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Ben J Glasgow; Fluorescence Quenching at Fluorescein Concentrations Used for Diagnosis in Ocular Surface Disease. Invest. Ophthalmol. Vis. Sci. 2016;57(12):428.
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© ARVO (1962-2015); The Authors (2016-present)
Punctate fluorescein corneal staining is a widely used criterion for dry eye disease but the mechanism is still debated. A population of presumed abnormal fluorescent epithelial cells (FLECs) outshines the background fluorescence of other epithelial cells, which may be unnoticed clinically but are seen easily with fluorescence microscopy. The recommended concentration of fluorescein for topical application is substantial. The hypothesis tested is that diagnostic concentrations of fluorescein produce quenching in solution and in normal epithelium.
Fluorescein concentrations in serial gravimetric dilutions (6 sets at each point) were determined by absorbance spectrophotometry. Amplitude averaged fluorescence lifetimes (τamp) were measured with time resolved single photon counting in a Microtime 200 laser confocal instrument (Picoquant). A pulsed diode laser (lex=485 nm) was used in tandem with a dichroic mirror (DCXR 500) and 525/545 nm band pass filter. τamp was measured in tears (n=4) and corneal epithelial cells (n= 10) removed by membrane cytology 5 min following application of fluorescein at various concentrations on the ocular surface. Individual lifetime decays were fit and analyzed in a multi-exponential model with least squares analysis. Stern-Volmer (S-V) plots for both intensity (I) and τamp were determined beginning with means of concentration values at the self-quenching threshold and fit to linear or exponential models by least squares analysis.
τ was more sensitive than I in determining the threshold concentration (500μM) of quenching, which is well below fluorescein concentrations generally used. S-V plots demonstrated both dynamic (R2=.98 exponential fit, I0/I) and static quenching components (R2=.94 linear,τ0/τ). Resonance energy transfer is implicated. Quenching (τamp<1ns) was evident in tears sampled immediately after instillation, versus dilute concentrations and tears sampled after 5 minutes (τamp≈ 4ns). Corneal epithelial cells showed quenching (τamp≤2ns) at 1-16 min after application.
Clinical concentrations of fluorescein show self-quenching by a set of complex mechanisms, but rapidly dilute as tears turnover. Moderate intracellular quenching in the normal cornea presents one rational explanation for the low fluorescence background observed clinically. Further studies are indicated to determine the role of intracellular quenching in FLECs.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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