June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
Enhancements to a confocal microfluorometer for lifetime spectroscopy of the cornea based on a digital frequency domain
Author Affiliations & Notes
  • Kushal Shah
    Optometry, Indiana University, Bloomington, Indiana, United States
  • Sahana Damale
    Computer Science, DSCE, Bangalore, Karnataka, India
  • Ramesh Babu
    Computer Science, DSCE, Bangalore, Karnataka, India
  • Uday B Kompella
    Pharmaceutical Sciences, University of Colorado, Bangalore, Karnataka, India
  • Beniamino Barbieri
    ISS, Champaign, Illinois, United States
  • Sangly P Srinivas
    Optometry, Indiana University, Bloomington, Indiana, United States
  • Footnotes
    Commercial Relationships   Kushal Shah, None; Sahana Damale, None; Ramesh Babu, None; Uday Kompella, None; Beniamino Barbieri, ISS (E); Sangly Srinivas, None
  • Footnotes
    Support  CTSI and FRSP (PI- SP)
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 3110. doi:
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      Kushal Shah, Sahana Damale, Ramesh Babu, Uday B Kompella, Beniamino Barbieri, Sangly P Srinivas; Enhancements to a confocal microfluorometer for lifetime spectroscopy of the cornea based on a digital frequency domain. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3110.

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

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Purpose : We report on enhancements to a confocal scanning microfluorometer [CSMF; Srinivas and Maurice, IEEE Trans Biomed Eng. 1992 Dec; 39(12):1283-91] for depth-resolved frequency domain (FD) lifetime spectroscopy across the cornea.

Methods : In the FD approach, the excitation is modulated. This results in a modulated fluorescence with phase and modulation depth dependent on the lifetime of the excited fluorophore. Here we have employed a blue LED (460 nm) that can be pulsed up to 200 MHz (ISS Model N742) as the excitation source. The fluorescence emission is detected by a photomultiplier (R928). The output of the photomultiplier is directed to the signal input on a digital FD (DFD) hardware (ISS model: FastFLIM). DFD unit outputs the modulation signal to the LED (5 Volts; peak-to-peak with 2 ns pulse width) and also performs Time-Tagged-Time-Resolved data acquisition for calculation of phase and modulation depth in the emission. The operation of the DFD is synced to the depth scanning subsystem via a frame CLK input on the DFD. As per the FD approach, fluorescence lifetime (t) is calculated by tan φ = ωΤ, where ω = 2πf and f is the excitation harmonic in Hz.

Results : Several modifications to the CSMF have vastly improved its usability. First, an adaptive algorithm for depth scanning has doubled the speed of scanning so that depth scans of human/rabbit corneas can be performed within a minute. Secondly, the addition of a sighting optic, situated before the detector, has enabled easy alignment of the excitation and emission slits to be confocal. With this improvement, we routinely get a depth-resolution better than 10 µm with a 40x objective. Finally, the addition of FastFlim has made the CSMF to be useful for lifetime measurements with 50-100 ps resolution. Our initial experiments with gave measurements of lifetime at 3.9 ns for fluorescein as expected.

Conclusions : CSMF can now make lifetime measurements at the sub-nanoseconds level and hence is now poised for applications with a wider array of fluorophores of interest. Accordingly, we will be able to measure physiological parameters such as pO2, Na+, Cl-, and pH across the depth of the cornea with a high depth resolution.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.


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