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Deanna Rankin, V Akshata, MY Thanuja, S Lingesh, Yevgen Povrozin, Beniamino Barbier, Subashree Murugan, Sudhir Ranganathan, Sangly P Srinivas; Digital frequency domain approach for pO2 sensing with a microfluorometer developed for transcorneal measurements. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4167.
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To further establish a computer-controlled data acquisition module in the frequency domain for fluorescence lifetime measurements with our custom-built confocal scanning microfluorometer (CSMF). In addition, we have evaluated the suitability of various Ru2+ formulations for sensing the pO2 dynamics induced by changes in corneal O2 metabolism or consumption of O2 during collagen cross-linking.
We adapted CSMF to measure fluorescence lifetime in the frequency domain using a computer-controlled data acquisition module (K520, Digital Frequency Domain, ISS Inc., Champaign, IL). The digital frequency domain (DFD) module enabled acquisition of pulse output of a photomultiplier tube (PMT; R928P, Hamamatsu) in response to a pulsed excitation from a blue laser (450 nm; ISS Model N742). A sighting optic, positioned along the emission path of CSMF, enabled precise confocal alignment of the excitation and emission slits for depth-resolved measurements. All measurements were performed using a 20x objective (Olympus; WD = 20 mm, NA = 0.4).
The instrument was first validated with fluorescein in solution and then in the porcine corneal stroma. The lifetime was ~ 4 nanoseconds as expected across the depth of the stroma although fluorescein was unequally distributed. The instrument was then employed to assess lifetime of Ru2+ phenanthroline and its analogs in (a) solution with PBS, (b) bound to silica nanoparticles (7 nm; Sigma Chemicals) and subsequently dispersed in silicone rubber (Clear RTV), and (c) RedEye oxygen sensor patches (Ocean Optics). These different formulations of Ru2+ showed a high pO2 sensitivity in gas and in liquid with fluorescence lifetime ranging from 800 ns to 40 µs as estimated from the fluorescence decay curves by non-linear least squares and by rapid lifetime determination (RLD). Responses of some Ru2+ formulations were validated by lifetime measurements using a custom-made benchtop fluorometer which was equipped with a multichannel scaler (MSA-300, Becker & Hickl, GmbH).
Our custom-built CSMF is now capable of measuring fluorescence lifetime with a wide array of Ru2+ formulations exhibiting lifetimes in the range of ns to µs. The interface of CSMF with the computer controlled DFD has enabled sensitive pO2 measurements, making our instrument suitable for measurements of local pO2 at the ocular surface or across the cornea.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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