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What is EM Sample Preparation?

To achieve the best results during examination in the Electron Microscope (EM), the perfect EM Sample Preparation (for TEM, SEM, AFM) is a prerequisite. The required techniques depend on the samples (biological samples, material samples) as well as on the application. EM Sample Preparation incudes all methods of preparations from embedding, tissue processing, coating, immunogold labeling through ultrathin sectioning with ultramicrotomes, cryo-ultramicrotomy, cryosectioning, critical point drying, plunge freezing, freeze substitution, freeze fracturing, freeze drying, contrasting, cryofixation, high pressure freezing, cryo transfer, freeze etching, freeze fracture to ion beam milling, ion beam etching, and target preparation – mechanical grinding and polishing. Only if each step of sample preparation is of the highest quality, can optimum results be obtained from a high resolution electron microscope.

  • Free Webinar On-Demand: Practical Applications of Broad Ion Beam Milling

    Mechanical polishing can be time consuming and frustrating. It can also introduce unwanted artifacts when preparing cross-sectioned samples for electron backscatter diffraction (EBSD) in the scanning electron microscope (SEM) or light microscope investigation. In contrast, ion beam milling can eliminate undesirable artifacts that will hamper your analysis and interpretation.
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  • Free Webinar On-Demand: Revealing Cellular Dynamics with Millisecond Precision – The New Tool That Turned Electron Micrographs into Motion Pictures of Neural Communication

    What if you can dissect the cellular dynamics with millisecond precision? What if you can unravel the morphological transformation of a neuron millisecond by millisecond using electron microscopy?
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  • Interview with Dr. Shigeki Watanabe on Research in Synaptic Membrane Dynamics

    Dr. Shigeki Watanabe, principle investigator of the department of Cell Biology at the Johns Hopkins University School of Medicine in Baltimore, held a workshop in Zürich, Switzerland on methods to study synaptic dynamics with millisecond precision. In collaboration with Dr. Andres Käch from the University of Zurich all workshop attendees enjoyed presentations and hands-on sessions on the EM ICE by Leica Microsystems with Light and Electrical Stimulation, revealing the latest developments in brain research. During this workshop Dr. Bernd Sägmüller from Leica Microsystems had the chance for an interview with Dr. Watanabe.
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  • Micro-CT of Insect Larva Protocol

    Species: red blood worm (midge larva) Critical point drying of midge larvae with subsequent X-ray micro-computed tomography (micro-CT) to reconstruct the inner anatomy.
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  • Contrast Enhancement of Polycrystalline Metals - Sample Preparation for SEM

    Application Note - Ion milling is a perfect alternative for chemical etching, especially for polycrystalline metals, such as copper. Ion milling can be used to increase the contrast of the grain structure and their interfaces. In contrast to chemical etching the milling process is clean, safe and easy to operate. Ion energy and milling time depend on the milling rate of the metal.
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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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  • Drosophila larvae - Sample Preparation for Cryo-SEM

    Application Note for Leica EM ACE900 - Drosophila larvae were sandwiched between two 3 mm aluminum slit carriers with the 100 μm cavities facing each other and high-pressure frozen with a Leica EM HPM100. No ethanol as synchronization media was used, 1-hexadecene was used as filler. The wholes of the slit carriers were filled with filter tips dipped in 1-hexadecene to keep the carrier sandwich complete after freezing.
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  • Giardia lamblia - Sample Preparation for Cryo-SEM

    Application Note for Leica EM ACE900 - A 100 mesh copper grid (12 um thickness) was dipped into a concentrated Giardia suspension and sandwiched between two flat 3 mm aluminum specimen carriers with scratched surfaces. Subsequently, the sandwich was transferred to the widened hole of a middle plate (3.1 mm diameter). A 50 um spacer ring was added on top and the specimen immediately frozen with an HPM100 high-pressure freezing machine without using alcohol as synchronization fluid.
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  • Cross Sectioning of Cadmiumsulphide (CdS) for Cathodoluminescence

    Cathodoluminescence can be used to achieve spectra and high resolution images of impurity and structural defects in semicondoctors, minerals and insulating materials. This application note explains how to prepare a perfect sample surface for carhodoluminescence and how to use ion beam slope cutting to prepare the sample surface free of any preparation artefacts.
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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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  • Cross Sectioning of Copper for Electron Backscattered Diffraction (EBSD)

