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Methods in Cell Biology

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https://www.readbyqxmd.com/read/28528643/a-new-method-for-cryo-sectioning-cell-monolayers-using-a-correlative-workflow
#1
Androniki Kolovou, Martin Schorb, Abul Tarafder, Carsten Sachse, Yannick Schwab, Rachel Santarella-Mellwig
Cryo-electron microscopy (cryo-EM) techniques have made a huge advancement recently, providing close to atomic resolution of the structure of protein complexes. Interestingly, this imaging technique can be performed in cells, giving access to the molecular machines in their natural context, therefore bridging structural and cell biology. However, in situ structural electron microscopy faces one major challenge, which is the ability to focus on specific subcellular regions to capture the objects of interest...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528642/minimal-resin-embedding-of-multicellular-specimens-for-targeted-fib-sem-imaging
#2
Nicole L Schieber, Pedro Machado, Sebastian M Markert, Christian Stigloher, Yannick Schwab, Anna M Steyer
Correlative light and electron microscopy (CLEM) is a powerful tool to perform ultrastructural analysis of targeted tissues or cells. The large field of view of the light microscope (LM) enables quick and efficient surveys of the whole specimen. It is also compatible with live imaging, giving access to functional assays. CLEM protocols take advantage of the features to efficiently retrace the position of targeted sites when switching from one modality to the other. They more often rely on anatomical cues that are visible both by light and electron microscopy...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528641/preserving-the-photoswitching-ability-of-standard-fluorescent-proteins-for-correlative-in-resin-super-resolution-and-electron-microscopy
#3
Errin Johnson, Rainer Kaufmann
There are many different correlative light and electron microscopy (CLEM) techniques available. The use of super-resolution microscopy in CLEM is an emerging application and while offering the obvious advantages of improved resolution in the fluorescence image, and therefore more precise correlation to electron microscopy (EM) ultrastructure, it also presents new challenges. Choice of fluorophore, method of fixation, and timing of the fluorescence imaging are critical to the success of super-resolution CLEM and the relative importance, and technical difficulty, of each of these factors depends on the type of super-resolution microscopy being employed...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528640/ec-clem-a-multidimension-multimodel-software-to-correlate-intermodal-images-with-a-focus-on-light-and-electron-microscopy
#4
Xavier Heiligenstein, Perrine Paul-Gilloteaux, Graça Raposo, Jean Salamero
Correlative light and electron microscopy (CLEM) is a scientific method covered by a broad range of techniques. The path taken to explore a scientific question is often driven both by the question and the technology available. Yet, one common step to all CLEM workflows is the registration of the multimodal images to assign a fluorescent signal to an ultrastructure. The manual relocation and registration of light microscopy and electron microscopy images can be challenging and time-consuming (Muller-Reichert & Verkade, 2014)...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528639/matrix-maps-an-intuitive-software-to-acquire-analyze-and-annotate-light-microscopy-data-for-clem
#5
Martin Schorb, Frank Sieckmann
Matrix MAPS provides an intuitive interface for acquiring light microscopy data during a correlative light and electron microscopy experiment either at room or cryogenic temperatures. First, the user graphically defines the geometry of the acquisition region on top of preview images. Multiple independent regions can then be imaged in an automated way, each with individual settings. The same interface allows the user to mark and select points or regions of interest whose coordinates can subsequently be transferred directly to the electron microscope...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528638/triclem-combining-high-precision-room-temperature-clem-with%C3%A2-cryo-fluorescence-microscopy-to-identify-very-rare-events
#6
Nicholas R Ader, Wanda Kukulski
Fiducial-based correlation of fluorescence and electron microscopy data from high-pressure frozen and resin-embedded samples allows for high-precision localization of fluorescent signals to subcellular ultrastructure. Here we introduce the triCLEM procedure to facilitate the identification of very rare events for high-precision correlation. We present a detailed protocol to screen high-pressure frozen cell monolayers on sapphire disks for very rare signals by cryo-fluorescence microscopy, relocate the cells of interest after freeze substitution and Lowicryl embedding, and perform fiducial-based correlation of the identified fluorescent signals to high-magnification electron tomograms...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528637/find-your-way-with-x-ray-using-microct-to-correlate-in-vivo-imaging-with-3d-electron-microscopy
#7
Matthia A Karreman, Bernhard Ruthensteiner, Luc Mercier, Nicole L Schieber, Gergely Solecki, Frank Winkler, Jacky G Goetz, Yannick Schwab
