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

Autor: Kushal Shah, D. R. Ramesh Babu, Sangly P. Srinivas, Sahana Damale, Beniamino Barbieri, Yevgen Povrozin
Rok vydání: 2019
Předmět:
Zdroj: Materials Today: Proceedings. 10:87-91
ISSN: 2214-7853
DOI: 10.1016/j.matpr.2019.02.192
Popis: 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= 2πf 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.
Databáze: OpenAIRE
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