Automatic segmentation of high pressure frozen and freeze-substituted mouse retina nuclei from FIB-SEM tomograms

Abstract

Focused Ion Beam milling combined with Scanning Electron Microscopy is a powerful tool to determine the 3-D organization of whole cells and tissue at an isotropic resolution of 3-5nm. This opens the possibility to quantify several cellular parameters and to provide detailed phenotypic information in normal or disease states. Here we describe Biocomputing methods to extract in an automated way characteristic features of mouse rod photoreceptor nuclei such as the shape and the volume of the nucleus; the proportion of heterochromatin; the number, density and distribution of nuclear pore complexes (NPC). Values obtained on five nuclei show that the number of NPC (348±8) is the most conserved feature. Nuclei in higher eukaryotes show large variations in size and rod nuclei are amongst the smallest reported (32±3μm³). Despite large species- and cell-type-specific variations in size, the density of NPC (about 15/μm²) is highly conserved.

Keywords: FIB/SEM tomography; Heterochromatin; Nucleus; Segmentation.