LIVE-CELL, LABEL-FREE IMAGING OF MICROTUBULE STRUCTURE AND DYNAMICS IN HUMAN IPSC-DERIVED NEURONS

Microtubules (MTs) are highly dynamic structures that are an integral part of the cytoskeleton of neurons. Defects in the MT network and dynamics have been associated with multiple neurodegenerative diseases, such as Parkinson’s and Alzheimer’s disease. Due to their molecular organization, axonal MTs are capable of generating second harmonic (SH) signals, allowing for label-free imaging of MTs and their dynamics in neurons. Here, we show for the first time that human iPSC-derived neurons are capable of generating SH signals. Using SH microscopy, we are able to visualize changes in MT dynamics in live cells, both at the population level in response to MT (de)stabilizing agents (Taxol, Nocodazole, Colchicine), as well as in response to the local destabilization of MTs via a high-power laser pulse delivered to a small area of a neurite or neurite bundle. Furthermore, we show that there are differences in SH generation in the different neuronal compartments. More specifically, the axon initial segment (AIS), with its parallel, bundled MTs, generates strong SH signals, allowing for the label-free detection of the AIS in live samples. Our data shows that SH imaging offers the unique possibility to visualize specific neuronal structures, as well as changes in MT dynamics in live cells in a label-free manner.