Yellow Cameleon

Several other approaches to solve the quantitation problem have been proposed. Hoppe et al. [2] determined y/ by calibrating it against constructs with known FRET efficiency. We and others [3, 6] have used data from a cell before and after acceptor photobleaching to relate the FRET-induced sensitized emission in the S channel to the loss of donor emission in the D channel by factors termed or G, respectively. For the CFP/YFP pair this works very well on confocal microscopes with a 514-nm Argon ion laser line, but on wide-held systems, selective acceptor photobleaching reportedly causes problems [ 14]. F inally, G can also be determined by comparison of several constructs that differ in FRET efficiency, a bit analogous to the Yellow Cameleon calibration described above [10,14],... 

Sensor any single-polypeptide construct (containing the fluorophores to be used in the experiment) that can be induced to change FRET significantly and homogeneously e.g., Yellow Cameleon in combination with ionomycin. 

Figure 2. Improvement of yellow cameleon by using a circularly permuted YFP variant. (A) The three-dimensional structure of GFP with the positions of the original (Metl) and new N-termini (Aspl73). (B) Domain structures of YCS.12 and YC3.60. CaM, Xenopus calmodulin Ml3, a CaM binding peptide derived from myosin light chain kinase (C, D) Emission spectra of YCS. 12 (C) and YC3.60 (D) (excitation at 435 nm) at zero (doted line) and saturated Ca (solid line). (E) A series of confocal pseudo-B/W images showing propagation of [Ca c- These images were taken at...

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