    Application Note for Leica EM TIC 3X - Electron Backscattered Diffraction (EBSD) is a surface technique creating diffraction patterns (Kikuchi-bands). It can be used for crystal orientation mapping, defect studies, phase identification, grain boundary studies and morphological studies. The information depth is just a few nm, therefore good sample preparation is very important to avoid damages.
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  • Porous Ceramics - Sample Preparation for SEM

    Application Note for Leica EM RES102 - Ceramic membrane filters with pore sizes down to a few nanometres must be investigated in cross-section with regard to the structure of the pores. The smallest pores are of special interest. In most cases, conventional grinding methods cannot be used for such problems, as the pore structure would be distorted. This applies in particular to the pores in the nanometre range.
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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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  • Semiconductor Structures with Large Differences in Hardness - Sample Preparation for SEM

    Application Note for Leica EM RES102 - In most cases, multi-layer structures or material combinations with large differences in hardness cannot be processed with conventional polishing techniques, or can only be very poorly processed. Due to the large differences in hardness, blurring or edge-rounding occurs, which distorts the original structure. In the application example shown, we are dealing with a solder ball structure. The goal was to determine the solder structure. The use of conventional grinding and polishing techniques was not possible due to the large differences in hardness between the solder ball and other materials in the sample.
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  • Cross Section of Solar Cells

    Application Note for Leica EM TIC020, Leica EM TIC 3X - Cross section of a complete solar cell.
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  • Cross Sectioning of a Multilayer System - Preparation of a Perfect Sample Surface for EBSD

    Application Note for Leica EM TIC 3X - Electron Backscattered Diffraction (EBSD) is a surface technique creating a diffraction pattern (Kikuchi-bands). It can be used for crystal orientation mapping, defect studies, phase identification, grain boundary studies and morphology studies. The information depth is just a few nm. Therefore good sample preparation is very important to avoid any damage. This is very difficult in case of multilayer system with big differences in hardness.
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  • High-Pressure Freezing and Freeze Substitution of Hep-2 Cells Infected with Chlamydia pneumoniae

    Application Note for Leica EM HPM100 - Hep-2 cells infected with Chlamydia pneumoniae were cultured on carbon-coated 6 mm Sapphire discs. Cells were high-pressure frozen in an EM HPM100 using the 6 mm CLEM middle plate with following setup: Sapphire disc with cells, spacer 200 μm, bare Sapphire disc, 2 spacers 200 μm. Ethanol was used as a synchronization fluid to transfer pressure at room temperature prior to cooling.
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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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  • Carbon Coating for Polymeric Materials

    Application Note fo Leica EM ACE600 - A solid understanding of polymer property-structure relationships is critical to improve and shorten development routes to new products. A direct way to determine correlations between structure and mechanical properties is provided by electron microscopy. Electron microscopy techniques have an important advantage over other methods, as they can provide local information at high spatial resolution. However, a major problem with polymers is their inherent lack of contrast.
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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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  • Commercially Available Hand Creams - Sample Preparation for Cryo-SEM

    Application Note for Leica EM HPM100 - Hand creams having different water contents were applied into the 100 μm cavities of two 3 mm type A sample carriers which were then closed cream sides inwards. The sample assembly was high pressure frozen with a Leica EM HPM100 and moved to a cooled Leica EM VCT100 loading station.
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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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  • Tobacco Leaf - Critical Point Drying Protocol for SEM

    Application Note for Leica EM CPD300 - Critical point drying of tobacco leafs with subsequent platinum coating and SEM analysis.
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  • Paper Samples - Sample Preparation for SEM

    Application Note for Leica EM RES102 - A coated paper sample has been prepared with ion beam slope cutting in order to test the procedure with regard to its applicability. With the use of ion beam slope cutting a cross section of paper could be prepared. On the basis of this sample processing, it was possible to show the largely unaffected original structure of the thermally-sensitive paper in the scanning electron microscope.
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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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  • Nematode E. dianae - Critical Point Drying Protocol for SEM

    Application Note for Leica EM CPD300 - Critical point drying of nematode Eubostrichus dianae to detect the ectosymbiotic bacteria layer with subsequent gold coating ans SEM analysis.
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