In this workshop we will describe the advantages of using photo switchable tags in combination with two-photon microscopy using transgenic Arabidopsis plants stably expressing DRONPA. Imaging of thick samples present the problem of light absorption and light scattering, making high-resolution deep imaging impossible for traditional, including confocal, fluorescence microscopy. Two-photon microscopy allows imaging in vivo tissues up to one millimeter depth because it uses near infra-red excitation, which minimizes light absorption and scattering. At the same time, the need of absorption of two photons simultaneously greatly reduce the out of focus excitation, and thus limits photo bleaching and reduce background. Two photon excitation is in accordance with most of the conventional fluorophores; we, however, want to introduce the photo switchable cytosolic molecule DRONPA, a coral-derived 28-kDa fluorescent protein that may be reversibly switched between a fluorescent on-state and a non-fluorescent off-state by irradiation with light. By combining DRONPA and the high resolution excitation of two-photon microscopy, we will activate DRONPA fluorescence in one single cell of the Arabidopsis root and follow it diffusion over time into neighboring cells. We will describe the sample preparation and imaging protocol, then participants will practice preparing and mounting the samples themselves. We will compare the results obtained by two-photon vs confocal microscopes and discuss advantages and disadvantages. At the end of the workshop we will discuss other possible applications of this method to answer to other biological questions.