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  • 3-Dimensional Imaging of Macroscopic Defects in Aluminum Alloys

    The investigation of macroscale defects in aluminum (Al) alloys with a rapid 3-dimensional (3D) imaging approach is described in this report. Aluminum (Al) alloys play an important role in the production of aircraft and vehicles, as well as products in other industries. Defects present in the Al alloy used for the production of aircraft, vehicles, or other products can have a significant effect on their quality, performance, and lifetime.
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  • Visualization of DNA Molecules

    Precise low angle rotary shadowing with heavy metals (like platinum) can be used in transmission electron microscopy (TEM) to observe molecular details of objects previously absorbed on a thin, low grain and electron-transparent carbon film. To achieve the highest contrast and better image quality, it is essential that the coating is directional, and it is given at a precise angle toward the sample. The fine grain of the metal layers and the homogeneous thickness of the coating material all over the sample surface are also crucial requirements to achieve high quality TEM images. This requires the method of e-beam evaporation a stream of evaporated material which is very directional, extremely homogeneous, cool and fine grained.
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  • Variety of RNAs in Peripheral Blood Cells, Plasma, and Plasma Fractions

    Human peripheral blood contains RNA in cells and in extracellular membrane vesicles, microvesicles and exosomes, as well as in cell-free ribonucleoproteins. Circulating mRNAs and noncoding RNAs, being internalized, possess the ability to modulate vital processes in recipient cells. In this study, with SOLiD sequencing technology, we performed identification, classification, and quantification of RNAs from blood fractions: cells, plasma, plasma vesicles pelleted at 16,000
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  • Picea abies (L.) KARST - Sample Preparation for TEM

    Application Note for Leica EM AMW - Plants (5-years old) were grown in pots filled with soil and kept in greenhouse conditions. Five weeks before harvesting the plants were transferred into growth chambers and cultivated at a temperature of 20°C during daytime and 12°C overnight. The relative humidity was set at 60% and the photoactive radiation was 500 μmol m-2 s-1 during daytime. Sample preparation for transmission electron microscopy (TEM) was performed in order to develop a standard protocol that would reduce sample preparation time for TEM-investigations. Therefore the overall and fine structure of leaf cells prepared with the Leica EM AMW were compared with leaf cells that were prepared with a conventional fixation protocol at room temperature.
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  • Maple (Acer saccharum) Leaves - High Pressure Freezing and Freeze Substitution for TEM

    Application Note for Leica EM HPM100 - Leaves were immersed in hexadecene and placed under a gentle (0.3 bar) vacuum for 10 minutes to evacuate the internal air spaces. The leaves were then trimmed to fit the carriers and placed in the 200 μm side of a 6 mm Type A specimen carrier. Free space was filled with additional hexadecene after which a 6 mm Type B specimen carrier was placed on top with the flat side down.
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  • Removal of Surface Layers - Sample Preparation for SEM and TEM

    Application Note for Leica EM RES102 - Sometimes it is necessary to remove surface layers to gain access to the real surface structure. That can be a native oxide, or layers coming from the preparation process itself, like re-deposition. Depending on the layers thickness and the energy used for the cleaning process, it takes between a few seconds and half an hour. The energy depends on the milling rate of the material.
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  • Multilayer Systems with Widely Different Sputter Rates - Sample Preparation for TEM

    Application Note for Leica EM RES102 - The multi-layer system to be prepared in cross-section consists of a Si substrate, a TiN layer with a thickness of a few nm and a 500 nm W layer. All these components have extreme differences in their hardness, their atomic weight and in their sputter rates. A preparation of this kind of samples with sample rotation would lead to a wall overlying the area of the layers.
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  • Correlative Cryo-Fluorescence and Cryo-Scanning Electron Microscopy as a Straightforward Tool to Study Host-Pathogen Interactions

    Correlative light and electron microscopy is an imaging technique that enables identification and targeting of fluorescently tagged structures with subsequent imaging at near-to-nanometer resolution. We established a novel correlative cryo-fluorescence microscopy and cryo-scanning electron microscopy workflow, which enables imaging of the studied object of interest very close to its natural state, devoid of artifacts caused for instance by slow chemical fixation. This system was tested by investigating the interaction of the zoonotic bacterium Borrelia burgdorferi with two mammalian cell lines of neural origin in order to broaden our knowledge about the cell-association mechanisms that precedes the entry of the bacteria into the cell.
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  • In-Containing Compound Semiconductors - Sample Preparation for TEM

    Application Note for Leica EM RES102 - Previous studies showed that surface accumulation of In occurs when InP was milled in a conventional way with Ar ions. The consequence is In islands on the sample surface. This leads to low quality of TEM samples. To remove these islands, reactive ion milling with iodine ions (RIBE / CAIBE) can be used. This method has the disadvantage of polluting the ion guns and the vacuum system of the ion milling device and leads to chemical reactions with the sample material. To avoid these problems we prepared these samples very gently with low energy Ar ions.
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  • High-Resolution Carbon Coating: How much Carbon is too much?

