We used multifocal two-photon laser scanning microscopy for local and selective protein activation and quantitative investigation of intracellular protein dynamics. The localized activation was realized with photo-activatable green-fluorescent-proteins (pa-GFP) and optical two-photon excitation in order to investigate the real-time intracellular dynamics in vivo. Such processes are of crucial importance for a deep understanding and modelling of regulatory and metabolic processes in living cells. Exemplarily, the intracellular dynamics of the Arabidopsis MYB transcription factor LHY/CCA1-like 1 (LCL1) that contains both a nuclear import and a nuclear export signal was quantitatively investigated. We used tobacco BY-2 protoplasts co-transfected with plasmids encoding photo-activatable green fluorescent protein (pa-GFP) fusion proteins and a red fluorescing transfection marker and measured the rapid nuclear export of pa-GFP-LCL1 I after its photo-activation in the nucleus. In contrast, an export-negative mutant of LCL1 remained trapped inside the nucleus. We detemined average time constants of 51s and 125s for the decrease of fluorescence in the nucleus due to active bi-directional nuclear transport of pa-GFP-LCL1 and diffusion of pa-GFP, respectively. (c) 2007 Elsevier Inc. All rights reserved.