Advantages and Limitations of Photoconvertible Probes to Study Subcellular Dynamics in Epithelial Cells

The recent development of a wide variety of genetically encoded photoconvertible fluorescent proteins has made it possible to study unprecedented dynamic processes by monitoring sub-populations of cells or labeled proteins. The use of photoconvertible fluorescent proteins, such as Eos, KAEDE, mMaple3, Dendra2 is a major advance. However, the conditions of their use in vivo and the inherent potential side-effects remain poorly characterized. Here, we used Drosophila pupal notum to characterize in vivo the conditions for photoconversion (PC) at the subcellular level. We compared the ability to photoconvert proteins exhibiting distinct localization and dynamics, namely, cytosolic and transmembrane proteins fused to photoconvertible probes and expressed at physiological levels. We report that the restriction of PC to a predefined region of interest depends on the mobility of the tagged protein, the power of the PC laser and the number of iterations. We characterized the axial spreading inherent to one-photon microscopy, which results in a PC cone that limits probe tracking on the z-axis. We discussed how the use of a two-photon laser can overcome this issue. We detail biases in the use of photoconvertible probes and propose strategies to circumvent them. Overall, our study provides a framework to study protein behavior at the subcellular level in living organisms.