Digital frequency domain approach for fluorescence lifetime spectroscopy of the cornea using an enhanced confocal scanning microfluorometer

Abstract We report on enhancement to a confocal scanning microfluorometer (CSMF) for depth-resolved frequency domain lifetime spectroscopy across the cornea. Fluorescence was detected by a photomultiplier tube (PMT; R928P, Hamamatsu) and coupled to computer-controlled data acquisition module (K520, Digital Frequency Domain, ISS Inc.).The digital frequency domain (DFD) module performed acquisition of PMT pulses for further calculation of phase ( ϕ ) and modulation (M) in the emission of the sample, relative to excitation pulses, at 30 harmonics. As per the DFD approach, fluorescence lifetime ( τ ) was calculated by tanϕ =ωτ , where ω= 2 πf with f being the excitation harmonic in Hz. The modulation depth can be calculated by M = 1 / √ ( 1 + ω 2 τ 2 ) . The DFD module was also used to pulse the excitation laser (450 nm; ISS Model N742) with the repetition rate of up to 10 MHz (fundamental frequency). Apart from coupling to DFD module, two other modifications were incorporated to enhance the CSMF: first, a sighting optic was constructed and placed before the PMT for rapid and precise confocal alignment of the excitation and emission slits, and second, a stepper motor was interfaced to the vertical nanostage for rapid depth scanning. These enhancements have enabled the CSMF to consistently produce a depth resolution of 7 µm during transcorneal scans with a 40x water immersion objective (Zeiss; WD = 1.2 mm, NA= 0.75). Overall, the CSMF is now capable of measuring transcorneal fluorescence lifetime with a wide array of fluorophores exhibiting lifetimes in the range of µs to nanoseconds.