    Application Note for Leica EM ACE600 - Carbon support films are routinely used for high resolution TEM. Thickness is one of the main criteria to assess its usefulness for a particular experiment. Within that respect graphene (oxide) layers are frequently used. However, charge dissipation and mechanical stability towards high probe currents and high voltages, including long term acquisition protocols are equally important.
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  • Each Atom Counts: Protect Your Samples Prior to FIB Processing

    Application Note for Leica EM ACE600 - Focused ion beam (FIB) technology has become an indispensable tool for site-specific TEM sample preparation. It allows to extract electron transparent specimens with nanometer precision using a focused Ga+ ion beam.
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  • "Shallow Trench Isolation" Structures - Sample Preparation for TEM

    Application Note for Leica EM RES102 - The cross-sectional preparation of structured semiconductor materials requires a very thorough mechanical pre-preparation. In doing this, it must be ensured that the structure of interest should be located as close to the centre of the sample as possible. As the sample will be ion milled from both sides, a specific preparation of the structure is necessary in most cases, which means that you must thin these structures from both sides.
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  • Metal Films and Sheets - Sample Preparation for TEM

    Application Note for Leica EM RES102 - Most metal films already have a thickness that requires no further mechanical pre-preparation. Frequently, however, they are also domed, which can lead to undefined milling angles. This is a disadvantage, particularly for films that contain inclusions, and that therefore mostly require very flat milling angles. Metal sheets are thicker than 100 µm. Mechanical pre-preparation is necessary to obtain an acceptable initial thickness and a good surface quality for ion milling.
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  • Cross-Sectional Preparation of Structured Semiconductor Materials for TEM

    Application Note for Leica EM RES102 - The vertical layer construction of a semiconductor structure should be examined as a TEM cross-sectional sample. In addition to the specific preparation of the desired structure, the widely different sputter rates and atomic weights of the individual components represent the level of difficulty involved with this preparation problem.
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  • Ultra-thin Carbon Support Films for Improved STEM-EELS Analysis of Nanoparticles

    Application Note for Leica EM ACE600 - Recent developments in aberration corrected transmission electron microscopes as well as further improvements in monochromaters and spectrometers have pushed the attainable energy resolution for Electron energy loss spectroscopy (EELS) to 100 meV and beyond. STEM-EELS of individual nanomaterials can be challenging due the necessity of a support film.
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  • Ways to Reveal More from your Samples: Ultra-Thin Carbon Films

    Application Note for Leica EM ACE600 - Much of the battle involved in obtaining good transmission electron microscopy data is in the specimen preparation itself. Even though some nanomaterials are already electron transparent (e.g. nanoparticles and proteins) and often do not require further thinning procedures, they need to be dispersed onto thin support films.
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  • Ion Beam Polishing of Sample Surfaces - Sample Preparation for SEM

    Application Note for Leica EM RES102 - Ion milling can be used to reduce the roughness of sample surfaces. Small angles less than 6° with respect to the sample surface are necessary. The high voltage depends on the material to be prepared. The reason for the levelling effect is the different milling angle of flat and rough surface areas. The milling rate is lower for small angles. The rough surface area will be faster milled. Ion polishing is often the final step of sample preparation. The prerequisite is a perfect mechanical prepreparation as samples with deep surface scratches cannot be ion polished. Soft materials usually have a smeared sample surface after mechanical polishing. It is necessary to remove this smeared material before ion polishing. Otherwise the above mentioned polishing effect does not work.
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  • Ceramics - Sample Preparation for TEM

    Application Note for Leica EM RES102 - Ceramic samples are mostly very brittle, and are therefore very difficult to thin mechanically to a low starting thickness for ion beam milling. The ion beam milling of insulators often leads to static charging of the surface of the sample. This, in turn, reduces the sputter rate. When using the Ti standard holder (standard TEM holder), however, sufficient secondary electrons are created by the ion beam also falling on the sample holder to largely compensate for the static charging.
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  • Arabidopsis thaliana(L.) Accession Col

    Application Note for Leica EM AMW - Plants were grown in growth chambers under defined conditions. After stratification for 4 days at 4°C seeds were grown in pots with soil with 9/15 hours day/night photoperiod. Day and night temperatures were 22°C and 18°C, respectively, the relative humidity was 60% and the plants were kept at 100% relative soil water content. Light intensity varied between 110 and 140 μmol m-2 s-1.
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  • Imaging of Host Cell-bacteria Interactions using Correlative Microscopy under Cryo-conditions

