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Yueren Wang, Sven Peters, Martin Hammer, Yiran Jiang, Tom Kemerly, Uday Kompella, Sangly Srinivas; Depth resolved fluorescence lifetime of fluorescein across the cornea. Invest. Ophthalmol. Vis. Sci. 2013;54(15):2607.
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Equilibrium distribution ratio of fluorescein between a/h and stroma, which is an important parameter required for estimation of corneal endothelial permeability in vivo, is affected by pH and adsorption to macromolecules. To examine the stromal microenvironment, we have established a confocal scanning microfluorometer (CSMF) for measurement of fluorescence lifetime of fluorescein across the cornea.
A custom-built CSMF was interfaced with components for lifetime measurements in the time domain based on time-correlated single photon counting (TCSPC). The excitation source was a picosecond pulsed laser (Becker & Hickl, Berlin, Germany, BDL-445-SMC) operated at a repetition rate of 50 MHz with the output power at the focal plane set to 1.4 μW. The emission photons (>530 nm) were detected by a high-speed hybrid photon counter (HPM-100-40) with a resolution of 120 ps. The detector output was coupled to a TCSPC module (SPC-130-EM, Becker & Hickl) with reference derived from the laser pulses. The decay functions were analyzed by non-linear least squares using the SPCM Imaging software. Depth scanning was performed with a nano-positioning scanning assembly (M126-DG; PI Polytec) coupled to the microscope body. Fresh porcine eye balls were mounted underneath the objective (40x W; 0.75 NA Zeiss), and fluorescein was loaded into the cornea by scratching the epithelium.
The instrument response function, obtained using Rose Bengal, which is reported to have a lifetime of 16 ps, indicated a full width at half maximum of 150 ps. The fluorescence decay curve for fluorescein (1 μM in PBS at pH = 8.0), which could be fitted to a mono-exponential decay, yielded a lifetime of 3.99 ns. Increasing the fluorescein concentration 10-fold did not affect the lifetime. Lifetime of fluorescein across the stroma, ~30 min after instillation of 10 μM, varied from 3.6 to 3.9 ns at the anterior and posterior stroma, respectively. The depth resolution of all the measurements was 12 μm.
Previously, intensity measurements have shown significant uneven equilibrium distribution of fluorescein across the stroma, with the ratio of fluorescence emission between aqueous humor and stroma increasing from 3 at the anterior stroma to 2 at the posterior. However, a significantly higher decrease in the lifetime of fluorescein in the anterior stroma indicates lower pH and/or increased albumin in the anterior stroma.
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