Method to quantify accuracy of position feedback signals of a three-dimensional twophoton laser-scanning microscope Michael Kummer,1 Knut Kirmse,1 Otto W. Witte,1 Jens Haueisen,2 and Knut Holthoff1,* 1
Experimentelle Neurologie, Hans-Berger-Klinik für Neurologie, Universitätsklinikum Jena, Erlanger Allee 101, D07747 Jena, Germany 2 Institut für Biomedizinische Technik und Informatik, Technische Universität Ilmenau Gustav-Kirchhoff Str. 2, D98693 Ilmenau, Germany *
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Abstract: 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 contactfree 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
