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GFPs | GFPs

Key Words: confocal, Fluorescence, fluorophore, live cell imaging, FRAP, FLIP, FRET, AOM, CLEM, TIRF, photoactivatable-GFP

Definition:GFP is a naturally occurring fluorophore found in the jellyfish Aequorea Victoria, which fluoresces green when exposed to blue light

TECHNOLOGY:

The gene for GFP has been isolated and cloned. It is used in imaging to identify and monitor proteins in living cells. Using recombinant DNA technologies, the gene for GFP can be linked to a target gene expressing the protein of interest. The location and degree of protein expression can then be visualized in living cells using fluorescence microscopy.

A number of color variants of the GFP gene have been created including blue (BFP), cyan (CFP), and yellow (YFP) enabling multi probe studies. Further variants include GFPs with increased emission brightness (enhanced-GFPs or EGFPs), and photoactivatable-GFP. GFP has the advantage that it is stable and relatively non-toxic to cells (compared with some synthetic fluorophores) and in general does not appear to interfere with the function of co-expressed proteins.

APPLICATIONS:

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The availability of GFP and its derivatives has revolutionized fluorescence imaging in living cells. It is widely used to image proteins and their interactions using techniques such as TIRF, FRET, FLIP and FRAP. Used in combination with confocal and time lapse imaging dynamic events can be monitored over time with a high temporal and spatial resolution. It has been used to visualize chromosomes, proteins and their interactions, monitor membrane dynamics, mRNA trafficking, cytoskeletal dynamics, embryonic development, and more.

MICROSCOPE CONFIGURATION:

GFPs can be visualized on any upright or inverted microscope with epi-fluorescence capability or stereo microscope with stereo fluorescence illuminator (also known as GFP-illuminator). The full potential for imaging live cell dynamics using the GFPs is realized in confocal imaging.

RECOMMENDED SYSTEM:

For full flexibility in the imaging of GFPs in living cells, the Ti series microscopes configured with wide field fluorescence and/or TIRF illumination system, C1, C1Si spectral confocal or LiveScan Sweptfield confocal system offer a wide variety of imaging capabilities. CLEM can be used with the C1 to reduce photobleaching and provide greater flexibility in time-lapse studies. Nikon's Ti series microscopes include noise-terminator technology to improve signal-to-noise rations. A number of specialized fluorescence objectives are available (Plan Fluor, Super Fluor, Plan Apochromat, Plan Apochromat VC).

LINKS:

Fluorescence imaging: [microscopyu]

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