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  • FRAP with TCS SP8 Resonant Scanner

    Fast FRAP experiments need a sufficient number of measurement points for meaningful interpretation and fitting analysis. To study very fast translocational processes, the use of a resonant scanner (RS) is preferred. The advantage in using FRAP with the RS is that statistics are much better in experiments that require fast acquisition: If the half time of recovery is about 0.5 sec you may have only about 3 to 4 data points using the conventional scanner, whereas with the resonant scanner you can get about 20 data points.
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  • How to Measure FRET

    Here, I will expand, including what to measure when doing FRET. There are a number of approaches to FRET quantification: 1. Sensitized Emission – This two-channel imaging technique uses an algorithm that corrects for excitation and emission crosstalk.
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  • You May Not Know Theodor Förster but You Know His Work: FRET

    If you think FRET stands for Fluorescence Resonance Energy Transfer, you are wrong … in good company but wrong. FRET actually stands for Förster Resonance Energy Transfer. Find out why and more about FRET in this article.
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  • Step by Step Guide for FRAP Experiments

    Fluorescence recovery after photobleaching (FRAP) has been considered the most widely applied method for observing translational diffusion processes of macromolecules. State of the art laser scanning microscopes such as the Leica TCS SP8 have the advantage of using a high intensity laser for bleaching and a low intensity laser for image recording. The LAS AF application wizard offers different ways to carry out a FRAP experiment.
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  • Label-free FLIM

    Many biological samples exhibit autofluorescence. Its often broad spectra can interfere with fluorescent labeling strategies. This application letter demonstrates how autofluorescence can serve as an intrinsic contrast in fluorescence lifetime imaging microscopy (FLIM) resulting in multi-color image stacks.
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  • FRET with FLIM

    FLIM combines lifetime measurements with imaging: lifetimes obtained for each image pixel are color-coded to produce additional image contrast. Thus, FLIM delivers information about the spatial distribution of a fluorescent molecule together with information about its biochemical status or nano-environment. A typical application of FLIM is FLIM-FRET. FRET is a well-established technique to study molecular interactions. It scrutinizes protein binding and estimates intermolecular distances on an Angström scale as well.
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  • Fluorescence Correlation Spectroscopy

    Fluorescence correlation spectroscopy ( FCS ) measures fluctuations of fluorescence intensity in a sub-femtolitre volume to detect such parameters as the diffusion time, number of molecules or dark states of fluorescently labeled molecules. The technique was independently developed by Watt Webb and Rudolf Rigler during the early 1970s.
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  • Webinar: Techniques and Methods in Live-Cell Imaging

    Imaging technologies are ubiquitous in today's life science laboratory. From basic microscopy to high throughput modalities, most cell based research benefits from some tried and true methods for imaging.
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  • Quantitative Fluorescence

    Seeing is believing – and measuring is knowing. Microscopes generate images that are not only used for illustration, but are also subject to quantification. More advanced techniques use illumination patterns (without image formation) or do not generate an image at all – but are still microscopical techniques. These F-techniques are becoming increasingly important in current biosciences.
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  • FLCS – Advances in Fluorescence Correlation Spectroscopy

    The characterization of substances at the single molecule level has become part of the standard repertoire of scientific research institutes. One of the most common methods is Fluorescence Correlation Spectroscopy ( FCS ), which can be used to examine the dynamics and concentration of fluorescent molecules in solution.
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  • Fluorescence Recovery after Photobleaching (FRAP) and its Offspring

    FRAP (Fluorescence recovery after photobleaching) can be used to study cellular protein dynamics: For visualization the protein of interest is fused to a fluorescent protein or a fluorescent dye. A region of interest (ROI) can be monitored applying a high amount of light to bleach the fluorescence within the ROI. The following illumination with low light conditions provides insight into the redistribution of molecules via recovery of fluorescence.
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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 a variation of light-matter interactions.
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  • FLIM-FRET in Solutions

    FRET efficiency can be measured based on fluorescence lifetime microscopy (FLIM). FLIM-FRET allows analysis of molecular interactions both in vitro and in vivo. This article describes the use of FLIM in the time domain (TCSPC) to measure FRET in vitro in a biochemical assay using a Cerulean-Citrine construct.
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