March 2012
Volume 53, Issue 14
ARVO Annual Meeting Abstract  |   March 2012
Confocal Scanning Microfluorometer for Trans-corneal Fluorescence Lifetime Measurements
Author Affiliations & Notes
  • Yueren Wang
    School of Optometry, Indiana University, Bloomington, Indiana
  • Kyle Gabe
    School of Optometry, Indiana University, Bloomington, Indiana
  • Uday B. Kompella
    Pharmaceutical Sci & Ophthal, University of Colorado Denver, Aurora, Colorado
  • Beniamino Barbieri
    ISS Inc., Champaign, Illinois
  • Sangly P. Srinivas
    School of Optometry, Indiana University, Bloomington, Indiana
  • Footnotes
    Commercial Relationships  Yueren Wang, None; Kyle Gabe, None; Uday B. Kompella, None; Beniamino Barbieri, ISS Inc., Champaign, IL (E); Sangly P. Srinivas, None
  • Footnotes
    Support  None
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 3093. doi:
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    • Get Citation

      Yueren Wang, Kyle Gabe, Uday B. Kompella, Beniamino Barbieri, Sangly P. Srinivas; Confocal Scanning Microfluorometer for Trans-corneal Fluorescence Lifetime Measurements. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3093.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: : Specular microscopy, usually employed for pachymetry in studies of stromal deturgescence, has been previously modified to a confocal scanning microfluorometer (CSMF) for trans-corneal fluorescence measurements (Srinivas and Maurice, IEEE Trans Biomed Eng. 39, 1992). Here, the CSMF has been adapted for fluorescence lifetime measurements.

Methods: : Since halogen lamps cannot be modulated or switched at high speeds, measurement of fluorescence lifetimes could not be attempted. Hence, the halogen lamp was replaced with LED and laser diode (473 nm; ISS Inc, Champaign IL), which can be easily modulated to GHz. The outputs of these sources were coupled to the excitation slit of the CSMF using a fiber optic bundle with a circular and a rectangular end. To enable rapid depth scanning, the microscope body was mounted on a nanostage coupled to a DC motor (PI Polytech, Germany). The fluorescence output through the emission slit was coupled to a photomultiplier tube (R928, Hamamatsu) held in place of the eyepiece. Phase delay (Φ) and demodulation (M), which are needed to calculate fluorescence lifetimes, were detected using either frequency domain electronics (ChronosFD, ISS) or a lock-in amplifier (SRS830, Stanford Research Systems, Palo Alto, CA).

Results: : Fluorescence lifetimes in the range of ns to ms could be measured with fluorescein and Pd-porphyrin, respectively, with minimal collection and excitation slits widths required for trans-corneal fluorescence measurements at a depth resolution of 10 μm using a 40x objective (Zeiss, 0.75 NA, Water). For Pd-porphyrin, changes in the oxygen-sensitive Φ and M could be observed in response to changes in pO2 using the LED modulated at 1 KHz. For fluorescein at pH = 7.0, Φ and M were obtained across 5-120 MHz with the blue laser. Fitting Φ and M data to a single exponential decay indicated a fluorescence lifetime of 3.7 ns, which is in line with values reported in the literature.

Conclusions: : A fiber optic with a slit-end has been adapted to a specular microscope. This permitted the use of light sources such as LEDs and lasers, which can be easily modulated and hence ideal for the measurement of fluorescence lifetime. The success in measuring fluorescence in ns to ms with small excitation and emission slit widths indicates that the instrument would be suitable for depth-resolved fluorescence lifetime measurements across the cornea.

Keywords: microscopy: confocal/tunneling • cornea: basic science • hypoxia 

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