The pH of the corneal stroma can be measured utilizing the pH sensitive properties of the fluorescent dye fluorescein. Fluorescein is instilled into the cornea by drops or iontophoresis.

Fluorescein in cornea	Slit-Lamp Fluorimeter

The fluorescence of fluorescein in the cornea is measured by a modified slit-lamp fluorimeter. The fluorimeter will alternately measure fluorescence by exciting the dye at a pH sensitive wavelength (490 nm) and a pH insensitive wavelength (450 nm). The ratio of fluorescence emission is proportional to pH and unaffected by the concentration of dye over a wide range.

Fluorescence spectra of fluorescein at different pH values

A calibration of fluorescence ratio (490/450) vs. corneal pH is done by incubating corneas with fluorescein at the desired pH and then measuring the fluorescence ratio. See Reference #1 below for further details.

Calibration of Fluorescence Ratio vs. Corneal Stromal pH (see Reference #1 for details)

We have found that simple eye closure (Ref #1) and/or corneal hypoxia (Ref #2 & #3; e.g., while wearing contact lenses) will reduce corneal pH. In an upcoming project we plan to use the change in corneal pH produced by a standard hypoxic stimulus (i.e.; contact lens) as a measure of glycolytic activity in human subjects.

References on corneal pH

1. Bonanno JA and Polse KA: Measurement of in vivo human corneal pH: Open and closed eyes. Invest Ophthalmol Vis Sci 28:522-530 1987.
2. Bonanno JA and Polse KA: Corneal acidosis during contact lens wear: Effects of hypoxia and carbon dioxide. Invest Ophthalmol Vis Sci 28:1514-1520, 1987.
3. Bonanno JA and Polse KA: Effect of rigid gas-permeable contact lens oxygen transmissibility on stromal pH in the living human eye. Ophthalmology 94:1305-1309, 1987.
4. Cohen S, Polse K, Brand R, and Bonanno JA: Stromal acidosis affects corneal hydration control. Invest Ophthalmol Vis Sci 33(1): 134-142, 1992.
5. Polse KA, River R, Gan C, Bonanno J, and Cohen S.: Contact lens wear affects corneal pH: Implications and new directions for contact lens research. J British Contact Lens Assoc, 15:171-177, 1992.
6. Rivera R., Gan C, Polse K, Bonanno JA, and Fatt I: Contact lenses affect corneal stromal pH. Optometry and Vision Science 70(12):991-997, 1993.
7. McNamara, N., Polse, K.A. and Bonanno, J.A.: Stromal acidosis modulates corneal swelling. Invest. Ophthalmol. Vis. Sci.35(3):846-850, 1994.
8. Cohen, SR, Polse KA, Brand, RJ and Bonanno, JA. The association between pH level and corneal recovery from induced edema. Curr Eye Res 14:349-355, 1995

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