Abstract
Purpose :
As an intrinsic property, the fluorescence lifetime of a fluorophore is independent of its concentration but is influenced by electrolyte levels, pH, temperature, membrane potential, and refractive index. Thus, lifetime measurements can probe the dynamics of biophysical/biochemical parameters in transparent tissues. Here our goal is to establish a dedicated ophthalmic time-resolved confocal scanning microfluorometer (OTR-CSMF) for intensity and lifetime measurements across the cornea.
Methods :
We established a prototype of OTR-CSMF comprising a fiber-optic-based confocal fluorometer, corneal perfusion system, multiple solid-state pulsed lasers (445, 488, and 532 nm), nanostage, and electronics for rapid lifetime acquisition by digital frequency-domain approach. Next, we developed software for data acquisition, depth scanning, laser control, and data analysis, based on Vinci™, a standard tool for desktop fluorometers. Finally, we have tested and validated OTR-CSMF with references dyes and porcine corneas ex vivo.
Results :
By focusing 488 nm laser at the coverglass:water interface using the 40x (0.8 NA; water) objective, depth vs. scatter produced a peak with FWHM of ~1.3 µm. The instrument point spread function (PSF) was assessed by fluorescence correlation spectroscopy (FCS) measurements of fluorescein in water, which led to the axial FWHM and radial beam waist estimates of 1.2 and 0.33 µm, respectively. These confirm the axial resolution and demonstrate the system's single-molecule detection sensitivity. Using rhodamine 110 in water (lifetime = 4 ns) as the standard, we found the lifetime of rhodamine B, fluorescein, and Ruthenium phenanthroline (RuPhen) to be close to values reported previously. Finally, transcorneal measurements with freshly isolated porcine corneas and multiple dyes demonstrated the principal functionality of OTR-CSMF (Figs. 1-2). Since fluorescein and RuPhen are hydrophilic, we secured their accumulation across the cornea by injection into the anterior chamber.
Conclusions :
OTR-CSMF transcends the microfluorometer described earlier by Srinivas and Maurice (IEEE Trans Biomed Eng, 39(12): 1283-1291, 1992). It enables transcorneal lifetime measurements (resolution of 50 ps; dynamic range of 100 ps – 1 ms) at a high axial resolution (~1.3 µm) and fluorescence sensitivity (~100 pM of fluorescein).
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.