Acceptor Emission

Fig. 4 Fluorescence resonance energy transfer imaging of live cells. Pair of digital images representing collection of light from the donor emission (top left image) and acceptor emission (bottom left image) following illumination of a sample at the donor excitation wavelength. The intensity of pixels from the donor emission image is divided by the intensity of pixels from the acceptor emission image via software and a color-coded map of the ratios is generated (right). In this diagram there is a high FRET signal in the nucleus (low donor intensity/high acceptor intensity).

probes as well as the green, cyan, and yellow fluorescent proteins (GFP, CFP, and YFP), introduced in cells by DNA transfection, have permitted the study of dynamic events involving changes in the proximity of macro-molecules in live cells. Sensors based on GFP variants can be targeted to any compartment for which there is a known signal sequence, enabling local measurement of the activity under study. In FRET imaging, light required to excite the donor is focused by a microscope lens onto a live cell sample. Photons emitted from the donor and the acceptor are respectively collected as two digital images (Fig. 4). The intensity levels of the two images are then ratioed on a pixel-per-pixel basis, yielding a two-dimensional map of the D/A emission ratio for the particular time point. The sample can be imaged over time and thus the spatio-temporal features of the FRET signal can be analyzed. By enabling the analysis of phenomena in the range of Forster distances (1-10 nm), FRET imaging goes beyond the limits resolution of light microscopy (~200 nm).

The binding of two different proteins respectively labeled with CFP and YFP (the pair with the best spectral overlap characteristics for FRET) can be measured kinetically by measuring the amount of FRET they undergo. Protease activity can likewise be monitored in live cells by placing YFP and CFP at each extremity of a cleavable peptide. A powerful class of sensor inserts a conformationally active peptide between a CFP-YFP pair. The peptide can adopt different conformations depending on the environment, thus changing the distance and angle between the fluorescent proteins and yielding differential FRET signals. The detection of fluctuations in intracel-lular Ca2+ concentration was the first demonstrated application of this class of sensors, and the list has grown since (see Ref. [10] for a review).

For microscopy measurements, high levels of cellular autofluorescence can be compensated for by using an unlabeled region of the cell as an internal reference; this background is subtracted from the signal in the region of interest prior to calculating FRET. A more serious problem is photobleaching of the donor—basically, a destruction of the fluorophore caused by overillumination which is common in microscopy because the excitation light scans the same sample area repeatedly. Under photobleaching conditions, the efficiency of FRET undergoes an apparent decrease over time as the donor fluorophore is unable to repopulate its excited state. This is donor dependent, but can be corrected for by establishing the rate of photobleaching in a control sample, and normalizing the FRET signal obtained in the test sample to this baseline.[10] Bioluminescence RET measurements (discussed in ''In Vitro: Homogeneous FRET'') can also be performed in live cells; these experiments do not suffer from photobleaching because all of the light is generated via a bioluminescent reaction.[9]

Getting Started With Dumbbells

Getting Started With Dumbbells

The use of dumbbells gives you a much more comprehensive strengthening effect because the workout engages your stabilizer muscles, in addition to the muscle you may be pin-pointing. Without all of the belts and artificial stabilizers of a machine, you also engage your core muscles, which are your body's natural stabilizers.

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