Transforming FIB-SEM Systems for Large-Volume Connectomics and Cell Biology
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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 × 107 μm3 while maintaining an isotropic resolution of 8 × 8 × 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 × 4 × 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.
In this chapter, 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.
Key wordsFocused ion beam scanning electron microscopy (FIB-SEM) Volume electron microscopy 3D imaging Large volume 3D structure Isotropic resolution Connectomics Cell biology Drosophila Mouse brain Mammalian cell
We would like to thank David Peale and Patrick Lee for consulting support in system modification. We also thank Zhiyuan Lu, Gleb Shtengel, David Hoffman, Amalia H. Pasolli, Kathy Schaefer, Aubrey Weigel, Nadine Randel, Michael J. Winding, and Graham Knott for EM sample preparation. We gratefully acknowledge Patrick Naulleau, Ian A. Meinertzhagen, and Steve Plaza for reviewing the manuscript and providing timely feedback. Our gratitude extends to Janelia FlyEM connectome program, in particular Gerry Rubin and Steve Plaza for their leadership. We were solely funded by the Howard Hughes Medical Institute.
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