Linked-fluorophore FRET calibration and FRET studies of the cyclin-CDK switch in mammalian cells

Domin, Alexander; Hooper, Rowan J.; Rauch, Uwe; Lindon, A. Catherine; Kaminski, Clemens F.

doi:10.1117/12.500733

Cited by 2 publications

(1 citation statement)

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“…1). Previous attempts at obtaining graded probes were only partially successful since were based on short linker peptides from 6 to 22 amino acid residues in length and no defined structure (Domin et al, 2003). In that paper there was a small but significant inverse correlation between linker length and FRET efficiency, but the difference between the highest and lowest FRET rate was only about 13%.…”

Section: Discussionmentioning

confidence: 97%

Imaging FRET standards by steady‐state fluorescence and lifetime methods

Domingo

Sabariegos

Picazo

et al. 2007

Microscopy Res & Technique

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Imaging fluorescence resonance energy transfer (FRET) between molecules labeled with fluorescent proteins is emerging as a powerful tool to study changes in ions, ligands, and molecular interactions in their physiological cellular environment. Different methods use either steady-state fluorescence properties or lifetime to quantify the FRET rate. In addition, some provide the absolute FRET efficiency whereas others are simply a relative index very much influenced by the actual settings and instrumentation used, which makes the interpretation of a given FRET rate very difficult. The use and exchange of FRET standards in laboratories using these techniques would help to overcome this drawback. We report here the construction and systematic evaluation of FRET standard probes of varying FRET efficiencies. The standards for intramolecular FRET were protein fusions of the cyan and yellow variants of A. victoria green fluorescent protein (ECFP and citrine) joined by short linkers or larger protein spacers, or ECFP tagged with a tetracysteine motif and labeled with the biarsenical fluorochrome, FlAsH. Negative and positive controls of intermolecular FRET were also used. We compared these FRET standards with up to four FRET quantification methods: ratioing of acceptor to donor emission, donor intensity recovery upon acceptor photobleach, sensitized emission after spectral unmixing of raw images, and fluorescence lifetime imaging (FLIM). The latter was obtained with a frequency-domain setup able to provide high quality lifetime images in less than a second, and is thus very well suited for live cell studies. The FRET rates or indexes of the standards were in good agreement regardless of the method used. For the CFP-tetraCys/FlAsH pair, the rate calculated from CFP quenching was faster than that obtained by FLIM.

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Section: Discussionmentioning

confidence: 97%

Imaging FRET standards by steady‐state fluorescence and lifetime methods

Domingo

Sabariegos

Picazo

et al. 2007

Microscopy Res & Technique

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Quantitative Fluorescence Microscopy Techniques

Esposito

Schlachter

Schierle

et al. 2009

Cytoskeleton Methods and Protocols

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Fluorescence microscopy is a non-invasive technique that allows high resolution imaging of cytoskeletal structures. Advances in the field of fluorescent labelling (e.g., fluorescent proteins, quantum dots, tetracystein domains) and optics (e.g., super-resolution techniques and quantitative methods) not only provide better images of the cytoskeleton, but also offer an opportunity to quantify the complex of molecular events that populate this highly organised, yet dynamic, structure.For instance, fluorescence lifetime imaging microscopy and Förster resonance energy transfer imaging allow mapping of protein-protein interactions; furthermore, techniques based on the measurement of photobleaching kinetics (e.g., fluorescence recovery after photobleaching, fluorescence loss in photobleaching, and fluorescence localisation after photobleaching) permit the characterisation of axonal transport and, more generally, diffusion of relevant biomolecules.Quantitative fluorescence microscopy techniques offer powerful tools for understanding the physiological and pathological roles of molecular machineries in the living cell.

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Linked-fluorophore FRET calibration and FRET studies of the cyclin-CDK switch in mammalian cells

Cited by 2 publications

References 0 publications

Imaging FRET standards by steady‐state fluorescence and lifetime methods

Imaging FRET standards by steady‐state fluorescence and lifetime methods

Quantitative Fluorescence Microscopy Techniques

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