Martel, Jozef Adamcik, Matthew Cook, Richard H. R. Hahnloser Abstract --Serial section electron microscopy (ssEM) is a widely used technique for obtaining volumetric information of biological tissues at nanometer scale. However, accurate 3D reconstructions of identified cellular structures and volumetric quantifications require precise estimates of section thickness and anisotropy (or stretching) along the XY imaging plane. In fact, many image processing algorithms simply assume isotropy within the imaging plane. T o ameliorate this problem, we present a method for estimating thickness and stretching of electron microscopy sections using nonparametric Bayesian regression of image statistics. We verify our thickness and stretching estimates using direct measurements obtained by atomic force microscopy (AFM) and show that our method has a lower estimation error compared to a recent indirect thickness estimation method as well as a relative Z coordinate estimation method. Furthermore, we have made the first dataset of ssSEM images with directly measured section thickness values publicly available for the evaluation of indirect thickness estimation methods. I NTRODUCTION Electron microscopy (EM) has enabled imaging of nano-scale neuroanatomical structures such as synapses. Serial section Scanning Electron Microscopy (ssSEM) and serial section Transmission Electron Microscopy (ssTEM) are used to inspect tissue volumes on the scale of tens to hundreds of micrometers in each dimension. Tissue sections suitable for ssEM typically have a thickness that ranges from 30 nm to 70 nm .