Method to quantify accuracy of position feedback signals of a three-dimensional two-photon laser-scanning microscope

Two-photon laser-scanning microscopy enables to record neuronal network activity in three-dimensional space while maintaining single-cellular resolution. One of the proposed approaches combines galvanometric x-y scanning with piezo-driven objective movements and employs hardware feedback signals for position monitoring. However, readily applicable methods to quantify the accuracy of those feedback signals are currently lacking. Here we provide techniques based on contact-free laser reflection and laser triangulation for the quantification of positioning accuracy of each spatial axis. We found that the lateral feedback signals are sufficiently accurate (defined as <2.5 µm) for a wide range of scan trajectories and frequencies. We further show that axial positioning accuracy does not only depend on objective acceleration and mass but also its geometry. We conclude that the introduced methods allow a reliable quantification of position feedback signals in a cost-efficient, easy-to-install manner and should be applicable for a wide range of two-photon laser scanning microscopes.