webKnossos: efficient online 3D data annotation for connectomics.

We report webKnossos, an in-browser annotation tool for 3D electron microscopic data. webKnossos provides flight mode, a single-view egocentric reconstruction method enabling trained annotator crowds to reconstruct at a speed of 1.5 ± 0.6 mm/h for axons and 2.1 ± 0.9 mm/h for dendrites in 3D electron microscopic data from mammalian cortex. webKnossos accelerates neurite reconstruction for connectomics by 4- to 13-fold compared with current state-of-the-art tools, thus extending the range of connectomes that can realistically be mapped in the future.

Mitotic Diversity in Homeostatic Human Interfollicular Epidermis.

Despite decades of skin research, regulation of proliferation and homeostasis in human epidermis is still insufficiently understood. To address the role of mitoses in tissue regulation, we utilized human long-term skin equivalents and systematically assessed mitoses during early epidermal development and long-term epidermal regeneration. We now demonstrate four different orientations: (1) horizontal, i.e., parallel to the basement membrane (BM) and suggestive of symmetric divisions; (2) oblique with an angle of 45°-70°; or (3) perpendicular, suggestive of asymmetric division. In addition, we demonstrate a fourth substantial fraction of suprabasal mitoses, many of which are committed to differentiation (Keratin K10-positive). As verified also for normal human skin, this spatial mitotic organization is part of the regulatory program of human epidermal tissue homeostasis. As a potential marker for asymmetric division, we investigated for Numb and found that it was evenly spread in almost all undifferentiated keratinocytes, but indeed asymmetrically distributed in some mitoses and particularly frequent under differentiation-repressing low-calcium conditions. Numb deletion (stable knockdown by CRISPR/Cas9), however, did not affect proliferation, neither in a three-day follow up study by life cell imaging nor during a 14-day culture period, suggesting that Numb is not essential for the general control of keratinocyte division.

Subcellular Raman Microspectroscopy Imaging of Nucleic Acids and Tryptophan for Distinction of Normal Human Skin Cells and Tumorigenic Keratinocytes.

At present, tumor diagnostic imaging is commonly based on hematoxylin and eosin or immunohistochemical staining of biopsies, which requires tissue excision, fixation, and staining with exogenous marker molecules. Here, we report on label-free tumor imaging using confocal spontaneous Raman scattering microspectroscopy, which exploits the intrinsic vibrational contrast of endogenous biomolecular species. We present a chemically specific and quantitative approach to monitoring normal human skin cells (keratinocytes and fibroblasts) as well as the human HaCaT in vitro skin carcinogenesis model and the tumor-derived MET in vivo skin cancer progression model. Mapping the amplitudes of two spectrally well isolated Raman bands at 752 and 785 cm(-1) allowed for direct visualization of the distributions representative of tryptophan-rich proteins and nucleic acids, respectively, with subcellular spatial resolution. Using these Raman markers, it was feasible to discriminate between normal human epidermal keratinocytes (NHEK) and dermal fibroblasts (NHDF) and to confine all tumorigenic cells from both the NHEK and NHDF. First evidence for the successful application of the proposed intracellular nucleic acid and tryptophan Raman signatures for skin cancer diagnosis was further demonstrated in an organotypic cutaneous squamous cell carcinomas model, allowing for the identification of tumor cells and their surrounding stroma in the tissue context.

Functional and multiscale 3D structural investigation of brain tissue through correlative in vivo physiology, synchrotron microtomography and volume electron microscopy.

Understanding the function of biological tissues requires a coordinated study of physiology and structure, exploring volumes that contain complete functional units at a detail that resolves the relevant features. Here, we introduce an approach to address this challenge: Mouse brain tissue sections containing a region where function was recorded using in vivo 2-photon calcium imaging were stained, dehydrated, resin-embedded and imaged with synchrotron X-ray computed tomography with propagation-based phase contrast (SXRT). SXRT provided context at subcellular detail, and could be followed by targeted acquisition of multiple volumes using serial block-face electron microscopy (SBEM). In the olfactory bulb, combining SXRT and SBEM enabled disambiguation of in vivo-assigned regions of interest. In the hippocampus, we found that superficial pyramidal neurons in CA1a displayed a larger density of spine apparati than deeper ones. Altogether, this approach can enable a functional and structural investigation of subcellular features in the context of cells and tissues.

Dense connectomic reconstruction in layer 4 of the somatosensory cortex.

