Abstract
Isotropic high-resolution imaging of large volumes provides unprecedented opportunities to advance connectomics and cell biology research. Conventional Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) offers unique benefits such as high resolution (< 10 nm in x, y, and z), robust image alignment, and minimal artifacts for superior tracing of neurites. However, its prevailing deficiencies in imaging speed and duration cap the maximum possible image volume. We have developed technologies to overcome these limitations, thereby expanding the image volume of FIB-SEM by more than four orders of magnitude from 103 µm3 to 3 x 107 µm3 while maintaining an isotropic resolution of 8 x 8 x 8 nm3 voxels. These expanded volumes are now large enough to support connectomic studies, in which the superior z resolution enables automated tracing of fine neurites and reduces the time-consuming human proofreading effort. Moreover, by trading off imaging speed, the system can readily be operated at even higher resolutions achieving voxel sizes of 4 x 4 x 4 nm3, thereby generating ground truth of the smallest organelles for machine learning in connectomics and providing important insights into cell biology. Primarily limited by time, the maximum volume can be greatly extended.
Here we provide a detailed description of the enhanced FIB-SEM technology, which has transformed the conventional FIB-SEM from a laboratory tool that is unreliable for more than a few days to a robust imaging platform with long term reliability: capable of years of continuous imaging without defects in the final image stack. An in-depth description of the systematic approach to optimize operating parameters based on resolution requirements and electron dose boundary conditions is also explicitly disclosed. We further explore how this technology unleashes the full potential of FIB-SEM systems, revolutionizing volume electron microscopy (EM) imaging for biology by gaining access to large sample volumes with single-digit nanoscale isotropic resolution.
