Abstract
Abstract: :
Purpose: Fluorescence lifetime (τ) of Ru dye (ruthenium(II) tris((4,7-diphenyl)-1,10-phenanthr-oline) perchlorate) is sensitive to O2. When excitation for the dye is modulated by a sine wave, the corresponding emission is also a sine wave of the same frequency but with an O2-sensitive phase delay (θ). θ and τ are related by tan θ = 2πfτ. This principle has been applied to measure O2 levels at high temporal resolution and tested by examining rapid absorption and desorption of O2 into and out of soft contact lenses. Methods: Contact lenses were soaked in PBS containing Ru dye (ruthenium(II)tris((4,7-diphenyl)-1,10-phenanthroline) perchlorate) for 10 min. The lens was then blotted and placed on a glass slide held diagonally in a cuvette connected to a N2 gas line. A blue LED with a peak output at 460 nm and modulated at 95 KHz was used for excitation. Emission at 600+20 nm was detected by an avalanche photodiode. O2-sensitive θ was measured by a lock-in amplifier and then logged to a PC at 10 Hz and at 16 bits resolution. Desorption was initiated by flushing the cuvette rapidly with N2; absorption of O2 from the surrounding air followed upon shutting off N2. Results: Desorption upon exposure to N2 led to a rapid increase in both the phase delay and intensity of fluorescence consistent with a decrease in the extent of quenching by the loss of O2. The absorption step that began immediately after shutting off the N2 flow showed a slow decrease in both the phase delay and intensity of fluorescence consistent with an increase in quenching by the elevated O2 in the lens matrix. Based on a double exponential model for θ vs time, oxygen mass transfer coefficients for desorption and absorption were 0.46 sec-1 (n = 5) and 0.05 sec-1 (n = 5), respectively. Conclusion: (1) Desorption was rapid due to negligible gas film resistance, a consequence of high N2 flow rate. Thus, the resistance for O2 loss is determined only by its diffusion coefficient in the lens and thickness of the lens. Absorption, however, is slow because of large gas film thickness surrounding the lens in addition to diffusional resistance in the lens. (2). The method of phase-fluorometry offers a high temporal resolution and therefore, may be employed in dynamic situations such as exemplified by the contact lens application in this study.
Keywords: 367 contact lens • 369 cornea: clinical science • 428 hypoxia