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Live-cell imaging has been widely used to study autophagosome biogenesis and maturation. When combined with correlative electron microscopy, this approach can be extended to reveal ultrastructural details in three dimensions. The resolution of electron microscopy is needed when membrane contact sites and tubular connections between organelles are studied.

Metallic powders are commonly used in additive manufacturing processes. While their post-process consolidated properties are widely studied, there is little research on the properties of the powders prior to consolidation. Understanding the powder characteristics before use in additive manufacturing processes could lead to fine-tuning properties of additively manufactured materials. The three-dimensional grain structure of metals can be useful in predicting their properties and microstructure. Powder particles are much smaller and more difficult to fixture and polish than their bulk counterparts, hence typical protocols are difficult to use when serially sectioning them. This method describes a recommendation as to how to fixture, mill, and image metallic powder particles using a Xe P-FIB to mill and take EBSD measurements. It is based on milling and imaging techniques used for bulk materials, but with the specific advantage of how to fixture the powder sample. Our modifications include: •the method of fixturing the specimen to the holder.•the method of protecting the sample during milling.

In 1927, the German popular science magazine Die Koralle published an article entitled "The Library of Brains." The article was about the Kaiser Wilhelm Institute for Brain Research in Berlin, established in 1914 as the continuation of the "Neurological Central Station" founded by Oskar Vogt (1870-1959) in 1898. The library metaphor plays on the huge collection of human and animal brains Oskar and his wife Cécile (1875-1862) had gathered over several decades. For examination, the brains were cut into paper-thin slices and embedded in paraffin: from one single organ, up to 30,000 slices could be extracted, which were to be "read" and studied like the pages of a book. In this chapter, we take the metaphor at face value, arguing that Vogt's institute actually functioned as a library. Numerous publications have emphasized the role of the Vogts and, in particular, of the brain collection for the constitution of modern neuroscience. The "library," however, has never been closely investigated. How was it designed? How was it filled? According to which criteria were the brains collected and ordered? How did the order and the collection itself reflect the Vogts' research program? Through a detailed investigation of the collection and the Vogt Archive, we will examine this "library" and reconstruct the order of the Vogt brains. The mutual relationship between collecting, sorting, examining and publishing about the brain will be discussed., (© 2018 Elsevier B.V. All rights reserved.)

The chapter provides a concise account of collecting techniques, and basic histological techniques for investigating planarians, intended for use by practicing scientists and postgraduate students new to the field. Selected hands-on methods are described that will lead to well-stained preparations suitable for light microscopic examination for subsequent taxonomic identification or general investigations into planarian functional cytology and histology. Major equipment items that are minimal essential requirements of a functional histological laboratory are indicated. The chapter includes sectioning and staining troubleshooting guides that will be very helpful for those who must work in the laboratory without the opportunity to consult qualified and experienced technicians or microscopists.

The sigma-1 receptor (Sig1R) is an endoplasmic reticulum chaperonin that is attracting tremendous interest as a potential anti-neurodegenerative target. While this membrane protein is known to reside in the inner nuclear envelope (NE) and influences transcription, apparent Sig1R presence in the nucleoplasm is often observed, seemingly contradicting its NE localization. We addressed this confounding issue by applying an antibody-free approach of electron microscopy (EM) to define Sig1R nuclear localization. We expressed APEX2 peroxidase fused to Sig1R-GFP in a Sig1R-null NSC34 neuronal cell line generated with CRISPR-Cas9. APEX2-catalyzed gold/silver precipitation markedly improved EM clarity and confirmed an apparent intra-nuclear presence of Sig1R. However, serial sectioning combined with APEX2-enhanced EM revealed that Sig1R actually resided in the nucleoplasmic reticulum (NR), a specialized nuclear compartment formed via NE invagination into the nucleoplasm. NR cross-sections also indicated Sig1R in ring-shaped NR membranes. Thus, this study distinguishes Sig1R in the NR which could otherwise appear localized in the nucleoplasm if detected with low-resolution methods. Our finding is important for uncovering potential Sig1R regulations in the nucleus., Competing Interests: CONFLICTS OF INTEREST The authors have declared that no competing interests exist.

In this chapter the authors report on an automated hardware and software solution enabling swift correlative sample array mapping of fluorescently stained molecules within cells and tissues across length scales. Samples are first observed utilizing wide-field optical and fluorescence microscopy, followed by scanning electron microscopy, using calibration points on a dedicated sample-relocation holder. We investigated HeLa cells in vitro, fluorescently labeled for monosialoganglioside one (GM-1), across both imaging platforms within tens of minutes of initial sample preparation. This resulted in a high-throughput and high spatially resolved correlative fluorescence and electron microscopy analysis and allowed us to collect complementary nanoscopic information on the molecular and structural composition of two differently distinct HeLa cell populations expressing different levels of GM-1. Furthermore, using the small zebrafish animal model Danio rerio, we showed the versatility and relocation accuracy of the sample-relocation holder to locate fluo-tagged macromolecular complexes within large volumes using long ribbons of serial tissue sections. The subsequent electron microscopy imaging of the tissue arrays of interest enabled the generation of correlated information on the fine distribution of albumin within hepatic and kidney tissue. Our approach underpins the merits that an automated sample-relocation holder solution brings in support of results-driven research, where relevant biological questions can be answered, and high-throughput data can be generated in a rigorous statistical manner., (Copyright © 2017 Elsevier Inc. All rights reserved.)

A correction to the article "No change without a cause - why climate change remains the most plausible reason for shrubs growth dynamics in Scandinavia" by M. Hallinger and M. Wilmking, that was published in the 2011 issue is presented.

Correlative light and electron microscopy harnesses the best from each of the two modalities of microscopy it utilizes; while light microscopy provides information about the dynamic properties of the cellular structure or fluorescently labeled protein, electron microscopy provides ultrastructural information in an unsurpassed resolution. However, tracing a particular cell and its rare and small structures such as centrosomes throughout numerous steps of the experiment is not a trivial task. In this chapter, we present the experimental workflow for combining live-cell fluorescence microscopy analysis with classical transmission electron microscopy, adapted for the studies of the centrosomes and basal bodies. We describe, in a step-by-step manner, an approach that can be affordably and successfully employed in any typical cell biology laboratory. The article details all key phases of the analysis starting from cell culture, live-cell microscopy, and sample fixation, through the steps of sample preparation for electron microscopy, to the identification of the target cell on the electron microscope., (Copyright © 2015 Elsevier Inc. All rights reserved.)