Emission ratio imaging

Emission ratio imaging is extremely popular due to its simplicity and speed. In essence, cells expressing donors and acceptors are illuminated at the donor wavelength and fluorescence intensity data are collected both at donor (D) and at acceptor (S) channels. Collected data may be either images, or, in case high acquisition speed is crucial and spatial information is not required, dualchannel photometer readings (see Textbox 1). S and D are not overlap-corrected and FRET is simply expressed as the ratio of intensities1 as ratio = S/D. 

Ratio imaging nicely cancels out some of the main complications in the interpretation of wide-field images in that it normalizes fluorescence intensity differences caused by for example, cell height (Fig. 7.T1) as well as possible slow drift in excitation intensity. Light sources invariably are much less stable than detectors. Incidentally, for these reasons emission ratio imaging has been applied for over 3 decades by the Ca2+ imaging community. 

The first FRET-based biosensors employing fluorescent proteins were developed over 10 years ago. These protease sensors consisted of a BFP donor fused to a GFP acceptor by a protease-sensitive linker [44, 119]. BFP and GFP have well separated emission spectra, resulting in little fluorescence bleed-through (Figs. 5.5A and 5.6A). This facilitates data analysis for FRET ratio imaging... 

The sensitivity, dynamic range, selectivity and stability are the key factors that determine the sensor performance. The current PEBBLE technologies have relied on fluorescence emission ratios for signal transduction (though fluorescence anisotropy and frequency modulation were also tested) and the performance of the PEBBLEs was established using either an Olympus IMT-II (Lake Success, NY, USA) inverted fluorescence microscope or a Fluoro-Max 2 spectrofluorometer (ISA Jobin Yvon-Spex, Edison, NJ, USA). The spectra and confocal images for the intracellular measurements were acquired with the same inverted fluorescent microscope and a Perkin Elmer UltraView confocal microscope system equipped with an Ar-Kr laser. 

Launikonis, B. S. Zhou, J. Royer, L. Shannon, T. R. Brum, G. Rios, E. Confocal imaging of [Ca ] in cellular organelles by SEER, shifted excitation and emission ratioing of fluorescence. J. Physiol. 2005, 567, 523-543. 

Apart from the unquenched donor image providing a FRET estimate Ed, another FRET estimate can be deduced directly from the pure sensitized emission (/ ) and direct acceptor excitation (FJ) components. From Appendix Table 7.A1, the following ratio can be defined10 ... 

Hence the quantity of EA can be simply calculated from the corrected sensitized emission image and the acceptor only image provided the ratio of the molar extinction coefficients of the donor and acceptor at the donor excitation wavelength is known (ct). This quantity can be determined from absorption spectra of purified labeled components or can be experimentally determined as follows. First, let us define a factor v that relates the signal of N acceptors in the S channel to the signal of the same number of donors in the D channel ... 

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