Imaging ATUM ultrathin section libraries with WaferMapper: a multi-scale approach to EM reconstruction of neural circuits

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 software package that takes a multi-resolution approach to mapping and imaging select regions within a library of ultrathin sections. This automated method selects and directs imaging of corresponding regions within each section of an ultrathin section library (UTSL) that may contain many thousands of sections. Using WaferMapper, it is possible to map thousands of tissue sections at low resolution and target multiple points of interest for high resolution imaging based on anatomical landmarks. The program can also be used to expand previously imaged regions, acquire data under different imaging conditions, or re-image after additional tissue treatments

Correlative light and electron microscopy using cathodoluminescence from nanoparticles with distinguishable colours

Correlative light and electron microscopy promises to combine molecular specificity with nanoscale imaging resolution. However, there are substantial technical challenges including reliable co-registration of optical and electron images, and rapid optical signal degradation under electron beam irradiation. Here, we introduce a new approach to solve these problems: imaging of stable optical cathodoluminescence emitted in a scanning electron microscope by nanoparticles with controllable surface chemistry. We demonstrate well-correlated cathodoluminescence and secondary electron images using three species of semiconductor nanoparticles that contain defects providing stable, spectrally-distinguishable cathodoluminescence. We also demonstrate reliable surface functionalization of the particles. The results pave the way for the use of such nanoparticles for targeted labeling of surfaces to provide nanoscale mapping of molecular composition, indicated by cathodoluminescence colour, simultaneously acquired with structural electron images in a single instrument.Physic

Pervasive Synaptic Branch Removal in the Mammalian Neuromuscular System at Birth

SummaryUsing 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 fiber. At birth, each neuromuscular junction is coinnervated by approximately ten highly intermingled axons (versus one in adults). Extensive die off of terminal branches occurs during the first several postnatal days, leading to much sparser arbors that still span the same territory. Despite the extensive pruning, total axoplasm per neuron increases as axons elongate, thicken, and add more synaptic release sites on their remaining targets. Motor axons therefore initially establish weak connections with nearly all available postsynaptic targets but, beginning at birth, massively redistribute synaptic resources, concentrating many more synaptic sites on many fewer muscle fibers. Analogous changes in connectivity may occur in the CNS.Video Abstrac

Large-Area Fluorescence and Electron Microscopic Correlative Imaging With Multibeam Scanning Electron Microscopy

Recent improvements in correlative light and electron microscopy (CLEM) technology have led to dramatic improvements in the ability to observe tissues and cells. Fluorescence labeling has been used to visualize the localization of molecules of interest through immunostaining or genetic modification strategies for the identification of the molecular signatures of biological specimens. Newer technologies such as tissue clearing have expanded the field of observation available for fluorescence labeling; however, the area of correlative observation available for electron microscopy (EM) remains restricted. In this study, we developed a large-area CLEM imaging procedure to show specific molecular localization in large-scale EM sections of mouse and marmoset brain. Target molecules were labeled with antibodies and sequentially visualized in cryostat sections using fluorescence and gold particles. Fluorescence images were obtained by light microscopy immediately after antibody staining. Immunostained sections were postfixed for EM, and silver-enhanced sections were dehydrated in a graded ethanol series and embedded in resin. Ultrathin sections for EM were prepared from fully polymerized resin blocks, collected on silicon wafers, and observed by multibeam scanning electron microscopy (SEM). Multibeam SEM has made rapid, large-area observation at high resolution possible, paving the way for the analysis of detailed structures using the CLEM approach. Here, we describe detailed methods for large-area CLEM in various tissues of both rodents and primates

Tailoring of the magnetic properties of SmCo\u3csub\u3e5\u3c/sub\u3e:Nb\u3csub\u3e0.33\u3c/sub\u3eCr\u3csub\u3e0.67\u3c/sub\u3e nanocomposites using mechanical alloying

Nanocomposite structures composed of ferromagnetic particles dispersed in a matrix are systems in which the magnetic properties can be tailored by varying the size and spacing of the ferromagnetic particles. Nanocomposites of SmCo5 in a non-magnetic Nb0.33Cr0.67 matrix exhibit a wide variety of magnetic properties. SmCo5 powder is premilled prior to mechanical alloying. The premilliing results in a maximum coercivity of 16 kOe after 2 hours of milling, and an enhanced remanence ratio. Both features may be due to exchange anisotropy and/or exchange coupling between hard and soft ferromagnetic phases. The nanocomposite samples show that, when the SmCo5 particulates are small enough, the primary effect of alloying is to disperse them throughout the matrix with no further refinement of size

Silicon-Vacancy Color Centers in Nanodiamonds: Cathodoluminescence Imaging Markers in the Near Infrared

Nanodiamonds doped with silicon-vacancy (Si-V) color centers are shown to be a promising candidate for cathodoluminescence (CL) imaging at the nanoscale, providing bright, non-bleaching, narrow-linewidth emission at wavelengths within the near-IR window of biological tissue. CL emission intensity from negative charge-state Si-V centers is greatly enhanced by increasing the nitrogen concentration during nanodiamond growth.Molecular and Cellular Biolog

Data from: Reconstruction of genetically identified neurons imaged by serial-section electron microscopy

Resolving patterns of synaptic connectivity in neural circuits currently requires serial section electron microscopy. However, complete circuit reconstruction is prohibitively slow and may not be necessary for many purposes such as comparing neuronal structure and connectivity among multiple animals. Here, we present an alternative strategy, targeted reconstruction of specific neuronal types. We used viral vectors to deliver peroxidase derivatives, which catalyze production of an electron-dense tracer, to genetically identified neurons, and developed a protocol that enhances the electron-density of the labeled cells and while retaining quality of the ultrastructure. The high contrast of the marked neurons enabled two innovations that dramatically speed data acquisition: targeted high-resolution reimaging of regions selected from rapidly-acquired lower resolution reconstruction, and an unsupervised segmentation algorithm. This pipeline reduces imaging and reconstruction times by at least two orders of magnitude, facilitating directed inquiry of circuit motifs