Combining in vivo imaging with electron microscopy (EM) uniquely allows monitoring rare and critical events in living tissue, followed by their high-resolution visualization in their native context. A major hurdle, however, is to keep track of the region of interest (ROI) when moving from intravital microscopy (IVM) to EM. Here, we present a workflow that relies on correlating IVM and microscopic X-ray computed tomography to predict the position of the ROI inside the EM-processed sample. The ROI can then be accurately and quickly targeted using ultramicrotomy and imaged using EM...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528636/correlative-two-photon-and-serial-block-face-scanning-electron-microscopy-in-neuronal-tissue-using-3d-near-infrared-branding-maps
#8
Robert M Lees, Christopher J Peddie, Lucy M Collinson, Michael C Ashby, Paul Verkade
Linking cellular structure and function has always been a key goal of microscopy, but obtaining high resolution spatial and temporal information from the same specimen is a fundamental challenge. Two-photon (2P) microscopy allows imaging deep inside intact tissue, bringing great insight into the structural and functional dynamics of cells in their physiological environment. At the nanoscale, the complex ultrastructure of a cell's environment in tissue can be reconstructed in three dimensions (3D) using serial block face scanning electron microscopy (SBF-SEM)...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528635/relocation-is-the-key-to-successful-correlative-fluorescence-and-scanning-electron-microscopy
#9
Delfine Cheng, Gerald Shami, Marco Morsch, Minh Huynh, Patrick Trimby, Filip Braet
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...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528634/3d-subcellular-localization-with-superresolution-array-tomography-on-ultrathin-sections-of-various-species
#10
Sebastian M Markert, Vivien Bauer, Thomas S Muenz, Nicola G Jones, Frederik Helmprobst, Sebastian Britz, Markus Sauer, Wolfgang Rössler, Markus Engstler, Christian Stigloher
Array Tomography (AT) is a relatively easy-to-use and yet powerful method to put molecular identity in its full ultrastructural context. Ultrathin sections are stained with fluorophores and then imaged by light and afterward by electron microscopy to obtain a correlated view of a region of interest: its ultrastructure and specific staining. By combining AT with high-pressure freezing for superior structural preservation and superresolution light microscopy, even small subcellular structures can be mapped in 3D...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528633/correlative-light-electron-microscopy-in-liquid-using%C3%A2-an-inverted-sem-asem
#11
Chikara Sato, Takaaki Kinoshita, Nassirhadjy Memtily, Mari Sato, Shoko Nishihara, Toshiko Yamazawa, Shinya Sugimoto
In atmospheric scanning electron microscope (ASEM), the inverted scanning electron microscope (SEM) observes the wet sample from below, while an optical microscope observes it from above simultaneously. The ASEM sample holder has a disposable dish shape with a silicon nitride film window at the bottom. It can be coated variously for the primary-culture of substrate-sensitive cells; primary cells were cultured in a few milliliters of culture medium in a stable incubator environment. For the inverted SEM observation, cells and the excised tissue blocks were aldehyde-fixed, immersed in radical scavenger solution, and observed at minimum electron dose...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528632/clafem-correlative-light-atomic-force-electron-microscopy
#12
Sébastien Janel, Elisabeth Werkmeister, Antonino Bongiovanni, Frank Lafont, Nicolas Barois
Atomic force microscopy (AFM) is becoming increasingly used in the biology field. It can give highly accurate topography and biomechanical quantitative data, such as adhesion, elasticity, and viscosity, on living samples. Nowadays, correlative light electron microscopy is a must-have tool in the biology field that combines different microscopy techniques to spatially and temporally analyze the structure and function of a single sample. Here, we describe the combination of AFM with superresolution light microscopy and electron microscopy...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528631/a-fully-integrated-three-dimensional-fluorescence-to-electron-microscopy-correlative-workflow
#13
Claudia S López, Cedric Bouchet-Marquis, Christopher P Arthur, Jessica L Riesterer, Gregor Heiss, Guillaume Thibault, Lee Pullan, Sunjong Kwon, Joe W Gray
While fluorescence microscopy provides tools for highly specific labeling and sensitive detection, its resolution limit and lack of general contrast has hindered studies of cellular structure and protein localization. Recent advances in correlative light and electron microscopy (CLEM), including the fully integrated CLEM workflow instrument, the FEI CorrSight with MAPS, have allowed for a more reliable, reproducible, and quicker approach to correlate three-dimensional time-lapse confocal fluorescence data, with three-dimensional focused ion beam-scanning electron microscopy data...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528630/correlation-of-live-cell-imaging-with-volume-scanning-electron-microscopy
#14
Miriam S Lucas, Maja Günthert, Anne Greet Bittermann, Alex de Marco, Roger Wepf
