The fluorescent indicator pyranine is suitable for measuring stromal and cameral pH in vivo.

Three pH-sensitive fluorescent indicators (fluorescein, bis-carboxyethyl-carboxyfluorescein and pyranine) were studied to determine their usefulness as probes for the living cornea and anterior chamber. Of the three dyes, pyranine had properties which render it almost ideally suited for this purpose. Adequate concentrations are reached in the cornea and anterior chamber after topical administration; measurements can be made by fluorophotometry for many hours. The pH is calculated by measuring the ratio of fluorescent intensity at two excitation wavelengths, I463/I404, a measurement which is dependent on pH but independent of the concentration of the fluorophore and other variables which can alter the intensity. In the rabbit eye, pyranine in the cornea and anterior chamber was observed to undergo easily measurable changes in fluorescent ratios associated with lid closure and contact lens wear, indicating its sensitivity to mild changes in pH.

Modification and calibration of the Bigliano-Webb Tonometer for improved accuracy of tonometry in rabbits.

The Bigliano-Webb Tonometer (Pneumatonograph) was calibrated for adult and young rabbit eyes using open stopcock and closed stopcock methods. The performance of an old and new instrument were compared illustrating the importance of recent calibration of the instrument for most accurate use. The fact that this tonometer is a high displacement tonometer which requires specific calibration, not only for species but for age of the animal (or size of the eye) is illustrated. A minor modification of the instrument--reducing the setting of a pressure regulator which supplies gas to the tip from its nominal 9 PSI to 2.5 PSI--is illustrated as a method of reducing the difference between intraocular pressure before tonometry and during tonometry, Pt-Po.

Spectral shift of fluorescein and carboxyfluorescein in the anterior chamber of the rabbit eye following systemic administration.

Systemically administered fluorescein is converted to fluorescein glucuronide. The conjugate is fluorescent and interferes with the measurement of fluorescein in the anterior chamber. Carboxyfluorescein is a hydrophilic derivative of fluorescein. In the rabbit, carboxyfluorescein is not converted as readily to a fluorescent metabolite. Thus, carboxyfluorescein has potential advantages as a quantitative fluorophore for studies of aqueous humor dynamics.