Abstract
Serial block face scanning electron microscopy (SBEM) provides nanoscale 3D ultrastructure of eukaryotic cells and tissue volumes. It allows automated acquisition of thousands of sequential images at different depths within a specimen block without layer-to-layer warping problems and section loss. However, image drift, jitter and distortion can still occur due to mechanical instabilities and build-up of surface charge. These artifacts pose challenges to 3D image stack alignment, which is crucial for post data analysis, especially for computer aided image segmentation and volume rendering. In this work, we have investigated effective means of improving fine-alignment in three dimensions to facilitate 3D image data reconstruction, visualization and quantification. The focal charge compensation module on the Zeiss Sigma VP/Gatan 3View SBEM system greatly reduces charging artifacts and the need for coarse image stack alignment. However, some lesser but important misalignments remain, especially for backscattered electron imaging at multiple primary energies to extract sub-slice depth information, which require image stack alignment with single voxel precision. For this fine alignment, we have tested three available programs: scale-invariant feature transform (SIFT), patch-based cross correlation in IMOD, and TeraSticher, which can be applied to very large datasets. A workflow of fine alignment of SBEM image stacks is proposed.
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call