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Debra N Rankin, S Lingesh, V Akshata, Subashree Murugan, Prema Padmanabhan, Suddhir R Rachapalle, Ramesh Babu, Sangly P Srinivas; Enhanced ocular spot fluorometry in the frequency domain. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2140.
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Previously, we have demonstrated an ocular spot fluorometer custom-built around a standard slit lamp (Srinivas et al., PLoS One. 2018 Jun 19;13(6)). The instrument, based on frequency domain technology, is characterized by high depth resolution (~200 µm) and the ability to measure fluorescence lifetime (~ µs timescale). The goal of the current study was to enhance its temporal resolution while maintaining its depth resolution and fluorescence sensitivity.
For performing fluorescence spectroscopy in the frequency domain, the excitation was modulated as a sine wave (white LED of 2W at 1 kHz – 1 MHz), and the emission was detected with a high bandwidth analog photomultiplier tube (PMT; 1 MHz). The current output of the PMT was connected to a lock-in amplifier (~ 500 kHz). The reference input to the lock-in was obtained from a photodiode (10 MHz) in the excitation path. Both phase delay (Φ) and demodulation (M) of the emission, relative to the excitation, were recorded at 1 kHz. This high temporal resolution of the steady-state and lifetime measurements are demonstrated by standard measurements of (a) blink-induced fluorescein dynamics on the ocular surface, (b) dynamics of pO2 as indicated by the lifetime of a Ru2+ complex (~µs), and (c) measurement of aqueous flare without interference from blinks.
The fluorescence intensity from the ocular surface could be recorded in excess of 1 kHz with an excitation slit of 1.5 mm x 1.5 mm. The rapid acquisition enabled detection and removal of the blink-induced artifacts from measured fluorescence and aqueous flare. Thus, assessment of fluorescein dynamics immediately after blink could be registered unambiguously. In healthy subjects, the fluorescence typically increased with time, reaching a steady state in ~200 ms a few minutes after a 0.25% fluorescein drop on the bulbar conjunctiva. Both Φ and M in response to quenching/dequenching of Ru2+ fluorescence could be recorded at 1 kHz. These data were employed to calculate pO2 using the Stern-Volmer equation.
The increased temporal resolution in the acquisition of Φ and M made possible by higher bandwidths of excitation modulation, PMT, and the lock-in amplifier has enabled the realization of a high-speed ocular spot fluorometer. Thus, the device is now devoid of smearing of the fluorescence/scatter signals by the blink action and also permits rapid acquisition of pO2 dynamics that ensues upon opening of the eye.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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