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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)
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.
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.
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.
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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