Abstract
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.