Fluorescence Ratioing (Ocular pH and Quenching)

Slide from Dr. Kenneth Polse, University of California, Berkeley.  Used with permission.

Fluorescein fluorescence is fairly stable with pH when excited by 450 nm light, but changes with pH when excited by longer wavelengths. This fact can be used to measure pH by ratioing techniques using the Fluorotron.

By ratioing a fluorophore that is quenched against another that is not, it is possible to measure the concentration of the quencher. This technique has been used to measure glucose in a contact lens.

Selected References:
Bonanno J, Polse K. Measurement of in vivo human corneal stromal pH: Open and closed eyes. Invest Ophthalmol Vis Sci 28:522-530 (1987). PubMed

Bonanno J, Polse K. Corneal acidosis during contact lens wear: Effects of hypoxia and CO2. Invest Ophthalmol Vis Sci 28:1514-20 (1987). PubMed

McNamara NA, Polse KA, Bonanno JA. Fluorophotometry in contact lens research: The next step. Optom Vis Sci 75:316-22 (1998). PubMed

Rivera R, Gan C, Polse K, Bonanno J, Fatt I. Contact lenses affect corneal stromal pH. Optom & Vis Sci 70:991-997 (1993). PubMed

March WF, Mueller A, Herbrechtsmeier P. Clinical trial of a noninvasive contact lens glucose sensor. Diabetes Technol Ther 6:782-9 (2004). PubMed