[High-resolution fluorescence microscopy of retinal pigment epithelium using structured illumination].
BACKGROUND
The accumulation of autofluorescent bodies in retinal pigment epithelium (RPE) cells has an impact on the pathogenesis of retinal diseases, including age-related macular degeneration. While current in vivo fluorescence microscopy allows a lateral resolution of fluorophores in a micrometer range, with ex vivo microscopy a lateral resolution down to 200 nm is possible. For the first time, we used structured illumination microscopy for ex vivo high-resolution fluorescence microscopy of RPE cells.


METHODS
Histological sections were prepared from a 68-year-old patient. With epifluorescence microscopy, fluorescent RPE cells were detectable. Structured illumination uses inhomogeneous illumination for resolution of previously nonresolvable structures, similar to the Moiré effect. Images were taken from RPE cells at different excitation wavelengths (488, 568, and 647 nm) and were reconstructed with special software. The different excitation patterns of the fluorescent granules in the RPE cells were colour-coded and analysed.


RESULTS
With structured illumination microscopy, autofluorescence signals of RPE cells were detectable, and a lateral resolution of 110 nm could be achieved. Using varying wavelengths, different pigments were excitable. Lipofuscin gave the highest signals, at 488 and 568 nm. The improved resolution showed inhomogeneous intragranular fluorophore patterns.


CONCLUSIONS
Structured illumination microscopy enabled us to generate images of fluorescent structures in RPE cells ex vivo with a lateral resolution of 110 nm. With the use of different excitation wavelengths, intracellular fluorescence patterns in single cell compartments are visible and allow further differentiation.
Ach
T
T
Universitäts-Augenklinik Heidelberg, Im Neuenheimer Feld 400, Heidelberg, Deutschland.
Best
G
G
Ruppenstein
M
M
Amberger
R
R
Cremer
C
C
Dithmar
S
S
D046169:Dermoscopy / Q000379:methods*; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D008062:Lipofuscin / Q000378:metabolism; D008297:Male; D008543:Melanins / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods*; D008875:Middle Aged; D015903:Moire Topography / Q000379:methods; D010857:Pigment Epithelium of Eye / Q000473:pathology*; D012680:Sensitivity and Specificity; D012984:Software
D008062:Lipofuscin; D008543:Melanins