Abstract
Purpose :
The lifetime (τ) of Rhodamine B (RhB) is pH-insensitive but temperature and viscosity-sensitive. We assessed τ distribution of RhB across the cornea for potential thermometry using a custom-built ophthalmic time-resolved confocal scanning microfluorometer (OTR-CSMF).
Methods :
Freshly isolated porcine corneas (n>20) were exposed to RhB (10 µM) at the epithelial (Epi) for > 60 min, and then transcorneal fluorescence (F) and τ were coregistered using OTR-CSMF. The latter consisted of a confocal fluorometer, solid-state pulsed laser (488 nm), nanostage, and electronics for fast lifetime acquisition by a digital frequency-domain (DFD) approach (Colyer et al., Microsc. Res. Tech. 71, 201). The depth scanning was performed over 900 µm, and measurements were acquired every 1 µm step. We employed a 40x water-dipping objective (0.8 NA; wd=3.3 mm). The DFD unit pulsed the laser at the 20-MHz repetition rate, and the emission was collected after a 500-nm long pass . To assess the intracellular localization of RhB, we exposed the corneal endothelial monolayers to RhB (10 µM) for 10 min (n=5). The effect of temperature on the τ was calibrated independently in PBS 20-40 °C.
Results :
With the 40x objective, OTR-CSMF enabled an axial resolution of ~1.3 μm, single-molecule detection sensitivity, and a τ resolution of 50 ps. After ~60 min of RhB at Epi, a time-dependent increase in F across the cornea was observed. F exhibited discontinuities at the interfaces between Epi & stroma and between stroma & endothelium, along with sharp spikes in the stroma corresponding to accumulation in the keratocytes. Unlike F, τ across the Epi and stroma were constant at ~ 2.5 and ~ 3 ns but showed characteristic downward spikes. Moreover, τ spikes in the stroma correlated with those of F. The F and τ spikes were not apparent when the tissue was refrigerated for a long time. When RhB was exposed to endothelial monolayer, the dye accumulated in the mitochondria and showed τ of 2 ns. The calibration experiments indicated a decline in τ by 5.7%/°C
Conclusions :
The high F levels in Epi are consistent with the lipophilicity of RhB. The enhanced τ in the stroma can be attributed to the interaction of RhB with glycosaminoglycans (Mercadé-Prieto et al., Photochem & Photobio Sci, 16, 1727). The mitochondrial temperature of keratocytes potentially modulates the downward τ spikes in the stroma. Our findings indicate the feasibility of using RhB for transcorneal thermometry.
This abstract was presented at the 2023 ARVO Imaging in the Eye Conference, held in New Orleans, LA, April 21-22, 2023.