Abstract
Purpose :
In our prior study (Srinivas et al., PLoS One. 2018), we introduced a slit lamp-based ocular fluorometer for measuring fluorescence intensity at random sites within the anterior segment of the eye. This device, known as the 'spot fluorometer,' is being enhanced to enable effective fluorescence lifetime measurements within the anterior chamber.
Methods :
The FastFLIM interface from ISS Inc. replaced the lock-in amplifier of the Spot fluorometer. The latter employs digital frequency domain (DFD) technology, as detailed by Colyer et al. in Microsc. Res. Tech. 71, 201–213, for rapid lifetime measurements. Thus, the newly developed Ophthalmic Time-Resolved Spot Fluorometer (OTR-Spot) integrates the original spot fluorometer, based around a Nikon slit lamp (FS-3), with a solid-state pulsed laser (488 nm) serving as the excitation source, and a high-speed photomultiplier tube (PMT; Hamamatsu 9238 HA). The DFD unit operates the laser at a 20-MHz repetition rate and collects the emission after a 500-nm long pass emission filter. A dichroic cube was introduced in place of the flip mirror, facilitating the integration of a laser diode (Nichia Inc; 5 mW) for in-line excitation. While the LED in the fluorometer was turned for focusing, lifetime measurements were carried out using the laser diode.
Results :
Similar to our OTR-CSMF system (Srinivas et al., Proc Vol. 11965, Multiphoton Microscopy in Biomed Sci XXII; 1196509 (2022)), the OTR-Spot showed high axial resolution and a lifetime resolution of 50 ps. Employing Rhodamine 110 (with a lifetime of 4 ns) as a reference, we observed that the lifetimes of Rhodamine B (RhB) and fluorescein closely aligned with previously established values. Following a ~ 60-minute exposure to RhB, we could detect the temperature-dependent lifetime variations of the dye from the epithelium, averaging around 2 ns. To test our system's performance at the microsecond scale, we measured the oxygen-sensitive lifetime of ruthenium phenanthroline, either adsorbed on silica nanoparticles or infused in contact lenses. The laser was pulsed at a frequency of 2 kHz, maintaining a duty cycle of 2%. An increase in fluorescence lifetime was observed when exposed to a nitrogen stream, consistent with the Stern-Volmer equation.
Conclusions :
The DFD method, known for its broad dynamic range in lifetime measurements, has been integrated with the spot fluorometer. The resulting OTR-Spot can perform rapid lifetime measurements (>1 kHz) from 80 ps to ms.
This abstract was presented at the 2024 ARVO Imaging in the Eye Conference, held in Seattle, WA, May 4, 2024.