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McKinzie Daniels, T Sushma, S Amutha, Ramesh Babu, Uday B Kompella, Sangly P Srinivas; Trans-corneal Fluorescence Lifetime Spectroscopy in Time Domain using a Multichannel Scalar. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3111.
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© ARVO (1962-2015); The Authors (2016-present)
Multi-channel scalars (MCS) have been used to measure fluorescence lifetimes of long-lived fluorophores in the time domain (TD). In this project, we have interfaced a standard MCS (SR430; Stanford Research Systems) to a custom-built confocal scanning microfluorometer (CSMF; Srinivas SP, Maurice DM., A microfluorometer for measuring diffusion of fluorophores across the cornea. IEEE Trans Biomed Eng., 39(12):1283-91; 1992) to enable measurements of fluorescence lifetimes of exogenous fluorophores such as Ru phenanthroline for sensing pO2 at localized depths across the cornea ex vivo.
A high power LED, employed as the excitation source, was pulsed by a constant current generator (AV-156-A-B) at 12V DC. Pulse width and current amplitude were set at 10 µs and 2 A, respectively. Pulse frequency was varied (100 - 1500 Hz) depending on the number of bins/record in the MCS (SR430; Stanford Research Systems). A sync signal from the current generator was coupled to trigger data acquisition in the MCS. Depending on the formulation of the Ru salts or Pd-porphyrin, dwell time, record lengths, and records/scan in SR430 were varied as 40-160 ns, 1-8k, 5000-10000, respectively. Since SR430 is equipped with a pulse discriminator, output of the PMT could be coupled directly to SR430. Set up of SR430 and transfer of bin data to a PC were controlled by a LabVIEW program.
Our preliminary experiments, with and without CSMF, consisted of measuring changes in lifetime of porphyrins (Porphyrin R2 and G2) as well as Ru phenothroline in response O2 and N2 transitions. In all our experiments, changes in lifetime could be recorded with a temporal resolution of 5-10 seconds. Despite the small slit widths, changes in lifetime of Ru salts could be measured with CSMF at high pulse frequencies giving a temporal resolution of > 0.1 Hz. The lifetime could also be calculated by non-linear least squares and RLD (rapid lifetime determination) algorithms in realtime.
Although the TD approach offers poor temporal resolution, the new CSMF-MCS combination will enable accurate and direct measurement of trans-corneal lifetime of long-lived fluorophores of interest to our applications (e.g., pO2 sensing). Moreover, the success of the RLD method will enable us to limit the number of records/scan once lifetime is estimated to desired accuracy during a scan.
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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