Spatially Resolved Recording of Transient Fluorescence-Lifetime Effects by Line-Scanning TCSPC

We present a technique that records transient changes in the fluorescence lifetime of a sample with spatial resolution along a one-dimensional scan. The technique is based on scanning the sample with a high-frequency pulsed laser beam, detecting single photons of the fluorescence light, and building up a photon distribution over the distance along the scan, the arrival times of the photons after the excitation pulses and the time after a stimulation of the sample. The maximum resolution at which lifetime changes can be recorded is given by the line scan period. Transient lifetime effects can thus be resolved at a resolution of about one millisecond. We demonstrate the technique for recording photochemical and nonphotochemical chlorophyll transients in plants and transient changes in free Ca2+ in cultured neurons. Microsc. Res. Tech. 77:216-224, 2014. © 2014 Wiley Periodicals, Inc.