Fluorescence lifetime imaging microscopy reveals quenching of fluorescein within corneal epithelium

Abstract

Topical application of fluorescein results in background fluorescence of normal corneal epithelial cells. The fluorescence appears relatively weak and is often ignored clinically. The concentrations of fluorescein applied clinically exceed the threshold for self quenching. The possibility that exuberant topical concentrations of fluorescein result in quenching of fluorescence in tears and normal corneal epithelium is explored. Fluorescence lifetime measurements are sensitive to quenching and are less vulnerable to inner filter effect than steady state measurements. The types of fluorescence lifetime quenching often report informative molecular interactions. Therefore, fluorescence lifetime confocal imaging was performed in solutions, tears and corneal epithelium removed by membrane cytology following applied fluorescein. Amplitude averaged fluorescence lifetimes (τamp) were measured with time resolved single photon counting using a pulsed diode laser for excitation of fluorescein. Lifetime decays were fit to multi-exponential models with least squares analysis. Stern-Volmer plots for both intensity (I) and (τamp) were determined. Stern-Volmer plots demonstrated both dynamic and static quenching components (R2 = 0.98 exponential fit, I0/I). Plots of τamp versus concentration of fluorescein revealed a linear relationship. Immediately after fluorescein application, quenching was evident in tears (τamp < 1 ns) versus tears sampled after 5 min (τamp = 3.7 ns). Corneal epithelium showed quenching (τamp ≥ 2 ns) from 1 to 16 min post fluorescein instillation. Clinical concentrations of fluorescein show self-quenching but rapidly dilute as tears turnover. Intracellular quenching occurs in normal corneal epithelium. Lifetime decay curves suggest complex mechanisms are involved. Quenching is a plausible explanation for the low fluorescence background observed clinically.

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications

“…Therefore, FLIM is well-suited for accurate measurements of quenching dynamics. 75,76 Multiple configurations or states of a fluorophore can be detected with FLIM at a single location or pixel. For example, both bound and unbound fluorophores, as well as proteins with distinct folding states, will have different molecular environments that coexist within the same pixel.…”

Thiolated and PEGylated silica nanoparticle delivery to hair follicles

“…Significant reductions (P<0.05) in fluorescence intensity of all tested nanoparticles and sodium fluorescein was evident after 2 hours of incubation on the skin surface and prior to tape stripping. The fluorescence intensity reduced to 12, 52, 7 and 10% for sodium fluorescein, TSNPs, PEGylated 750 Da and PEGylated 5000 Da TSNPs respectively which could be attributed to quenching of fluorescein (Glasgow, 2016;Song et al, 1995) and penetration into the follicles. Teichmann et al reported recovery of 95% of sodium fluorescein dye from stratum corneum and 5% from follicular infundibula after differential stripping (Teichmann et al, 2005).…”

Using fluorescence lifetime dequenching to estimate the average quinary stoichiometry of proteins in living cells

“…The model trajectory is only significantly close to point A, and at this point t = 1.43 Q ns. In the context of Glasgow et al [23], this extent of quenching would correspond to a local concentration of fluorescein of 18 mM.…”

“…Therefore, the intracellular protein concentration is ~1.66 mM. Even if the labelling efficiency was 100% and all proteins had been tagged by one fluorophore, 1.66 mM of fluorescein would only be able to quench the average lifetime down to ~3.8 ns via random collisions [23]. This is insufficient to explain the 2.64 ns average lifetime obtained on day 1.…”