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Alex Meyer, Asnika Sridhar, Ramesh Babu, Uday B Kompella, Sangly P Srinivas; Phase-resolved fluorometer for fluorescence lifetime measurements in the human eye. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3537.
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To report on the enhancements to an ocular fluorometer for performing lifetime-based pO2 measurements in the human eye.
A slit lamp has been modified to perform ocular fluorometry. To enable depth-resolved measurements, an adjustable collection slit is placed confocal to the excitation slit (i.e., the slit of the illumination optics) along the emission axis. The measurements of fluorescence lifetime (Τ) are accomplished in the frequency domain (FD) so that tan φ = ωΤ, where ω = 2pf and φ is the phase delay in the emission relative to excitation; f is frequency of the excitation sine wave. To accomplish high-speed modulation of the excitation, we replaced the halogen lamp of the slit lamp with an LED. For measurements of j, we employed a lock-in amplifier (SR830) whose reference was coupled to the sync signal of a function generator which provided a sine wave input for driving the LED. The signal input of the lock-in was coupled to the output of a red-sensitive photomultiplier (R928). The excitation frequency was set upλ at f = 1/(2π (Τ1 * Τ2) where Τ1 and Τ2 are expected lifetimes in the presence and absence of O2, respectively [J Opt Soc Am A Opt Image Sci Vis. 20(2):368-79; 2003].
The fluorometer could be maneuvered to measure fluorescence from the tears, cornea, and a/c easily. The excitation and emission slits were positioned confocal to each other and could be ascertained readily with the sighting optics situated in between the detector and collection slit. We have performed initial experiments with formulations of Ru phenanthroline bound to silica and dispersed in silicone (λex = 440 nm, λem > 600 nm) and Pd-porphyrin (λex = 520 nm, λem > 600 nm). As is characteristic of the frequency-domain method, the pO2-sensitive phase delay, and modulation data could be acquired in excess of 100 Hz yielding a high temporal resolution in the measurements of pO2. Calibration with mixtures of O2 and N2 indicated high quenching constants for formulations of Ru and porphyrin. The optimal excitation frequencies for the two dyes were 33 and 4 kHz, respectively.
The new FD spot fluorometer is capable of pO2 measurements with high sensitivity and temporal resolution as required for measurements of rapid pO2 dynamics underneath contact lenses. The sighting-optics enables easy positioning of the excitation and emission slits to be confocal which is necessary for high depth resolution.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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