Abstract

DO.19.08

Structured illumination microscopy for detection of lysosomes in retinal pigment epithelium

Thomas Ach1, Gerrit Best2, Mira Ruppenstein1, Roman Amberger2, Cristoph Cremer2, Stefan Dithmar1
1Universitäts-Augenklinik Heidelberg, 2Kirchhoff-Institut für Physik, Universität Heidelberg

Objective
Lipofuscin as the main fluorophore group of the ocular fundus gives evidence of metabolic activity of the retinal pigment epithelium (RPE) in autofluorescence images. Widefield and Two-Photon-images of the RPE obtain a resolution in the range of 300 - 400 nm. For the first time, structured illumination microscopy with increased resolution for detection of fluorescent material in the RPE is presented.
Methods
Structured illumination microscopy can double the resolution of a common widefield microscope. Depending on excitation wavelengths and objective lenses, lateral resolution of 100 to 150 nm is feasible. In axial direction, resolution improvement is also possible (3 beam interference) and through increased frequency support the gap in optic transfer function („missing cone“) can be filled to achieve optical sectioning. Lysosomes in the RPE of deparaffined retinal slides (age of retina: 2 to 89 years) were illuminated with laser of different wavelengths (404 to 647 nm) with the structured illumination microscopy technique and evaluated with regard to the fluorescence behaviour.
Results
Intracelluar structures of the RPE can be detected with a lateral resolution of 150 nm. With aging retina, fluorescent material (FM) accumulates in the lysosomes. In overview, FM in young age retina seems to be more inhomogeneous (different wavelengths result in excitation of different fluorescent structures) while older retinas show homogeneous large-area fluorescence (different wavelengths excitates identical material). Examination of single lysosomes shows inverse effect. In comparison with young age retina, lysosomes in higher age are more inhomogenous.
Conclusions
For the first time, with the structured illumination microscopy, fluorescent structures in RPE can be detected with a lateral resolution of 150 nm. Age-dependent distribution and changes in fluorescent material in single lysosomes as well as wavelength-dependent intralysosomal distribution pattern of lipofuscein-fluorophores are observable. This allows new insights in RPE pathogenesis. 

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