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Deborah Antwi, Ashley Ryckman, Lindsey Becker, Jun Zhang, Richard J Braun, Peter Ewen King-Smith, Carolyn G Begley; Estimating Fluorescein Concentration in the Tear Film via Two Methods. Invest. Ophthalmol. Vis. Sci. 2016;57(12):6180.
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© ARVO (1962-2015); The Authors (2016-present)
Fluorescein (FL) concentration in the tear film is needed to better estimate relative changes in tear film thickness via changes in fluorescence during tear break-up (TBU). The purpose of this study was to develop methods to measure FL concentration via image analysis of the inferior meniscus and by direct measurement of very small samples
An eye meniscus model was constructed to mimic the inferior meniscus with an upper sphere mimicking the cornea with radius of curvature of 7.8mm and a lower sphere forming an angle of 76.5 degrees (Wu et al, 2015). Two microliters of various FL concentrations were imaged on a slit lamp biomicroscope using the same light levels and conditions used on human subjects. Images were converted to grayscale and FL (pixel) intensity measured by a custom MATLAB program. Fluorescence intensity of 5 microliter samples of the known concentrations was then measured on an RT- PCR (Real time polymerase chain reaction). Calibration curves for FL concentration were developed for the slit lamp biomicroscope and PCR data.
Fluorescence intensity measured by the slit lamp biomicroscope image analysis reached its maximum at the 0.04% concentration and then rapidly decreased due to concentration self-quenching. A similar pattern was reached with PCR data, with a maximum at 0.14%FL. FL concentrations of 0.6%, 0.4%, 0.3%, 0.14 and 0.04%, yielded average pixel intensity values of 25.5, 53.2, 76.6, 149.1 and 150.55 (image analysis) and Rpre (Initial fluorescence from pre-read) values of, 21561, 30531, 25574, 47002 and 16433
These methods show promise for measuring FL concentrations in the tear film from the inferior meniscus. During TBU, as visualized via tear film fluorescence, both FL concentration and tear film thickness are unknown. Knowledge of FL concentration within the tear film before TBU begins allows better estimation of relative tear film thickness changes during TBU.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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