Month: November 2011

Subcellular Localization of AKT and Tubulin using Super-Resolution Microscopy

Conditions for STED nanoscopy: A431 cells (ATCC CRL-1555) were incubated at 37 °C with 5 % CO2 in DMEM (Invitrogen, MO) with 10 % FBS (Rockland, PA) and Pen Strep (Invitrogen, USA), in 75 cm2 Corning CellBind cell culture flasks (Sigma, MO) unless otherwise noted. At 70 to 80 % confluence cells were detached using TrypLE (Invitrogen, MO), and then …

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Fluorescence Recovery after Photobleaching (FRAP) and its Offspring

Typically, the protein of interest is made fluorescent by expression as a GFP (green fluorescent protein cloned from the jellyfish A. victoria) fusion protein or by tagging the protein of interest with reactive ligands which then binds a fluorescent dye. The most common way to fluorescently tag  proteins in living cells is GFP technology. Of …

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Two-color STED Microscopy of Living Synapses using a Single Laser-beam Pair

The advent of superresolution microscopy has opened up new research opportunities into dynamic processes at the nanoscale inside living biological specimens. This is particularly true for synapses, which are very small, highly dynamic, and embedded in brain tissue. Stimulated emission depletion (STED) microscopy, a recently developed laser-scanning technique, has been shown to be well suited …

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Quantitative Imaging in Cell Biology: Light Sheet Microscopy

This chapter introduces the concept of light sheet microscopy along with practical advice on how to design and build such an instrument. Selective plane illumination microscopy is presented as an alternative to confocal microscopy due to several superior features such as high-speed full-frame acquisition, minimal phototoxicity, and multiview sample rotation. Based on our experience over …

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Modern Fluorescent Proteins and their Biological Applications

Modern fluorescent proteins: from chromophore formation to novel intracellular applications The diverse biochemical and photophysical properties of fluorescent proteins (FPs) have enabled the generation of a growing palette of colors, providing unique opportunities for their use in a variety of modern biology applications. Modulation of these FP characteristics is achieved through diversity in both the …

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Applications, Labeling Strategies and Fluorophores for Super-Resolution

Great insights can be obtained from conventional fluorescence microscopy, but studying the architecture and protein dynamics of sub-cellular compartments can be challenging, since a major portion of the information concerning the structural organization is lost due to the light diffraction limit. Several approaches to overcome this limitation have been developed and super-resolution has proven an …

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Technical Cleanliness in the Production of Automotive Components

Dr. Härtel, why is residual dirt analysis so important? We thoroughly check everything from parts to assembly groups for particle residue, as we now know that cleanliness is a critical factor for reliability and service life. The components are washed and cleaned several times to remove clinging particles. To do so, tools such as lances …

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Neurobiology and Microscopy

Widefield microscopic images, also when using high-resolution immersion techniques, contain information both from the focal plane and from all layers above and below that plane. If the sample is very thin, these contributions are negligible. In thick samples, especially with dense fluorescent staining of complex structures, the relevant information may be covered by out-of-focus haze. …

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Förster Resonance Energy Transfer (FRET)

The Förster Resonance Energy Transfer (FRET) phenomenon offers techniques that allow studies of interactions in dimensions below the optical resolution limit. FRET describes the transfer of the energy from an excited state of a donor molecule to an acceptor molecule. Unlike absorption or emission of photons, FRET is a non-radiative energy exchange and consequently not …

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