Phase-resolved Fluorometer for Fluorescence Lifetime Measurements in the Human Eye

A slit-lamp is a standard instrument in ocular diagnostics. It consists of an illumination assembly and viewing, binocular optics. The illumination assembly projects a beam of visible light into the eye as a thin sheet, and its interaction with intraocular structures is then viewed at an angle with binocular optics. To perform fluorescence lifetime spectroscopy, we have modified a slit lamp to form an ocular spot fluorometer in the frequency domain (FD). To ensure depth-resolved fluorescence measurements, an adjustable collection slit has been positioned confocal to the excitation slit (i.e., the slit of the illumination optics) at the image plane of the slit lamp. This has enabled measurements exclusively from any desired spot in the eye, which corresponds to the focal point of the excitation beam. Thus, the sampling volume, referred to as the focal diamond, is formed by the intersection of the emission and illumination/excitation beams. For purposes of implementation in FD, we modulated the excitation (10-100 kHz) by driving a white LED (10 W) with a power amplifier connected to a function generator. The emission was detected by an analog photomultiplier tube (PMT). The output of the PMT was connected to a lock-in amplifier to measure the phase delay (phi) and demodulation (M), which are related to the fluorescence lifetime in the emission relative to the excitation sine wave. Both phi and M, measured with Ru phenanthroline, were sensitive to pO(2) and could be acquired at frequencies greater than 100 Hz. Overall, the findings of this study indicate the potential of our instrument to sense rapid pO(2) dynamics in the eye. (C) 2019 Elsevier Ltd. All rights reserved. Selection and/or Peer-review under responsibility of International conference on NanoTechnology in Energy, Nano-bio interface & Sustainable Environment (INTENSE).