Fluorescence Lifetime Imaging Implemented with Resonant Galvanometer Scanners

Abstract

Fluorescence Lifetime Imaging (FLIM) typically utilises specialised image intensifiers to obtain a sequence of images at known times relative to a periodic excitation source. Either time-domain or frequency-domain gating characteristics of such devices have been used to derive fluorescence lifetime images on the nanosecond timescale. However, such devices can be problematical in terms of cost, robustness and complexity. This paper explores an alternative method of obtaining lifetime images by using continuously oscillating scanning elements at defined frequencies. Employing a frequency domain approach, sinusoidally modulated laser excitation at frequencies suitable for nanosecond timescale emission is scanned rapidly and symmetrically over a line by using such resonant scanners. By introducing a sampling frequency on the optical data stream critically related to both the excitation modulation frequency and the scanner frequency, it becomes possible to encode the lifetime-related phase delay and demodulation data as a function of position. The sampling achieves the necessary down shifting of the high frequency data in addition to imposing a continuous instrumental phase shifting function. By combining sampled data for a given pixel across repeated passes of the scanner action, formulas are derived for both the steady-state intensity and lifetime-related phase and demodulation data. The overall method is illustrated by simulations and by experiments on model systems.