Month: December 2016

Super-Resolution Optical Microscopy of Lipid Plasma Membrane Dynamics

Plasma membrane dynamics are an important ruler of cellular activity, particularly through the interaction and diffusion dynamics of membrane-embedded proteins and lipids. FCS (fluorescence correlation spectroscopy) on an optical (confocal) microscope is a popular tool for investigating such dynamics. Unfortunately, its full applicability is constrained by the limited spatial resolution of a conventional optical microscope. …

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Cross Sectioning of a Multilayer System – Preparation of a Perfect Sample Surface for EBSD

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 …

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High-Pressure Freezing and Freeze Substitution of Hep-2 Cells Infected with Chlamydia pneumoniae

After freezing, the Sapphire disc was removed from the middle plate in anhydrous acetone at -90°C and immediately transferred into a 2 ml Eppendorf tube containing 2% OsO4 in anhydrous acetone, precooled to -90 °C in an Leica EM AFS2 freeze-substitution unit. Samples were substituted for 8 h at -90°C, 8 h at -60°C, 8 …

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Multilayer Systems with Widely Different Sputter Rates – Sample Preparation for TEM

In case of the Leica EM RES102, an oscillating movement can be used in addition to the sample rotation. In doing this, both the zero point and the angle of the oscillation can be adjusted. The zero point should be set up so that the incidence of the ion beam is vertical with respect to …

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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 …

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In-Containing Compound Semiconductors – Sample Preparation for TEM

Preparation ConditionsMechanical pre-preparation:Preparation of InGaAsP / InP MQW (Multiple Quantum Well)- structures and InP-samples. The samples were double-sided dimpled. A milling angle lower than 7° was not possible. Ion milling: Sample holder: Quick-clamp-holder Acceleration voltage: 4 kV and 2 kV (final step) Milling angle: 7° (double-sided milling) Sample movement: Rotation and Oscillation (alternating) ResultsThe use …

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Methods to Calibrate and Scale Axial Distances in Confocal Microscopy as a Function of Refractive Index

Accurate distance measurement in 3D confocal microscopy is important for quantitative analysis, volume visualization and image restoration. However, axial distances can be distorted by both the point spread function (PSF) and by a refractive-index mismatch between the sample and immersion liquid, which are difficult to separate. Additionally, accurate calibration of the axial distances in confocal …

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Factors to Consider When Selecting a Research Microscope

An optical microscope is often one of the central devices in a life-science research lab. It can be used for various applications which shed light on many scientific questions. Thereby the configuration and features of the microscope are crucial for its application coverage, ranging from brightfield through fluorescence microscopy to live-cell imaging. This article provides …

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