Live-cell imaging is one of the most widely applied methods in live science. Here we describe two setups for live-cell imaging, which can easily be combined with volume SEM for correlative studies. The first procedure applies cell culture dishes with a gridded glass support, which can be used for any light microscopy modality. The second approach is a flow-chamber setup based on Ibidi μ-slides. Both live-cell imaging strategies can be followed up with serial blockface- or focused ion beam-scanning electron microscopy...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528629/correlative-light-and-electron-microscopic-detection-of-gfp-labeled-proteins-using-modular-apex
#15
Nicholas Ariotti, Thomas E Hall, Robert G Parton
The use of green fluorescent protein (GFP) and related proteins has revolutionized light microscopy. Here we describe a rapid and simple method to localize GFP-tagged proteins in cells and in tissues by electron microscopy (EM) using a modular approach involving a small GFP-binding peptide (GBP) fused to the ascorbate peroxidase-derived APEX2 tag. We provide a method for visualizing GFP-tagged proteins by light and EM in cultured cells and in the zebrafish using modular APEX-GBP. Furthermore, we describe in detail the benefits of this technique over many of the currently available correlative light and electron microscopy approaches and demonstrate APEX-GBP is readily applicable to modern three-dimensional techniques...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28528628/millisecond-time-resolution-correlative-light-and-electron-microscopy-for-dynamic-cellular-processes
#16
Ludek Stepanek, Gaia Pigino
Molecular motors propel cellular components at velocities up to microns per second with nanometer precision. Imaging techniques combining high temporal and spatial resolution are therefore indispensable to understand the cellular mechanics at the molecular level. For example, intraflagellar transport (IFT) trains constantly shuttle ciliary components between the base and tip of the eukaryotic cilium. 3-D electron microscopy has revealed IFT train morphology and position, but was unable to correlate these features with the direction of train movement...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28215341/dissection-and-characterization-of-microtubule-bundles-in-the-mitotic-spindle-using-femtosecond-laser-ablation
#17
R Buđa, K Vukušić, I M Tolić
The mitotic spindle is a highly organized and dynamic structure required for segregation of the genetic material into two daughter cells. Although most of the individual players involved in building the spindle have been characterized in vitro, a general understanding of how all of the spindle players act together in vivo is still missing. Hence, in recent years, experiments have focused on introducing mechanical perturbations of the spindle on a micron scale, thereby providing insight into its function and organization, as well as into forces acting in the spindle...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28215340/quantitative-approaches-for-the-study-of-microtubule-aster-motion-in-large-eggs
#18
H Tanimoto, N Minc
The proper positioning of microtubule (MT) asters underlies fundamental processes such as nuclear centration, cell polarity, division positioning, and embryogenesis. In large eggs and early blastomeres, MT asters may exhibit long range motions with atypical speed and precision to target their functional position. The biophysical mechanisms regulating such motions remain however largely unknown. The centration of sperm asters in sea urchin embryos is a stereotypical example of such aster long range motion. In this chapter, we describe methods developed in this system to (1) quantify sperm aster 3-D motion with confocal microscopy and automated image analysis and (2) severe a portion of astral MTs with a UV laser...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28215339/precise-tracking-of-the-dynamics-of-multiple-proteins-in-endocytic-events
#19
A Picco, M Kaksonen
Endocytosis is a complex and dynamic process that involves dozens of different proteins to define the site of endocytosis, form a membrane invagination, and pinch off a membrane vesicle into the cytoplasm. Fluorescent light microscopy is a powerful tool to visualize the dynamic behaviors of the proteins taking part in the endocytic process. The resolution of light microscopy is, however, a serious limitation. Here, we detail a fluorescence microscope method that we have developed to visualize the dynamics of the clathrin-mediated endocytic protein machinery in yeast cells...
2017: Methods in Cell Biology
https://www.readbyqxmd.com/read/28215338/investigating-symmetry-breaking-in-yeast-from-seeing-to-understanding
#20
P Singh, A Das, R Li
Diverse cellular functions require the establishment of cell polarity. Although the morphological manifestation of cell polarity can be complex, common principles and conserved regulatory pathways can be studied leveraging the powerful genetic tools in the budding yeast Saccharomyces cerevisiae. A vast array of genetic and cell biological tools and interdisciplinary approaches have been employed to understand the establishment and maintenance of cell polarity in budding yeast. These tools enabled the identification of key molecular components, their mechanisms of action, and the pathway governing the spatiotemporal properties of cell polarity...
2017: Methods in Cell Biology
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