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P. Kambadur, R. Mutharasan, S.P. Srinivas; Adaptation of a Spot Fluorometer for Lifetime Applications . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3671.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: A Spot Fluorometer which was developed by Maurice (see "Fluorometry of the Anterior Segment", Brubaker, Maurice and McLaren, In: Noninvasive Diagnostic Techniques in Ophthalmology, Springer 1996) for anterior segment fluorometry, has been adapted for enhanced sensitivity and phase domain fluorometry of the anterior segment. Methods: The halogen lamp source was replaced by blue/green light emitting diodes (LEDs) for excitation at 440-490, 530 nm, respectively. For measurements of lifetime by the frequency-domain approach, LEDs were intensity modulated as sine waves using a function generator at frequencies 300 Hz -100 KHz. The emission was detected by a high-speed, broad spectrum photomultiplier tube (GaAs technology; sensitive up to 750 nm). The photomultiplier output was fed to a lock-in amplifier (SRS 830; 120 KHz bandwidth) along with sync output from the function generator (as the reference) for sensitive emission detection. The outputs, amplitude and phase, were logged into a PC using a GPIB interface. Results: Replacement of the original light source did not require changes to the excitation optics. Steady state fluorescence measurements revealed improved sensitivity to fluorescein due to a combination of factors including high stability of the LED output, sensitive photomultiplier, and lock-in based detection. Sensitivity of the apparatus for lifetime measurement was briefly examined using three different oxygen-sensitive fluorescent/phosphorescent dyes: Tris (1, 10 phenatroline) ruthenium (II) chloride, Pd-meso-tetra (4-carboxyphenyl) porphyrin and Oxyphor R2. Using the collection window (designed for corneal measurements), pO2 sensitivity was determined to be about 1.5 degree phase shift per mm Hg with noise level being 0.05 deg. Frequency sweeping between 500 Hz- 100 KHz indicated that optimal frequencies for modulation were 95 KHz for the Ru salt, 8 KHz for the Pd-porphyrin, and 1.5 KHz for Oxyphor R2. Conclusion: LEDs are easy to modulate at high frequencies and, therefore, are suitable for phase-domain fluorometry. In addition, LEDs provide an extremely stable light source essential for sensitive steady state fluorometry. Thus, adaptation to LEDs has enabled the spot fluorometer to be used for lifetime measurement applications such as pO2 sensing.
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