High-speed FLIM data acquisition by time-correlated single-photon counting

Abstract

In this study, we describe a time-correlated single photon counting (TCSPC) technique for multi-wavelength lifetime imaging in laser-scanning microscopes. The technique is based on a four-dimensional histogramming process that records the photon density versus the time in the fluorescence decay, the x-y coordinates of the scanning area and the wavelength. It avoids any time gating or wavelength scanning and, therefore, yields a near-ideal counting efficiency. The decay functions are recorded in a large number of time channels, and the components of a multi-exponential decay can be resolved down to less than 30 ps. A single TCSPC imaging channel works with a high detection efficiency up to a photon count rate of about 5•10⁶s^-1. A modified version of the TCSPC fluorescence lifetime imaging (FLIM) technique uses several fully parallel detector and TCSPC channels. It operates at a count rate of more than 10⁷ photons per second and records double-exponential FLIM data within less than 10 seconds.