Green fluorescent protein (GFP) from Aqueorea Victoria  and its multicolored variations on the theme are among the most routinely fluorescent tracers used for biological visualization . Interest has grown in more precise localization studies of protein activity and movement within a cell and we could say that a new revolution started with the advent of photoactivatable fluorescent proteins [3, 4]. Fluorescence of proteins effectively brought a “new light” in molecular and cellular biology studies [5, 6], the “fluorescence toolbox” is growing  and steps towards macromolecular-scale resolution, using optical microscopes, are becoming reality . Within this pivotal scenario we focused on pa-GFP as photoactivatable fluorescent protein , and on the indispensable tools offered by confocal and two-photon microscopy [9–11]. Particle tracking inside the cell largely benefits from the ability to spatially and temporally mark specific structures to follow their “signalling” over a “dark” background as made possible since the advent of pa-GFP. Pa-GFP results from a site specific mutagenesis substituting threonine 203 with histidine in wild-type GFP. This leads an excellent photo-convertible molecule producing up to a 100 fold increase in 488 nm excited fluorescence after irradiation with high energy flux at 405 nm . Selective photoactivation by means of confocal microscopy immediately demonstrated that pa-GFP can be considered an optimal tool to study spatial and temporal dynamics of proteins in vivo, as tracking of lysosome and mitochondria [3, 4]. At the same time, when one is performing experiments using pa-GFP, one the first key aspects is related related to the ability to perform spectral fingerprinting . This is particularly useful when considering the “sea of autofluorescent molecules” present within living cells and tissues .
Now, in terms of spatial confinement of the photo activation process, the use of two-photon or even multiphoton excitation [14, 15] provides several favorable aspects compared to single photon confocal microscopy in photomarking biological structures to be tracked [16–18]. The highly confined excitation volumes, of the order of magnitude of subfemtoliter, due to the non-linear requirements provide a unique control of the excitation and consequently photoactivation in the 3D space.
Even though single photon confocal laser scanning microscopy can efficiently modulate excitation power in a planar sub-micron region, it fails to elicit the same control along the optical axis, being the excitation volume extended to the entire illumination cone of the objective . The ability to mark specific cells in living embryos by photoactivating biomolecular markers can provide a unique tool in developmental biology studies for understanding cell fate and mechanisms of differentiation .