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We describe automated technologies to probe the structure of neural tissue at nanometer resolution and use them to generate a saturated reconstruction of a sub-volume of mouse neocortex in which all cellular objects (axons, dendrites, and glia) and many sub-cellular components (synapses, synaptic vesicles, spines, spine apparati, postsynaptic densities, and(More)
Recent studies have shown that machine learning can improve the accuracy of detecting object boundaries in images. In the standard approach, a boundary detector is trained by minimizing its pixel-level disagreement with human boundary tracings. This naive metric is problematic because it is overly sensitive to boundary locations. This problem is solved by(More)
Conventional heavy metal poststaining methods on thin sections lend contrast but often cause contamination. To avoid this problem, we tested several en bloc staining techniques to contrast tissue in serial sections mounted on solid substrates for examination by field emission scanning electron microscopy (FESEM). Because FESEM section imaging requires that(More)
Electron-electron interactions and detector bandwidth limit the maximal imaging speed of single-beam scanning electron microscopes. We use multiple electron beams in a single column and detect secondary electrons in parallel to increase the imaging speed by close to two orders of magnitude and demonstrate imaging for a variety of samples ranging from(More)
VIDEO ABSTRACT Using light and serial electron microscopy, we show profound refinements in motor axonal branching and synaptic connectivity before and after birth. Embryonic axons become maximally connected just before birth when they innervate ∼10-fold more muscle fibers than in maturity. In some developing muscles, axons innervate almost every muscle(More)
The automated tape-collecting ultramicrotome (ATUM) makes it possible to collect large numbers of ultrathin sections quickly-the equivalent of a petabyte of high resolution images each day. However, even high throughput image acquisition strategies generate images far more slowly (at present ~1 terabyte per day). We therefore developed WaferMapper, a(More)
Cryo-electron microscopy can provide high-resolution structural information about cells and organelles in the nearly native, frozen-hydrated state. Applicability, however, is limited by difficulties encountered in preparing suitably thin, vitreously frozen biological specimens. We demonstrate, by cryo-electron tomography of Escherichia coli cells, that a(More)
The endoplasmic reticulum (ER) often forms stacked membrane sheets, an arrangement that is likely required to accommodate a maximum of membrane-bound polysomes for secretory protein synthesis. How sheets are stacked is unknown. Here, we used improved staining and automated ultrathin sectioning electron microscopy methods to analyze stacked ER sheets in(More)
A full understanding of brain function requires extensive knowledge of the intricate patterns of axons and dendrites that connect neurons at synapses. Such wiring diagrams (" connectomes ") are in short supply owing to the enormous number of synaptic connectivities that need to be catalogued and the very high resolution necessary to trace them [1]. The key(More)
This paper describes the use of several methods of template stripping (TS) to produce ultraflat films of silver, gold, palladium, and platinum on both rigid and polymeric mechanical supports: a composite of glass and ultraviolet (UV)-curable adhesive (optical adhesive, OA), solder, a composite of poly(dimethyl siloxane) (PDMS) and OA, and bare OA. Silicon(More)