FastFIB: Self-Supervised Denoising Enables Faster, High-Resolution, Large-Volume FIB-SEM Imaging

High-pressure freezing/freeze substitution combined with focused ion beam–scanning electron microscopy (FIB–SEM) provides three-dimensional views of cellular ultrastructure at nanometer resolution, but slow acquisition limits routine imaging at the voxel sizes needed for reliable interpretation and segmentation. We developed FastFIB, a self-supervised denoising pipeline that uses simultaneously acquired secondary-electron (InLens) and energy-selective backscattered-electron (ESB) images from each milled surface as paired noisy observations in a Noise2Noise framework. FastFIB restores high-quality ESB images from low-SNR data collected with 10–30-fold shorter dwell times, making 2×2×1-nm sampling practical.

Applied to mammalian cells, FastFIB resolved microtubule lumens, polygonal clathrin lattices on coated pits and vesicles, and extended hexagonal lattices within flat endosomal clathrin patches. These endosomal lattices were not spatially associated with intraluminal-vesicle buds. FastFIB also revealed vesicle clusters at endoplasmic reticulum exit sites and separate Golgi stacks with no detectable membrane continuity between them. Morphologically distinctive ectosomes were present extracellularly and within endosomes. Budding ectosome profiles occurred at the plasma membrane but not at endosomal membranes, consistent with uptake from the cell surface. Thus, FastFIB makes high-resolution volume imaging practical and reveals structural intermediates difficult to resolve with conventional 5×5×5-nm FIB–SEM sampling.