The dense circuit structure of mammalian cerebral cortex is still unknown. With developments in three-dimensional electron microscopy, the imaging of sizable volumes of neuropil has become possible, but dense reconstruction of connectomes is the limiting step. We reconstructed a volume of ~500,000 cubic micrometers from layer 4 of mouse barrel cortex, ~300 times larger than previous dense reconstructions from the mammalian cerebral cortex. The connectomic data allowed the extraction of inhibitory and excitatory neuron subtypes that were not predictable from geometric information. We quantified connectomic imprints consistent with Hebbian synaptic weight adaptation, which yielded upper bounds for the fraction of the circuit consistent with saturated long-term potentiation. These data establish an approach for the locally dense connectomic phenotyping of neuronal circuitry in the mammalian cortex.

SynEM, automated synapse detection for connectomics.

Nerve tissue contains a high density of chemical synapses, about 1 per µm3 in the mammalian cerebral cortex. Thus, even for small blocks of nerve tissue, dense connectomic mapping requires the identification of millions to billions of synapses. While the focus of connectomic data analysis has been on neurite reconstruction, synapse detection becomes limiting when datasets grow in size and dense mapping is required. Here, we report SynEM, a method for automated detection of synapses from conventionally en-bloc stained 3D electron microscopy image stacks. The approach is based on a segmentation of the image data and focuses on classifying borders between neuronal processes as synaptic or non-synaptic. SynEM yields 97% precision and recall in binary cortical connectomes with no user interaction. It scales to large volumes of cortical neuropil, plausibly even whole-brain datasets. SynEM removes the burden of manual synapse annotation for large densely mapped connectomes.

SegEM: Efficient Image Analysis for High-Resolution Connectomics.

Progress in electron microscopy-based high-resolution connectomics is limited by data analysis throughput. Here, we present SegEM, a toolset for efficient semi-automated analysis of large-scale fully stained 3D-EM datasets for the reconstruction of neuronal circuits. By combining skeleton reconstructions of neurons with automated volume segmentations, SegEM allows the reconstruction of neuronal circuits at a work hour consumption rate of about 100-fold less than manual analysis and about 10-fold less than existing segmentation tools. SegEM provides a robust classifier selection procedure for finding the best automated image classifier for different types of nerve tissue. We applied these methods to a volume of 44 × 60 × 141 µm(3) SBEM data from mouse retina and a volume of 93 × 60 × 93 µm(3) from mouse cortex, and performed exemplary synaptic circuit reconstruction. SegEM resolves the tradeoff between synapse detection and semi-automated reconstruction performance in high-resolution connectomics and makes efficient circuit reconstruction in fully-stained EM datasets a ready-to-use technique for neuroscience.

Three-Dimensional In Vitro Skin and Skin Cancer Models Based on Human Fibroblast-Derived Matrix.

Three-dimensional in vitro skin and skin cancer models help to dissect epidermal-dermal and tumor-stroma interactions. In the model presented here, normal human dermal fibroblasts isolated from adult skin self-assembled into dermal equivalents with their specific fibroblast-derived matrix (fdmDE) over 4 weeks. The fdmDE represented a complex human extracellular matrix that was stabilized by its own heterogeneous collagen fiber meshwork, largely resembling a human dermal in vivo architecture. Complemented with normal human epidermal keratinocytes, the skin equivalent (fdmSE) thereof favored the establishment of a well-stratified and differentiated epidermis and importantly allowed epidermal regeneration in vitro for at least 24 weeks. Moreover, the fdmDE could be used to study the features of cutaneous skin cancer. Complementing fdmDE with HaCaT cells in different stages of malignancy or tumor-derived cutaneous squamous cell carcinoma cell lines, the resulting skin cancer equivalents (fdmSCEs) recapitulated the respective degree of tumorigenicity. In addition, the fdmSCE invasion phenotypes correlated with their individual degree of tissue organization, disturbance in basement membrane organization, and presence of matrix metalloproteinases. Together, fdmDE-based models are well suited for long-term regeneration of normal human epidermis and, as they recapitulate tumor-specific growth, differentiation, and invasion profiles of cutaneous skin cancer cells, also provide an excellent human in vitro skin cancer model.

Two distinct channels of olfactory bulb output.

Rhythmic neural activity is a hallmark of brain function, used ubiquitously to structure neural information. In mammalian olfaction, repetitive sniffing sets the principal rhythm but little is known about its role in sensory coding. Here, we show that mitral and tufted cells, the two main classes of olfactory bulb projection neurons, tightly lock to this rhythm, but to opposing phases of the sniff cycle. This phase shift is established by local inhibition that selectively delays mitral cell activity. Furthermore, while tufted cell phase is unperturbed in response to purely excitatory odorants, mitral cell phase is advanced in a graded, stimulus-dependent manner. Thus, phase separation by inhibition forms the basis for two distinct channels of olfactory processing.