Digital frequency domain approach for pO₂ sensing with a microfluorometer developed for transcorneal measurements

Abstract

Purpose : 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.

Methods : 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).

Results : 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).

Conclusions : 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.