for Photonic Technologies, Jena, Germany
    Institute of Physical Chemistry and Abbe Center of Photonics, Friedrich-Schiller University Jena, Jena, Germany
  • Andreas Berlin
    Dept of Ophthalmology, University Hospital Wuerzburg, Wuerzburg, Germany
  • Footnotes
    Commercial Relationships   Thomas Ach, None; Ioana-Sandra Tarau, None; Jost Hillenkamp, None; Sebastian Unger, None; Christian Karras, None; Christine Curcio, Genentech/Roche (F), Heidelberg Engineering (F), MacrRegen Inc. (I); Rainer Heintzmann, None; Andreas Berlin, None
  • Footnotes
    Support  1R01EY027948 (TA; CAC)
Investigative Ophthalmology & Visual Science June 2020, Vol.61, 4138. doi:
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      Thomas Ach, Ioana-Sandra Tarau, Jost Hillenkamp, Sebastian Unger, Christian Karras, Christine Curcio, Rainer Heintzmann, Andreas Berlin; Near infrared (NIR) imaging of the human retinal pigment epithelium (RPE) using super-resolution structured illumination microscopy (SIM). Invest. Ophthalmol. Vis. Sci. 2020;61(7):4138.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose : Histologically, three major granule types with distinct autofluorescence (AF) properties are found within the cell bodies of human RPE: lipofuscin (L), melanolipofuscin (ML), and melanosomes (M). In contrast to L and ML, Ms block short wavelength light but may exhibit AF in the NIR range. Clinically, NIR FAF is believed to reflect M abundance, but histological data are sparse. This study reports the subcellular origin of NIR AF within human RPE cells using super-resolution microscopy.

Methods : RPE flatmounts and retinal cross sections from human donors were microscopically imaged using a custom built NIR SIM device (excitation: 785 nm, emission >800 nm; plan-apochromatic 63X 1.46 oil immersion objective), a commercially available SIM device (Elyra S1, Zeiss; excitation: 488 nm, 633 nm; plan-apochromatic 63x 1.4 oil immersion objective), and a light microscope. Tissues were imaged at the fovea, perifovea, and near-periphery. The NIR SIM signals were further magnified using a 1.6 magnification changer (Optovar, Zeiss) and detected using an Orca Flash4.0 V3 camera (Hamatsu). The SIM data was reconstructed using custom written algorithms (NIR SIM) or the device's internal software (Elyra S1). For signal co-localization, SIM images were superimposed and the light microscope images confirmed the melanin within the pigmented granules.

Results : Based on AF phenotype, SIM imaging enables clear distinction between intracellular RPE granule types. 488 nm SIM shows bright signals from L and the L part within ML. The same signal pattern, though less intense, is detectable at 633 nm excitation. At 785 nm excitation, AF pattern changes and AF arises from the pigmented part of ML. M are only sporadically visible due to preparation artefacts that remove or damage apical processes, and do not contribute substantially to the AF signal in cell bodies.

Conclusions : ML seems to be the major source in NIR imaging of RPE cell bodies. This is in line with recent histological findings showing ML to be the leading granule type at the fovea. Therefore, clinical NIR AF imaging of the posterior pole with its high AF signal arising from the fovea might be a good indicator for intracellular RPE ML abundance.

This is a 2020 ARVO Annual Meeting abstract.

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