    Pathogenic bacteria have developed intriguing strategies to establish and promote infections in their respective hosts. Most bacterial pathogens initiate infectious diseases by adhering to host cells surface. Knowledge of the interplay between the pathogenic organism and the host cells can provide fundamental new insight into the underlying mechanisms of the infectious process and therefore of the disease. Various microscopy techniques have proven to be critical tools to study these events.
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  • TEM Sample Preparation Made Easy - Prepare TEM Specimen by Broad Beam Argon Ion Milling

    Quantitative and analytical analysis at high spatial resolution places stringent demands on the quality of the produced TEM specimens. Pristine and high-quality samples are indispensible for atomic resolution TEM analysis. In this application note a general procedure for obtaining cross-sectional and plan-view TEM specimens using the Leica EM RES102 ion milling system is outlined. The procedure described below can be easily adapted for a large range of materials e.g. thin film materials, semiconductors, multilayered materials, ceramics, superconductors, …
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  • Transmission Electron Microscopy Sample Preparation Protocols for the Ultrastructural Study of Cysts of Free-living Protozoa

    Cysts of free-living protozoa have an impact on the ecology and epidemiology of bacteria because they may act as a transmission vector or shelter the bacteria against hash environmental conditions. Detection and localization of intracystic bacteria and examination of the en- and excystment dynamics is a major challenge because no detailed protocols for ultrastructural analysis of cysts are currently available.
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  • Correlative In-Resin Super-Resolution and Electron Microscopy Using Standard Fluorescent Proteins

    We introduce a method for correlative in-resin super-resolution fluorescence and electron microscopy (EM) of biological structures in mammalian culture cells. Cryo-fixed resin embedded samples offer superior structural preservation, performing in-resin super-resolution, however, remains a challenge.
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  • Detailed Morphological Characterisation of Hendra Virus Infection of Different Cell Types Using Super-Resolution and Conventional Imaging

    Hendra virus (HeV) is a pleomorphic virus belonging to the Paramyxovirus family. Our long-term aim is to understand the process of assembly of HeV virions. As a first step, we sought to determine the most appropriate cell culture system with which to study this process, and then to use this model to define the morphology of the virus and identify the site of assembly by imaging key virus encoded proteins in infected cells.
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  • Correlating Intravital Multi-Photon Microscopy to 3D Electron Microscopy of Invading Tumor Cells Using Anatomical Reference Points

    Cancer research unsing multiphoton microscopy and 3D electron microscopy. Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task.
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  • Brief Introduction to Freeze Fracture and Etching

    Freeze fracture describes the technique of breaking a frozen specimen to reveal internal structures. Freeze etching is the sublimation of surface ice under vacuum to reveal details of the fractured face that were originally hidden. A metal/carbon mix enables the sample to be imaged in a SEM (block-face) or TEM (replica). It is used to investigate for instance cell organelles, membranes, layers and emulsions.
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  • Brief Introduction to Specimen Trimming

    Before ultrathin sectioning a sample with an ultramicrotome it has to be pre-prepared. For this pre-preparation, special attention must be paid to the sample size (size of the section), location of the sample (targeting) and accuracy of the block-face edges. This process is generally called trimming, wherein the sample is shaped mainly to a frustum of a pyramid.
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  • Brief Introduction to Ultramicrotomy

    The usual thicknesses for transmission electron microscopic examinations range between 20 nm and 150 nm. Ultramicrotomy is a fast and clean method of producing ultra-thin sections of biological samples as well as polymers, rubber, ductile and even hard and brittle materials. A key advantage of ultramicrotomy is the size and homogeneity of the electron-transparent area of specimens prepared with this technique.
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  • Glycerol Spraying/Platinum Low Angle Rotary Shadowing of DNA with the Leica EM ACE600 e-beam

    Glycerol spraying/low angle rotary shadowing (Aebi and Baschong, 2006) is a preparation technique used in biology to visualize structures yielding insufficent contrast with other techniques, due to their small diameter. This method is commonly used for specimens which include proteins with coiled coil domains or DNA.
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  • Rapid Immunohistochemical Diagnosis of Tobacco Mosaic Virus Disease by Microwave-assisted Plant Sample Preparation

    Immunoelectron microscopy is a powerful method to diagnose viral diseases and to study the distribution of the viral agent within plant cells and tissues. Nevertheless, current protocols for the immunological detection of viral diseases with transmission electron microscopy (TEM) in plants take between 3 and 6 days and are therefore not suited for rapid diagnosis of virus diseases in plants. In this study, we describe a method that allows rapid cytohistochemical detection of tobacco mosaic virus (TMV) in leaves of tobacco plants.
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