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  • Symmetric Synapse - Clathrin Coated Endocytosis Pit in the Postsynaptic Dendrite

    Application Note for Leica EM ICE - WT hippocampal neurons were plated at a density of 80,000 cells/cm2 on 6 mm sapphire disks for 14 days. Sample were frozen using a high-pressure freezer (Leica EM ICE) under a pressure of 2100bar by mounting it into a sandwich support with extracellular solution containing 15% of Ficoll 400, to assess ice crystal damage. The Cryo-fixation was achieved within milliseconds allowing simultaneous immobilization of all macromolecular components. After freezing, sam­ple was transferred into cryovials containing 1% glutaraldehyde, 1% osmium tetroxide, 1% milliQwater in anhydrous acetone and processed in an automated freeze-substitution device (Leica EM AFS2).
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  • Cryo CLEM – the Combination of Cryo Fluorescence Microscopy with Cryo Electron Microscopy

    Many biological insights can be obtained by combining the power of Fluorescence Microscopy (FM) with that of Electron Microscopy (EM) to study the same sample – this is called Correlative Light and Electron Microscopy (CLEM). In FM, specific proteins can be labelled and identified, and their dynamics and interactions can be visualized in fixed or living cells.
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  • Video Tutorials: Filling and Assembling of Different Carriers for High-Pressure Freezing

    High pressure freezing (HPF) is a cryo-fixation method primarily for biological samples, but also for a variety of non-biological materials. It is a technique that yields optimal preservation in many cell types and tissues or in organic and inorganic composites. Most commonly, the high pressure frozen samples are analyzed further with light or electron microscopy after appropriate processing.
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  • Capturing Cellular Dynamics with Millisecond Temporal Resolution

    The combination of two powerful techniques: optogenetics and high-pressure freezing now makes it possible to visualize a dynamic cellular activity with temporal resolution of 5 milliseconds. By coupling a flash of light with high-pressure freezing, the process of vesicle recycling at the synapses can now be imaged by electron microscopy.
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  • Freeze Substitution of Trypanosoma brucei

    Chemical fixation of biological specimens for ultrastructural investigation is a relatively slow and selective process, and therefore a common source of artifacts. Freezing, on the other hand, is an excellent method to physically fix biological specimens in their entirety and without delay; the formation of ice crystals large enough to displace cellular material and destroy structures, would be, however, a major issue.
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  • Brief Introduction to Freeze Substitution

    Freeze-substitution is a process of dehydration, performed at temperatures low enough to avoid the formation of ice crystals and to circumvent the damaging effects observed after ambient-temperature dehydration. During freeze substitution the “frozen” water is dissolved by an organic solvent, which usually also contains chemical fixatives.
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  • From Dynamic Live Cell Imaging to 3D Ultrastructure: Novel Integrated Methods for High Pressure Freezing and Correlative Light-Electron Microscopy

    To correlate dynamic events in adherent cells with both ultrastructural and 3D information, we developed a method for cultured cells that combines confocal time-lapse images of GFP-tagged proteins with electron microscopy. With laser micro-patterned culture substrate, we created coordinates that were conserved at every step of the sample preparation and visualization processes. Specifically designed for cryo-fixation, this method allowed a fast freezing of dynamic events within seconds and their ultrastructural characterization.
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  • Brief Introduction to High-Pressure Freezing

    Water is the most abundant cellular constituent and therefore important for preserving cellular ultra-structure. Currently the only way to fix cellular constituents without introducing significant structural alterations is by cryo-fixation. There are currently two common methods employed; plunge freezing and high pressure freezing.
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  • Advanced Correlative Light/Electron Microscopy: Current Methods and New Developments Using Tokuyasu Cryosections

    Microscopy is an essential tool for analysis of cellular structures and function. With the advent of new fluorescent probes and super-resolution light microscopy techniques, the study of dynamic processes in living cells has been greatly facilitated. Fluorescence light microscopy provides analytical, quantitative, and three-dimensional (3D) data with emphasis on analysis of live cells using fluorescent markers.
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  • CLEM: Combining the Strengths of Light and Electron Microscopy

    In recent years light microscopy studies have been dominated by live cell imaging while electron microscopy has been used for high-resolution studies. Latterly, there has been increasing interest in combining these techniques. This combination is called Correlative Light Electron Microscopy (CLEM). Due to the high resolution made possible by electron microscopy, artefacts induced during preparation of a sample can, however, also be clearly seen.
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