Staining and peeling of the internal limiting membrane using a fluorescent dye (Rhodamine 6 G)

Aim: To assess whether low concentrations of a fluorescent dye such
as Rhodamine 6G would help the unaided human eye visualise the
vitreous and the internal limiting membrane (ILM) under standard
halogen illumination.Material/methods: The UV/Vis absorption (E)
and fluorescence (I) spectra of Rhodamine 6G in water were measured
and compared with Indocyanine Green (ICG). Surgery was performed in
two rhesus monkeys and consisted of standard pars plana vitrectomy
with halogen light source used for illumination. Rhodamine 6G was
diluted in balanced salt solution (BSS). A few drops of the dye in
a concentration of 0.1% (307 mOsm) were applied over the posterior
pole in the air-filled globe and washed out by irrigation after 1
min. Immediately after surgery, the globes were enucleated, fixated
and prepared for histological evaluation.Results: In contrast to
ICG, both the maximum of the absorption and emission of Rhodamin 6G
are very much within the spectral sensitivity of the human eye. The
Rhodamine 6G--BSS itself appears red in colour. Using a dye
concentration of 0.1%, there was no visible red-staining of the ILM
as such. As the dye was irrigated out with BSS, a marked green
fluorescence of the fluid within the vitreous cavity was noted.
With halogen illumination through a standard 20-gauge light pipe,
the dye provided a sufficient green fluorescence to identify and
safely remove the ILM and to clearly differentiate areas of peeled
from non-peeled ILM. During light microscopy, eyes revealed a
peeled ILM demarcation with no signs of acute retinal
toxicity.Conclusion: The findings indicate that a fluorescent dye
can be used for ILM peeling. Assuming that the fluorophore provides
a high enough fluorescence quantum yield after adsorption to the
ILM, much lower dye concentrations could be used compared with
absorbent dyes, thereby minimising toxic effects.