Fluorescent Protein-Based Biosensors and Their Clinical Applications - Progress in Molecular Biology and Translational Science - page 331

Dr. Nagai and colleagues recently developed YCs with high Ca 2 þ affinity
( K d 0 ¼

15-140 nM, Cameleon-Nano) and 1450% signal changes by
replacing the polyglycine linker with a longer form. 99 More recently,
Dr. Matsuda and colleagues reported an optimized backbone for FRET-
based biosensors (the extension for enhanced visualization by evading
extra-FRET (Eevee) system). 100 In this system, introduction of a long,
flexible linker ranging from 116 to 244 amino acids in length makes FRET
biosensors almost completely dependent on the distance between the donor
(ECFP) and the acceptor (Ypet). The Eevee system has succeeded in
improving a variety of biosensors for tyrosine kinases, serine/threonine
kinases, and GTPases. 100

6. METHODS FOR EVALUATING FRET EFFICIENCY
The basis for obtaining FRET efficiency by the sensitized emission
method was described in the preceding section (see Section 5.1 ). Although
fluorescence plate readers, fluorescence spectrometers, or similar equip-
ment may be used, fluorescence microscopy is the most popular method
for evaluation; Using microscopy, dynamic cellular events, including pro-
tein interaction and activation, can be analyzed in living cells or organisms.
General requirements for such experiments include an epifluorescence mi-
croscope, a cooled charge-coupled device (CCD) for image acquisition,
and filters suited for the excitation and emission spectra of the donor
and the acceptor ( Fig. 8.3E ). Mechanical shutter(s) and filter wheels
(in which filters are set) under the control of imaging software may be es-
sential in time-lapse experiments to avoid excess exposure of cells to
light during the periods of acquisition and to acquire two or three images
for FRET evaluation, respectively. After image acquisition, image-
processing software may help to calculate FRET indices for each pixel.
As described above, in intramolecular FRET systems, FRET indices are
estimated by simply calculating the ratio of acceptor emission to donor
emission (that are obtained by donor excitation); in such systems, it is pos-
sible to obtain FRET indices from simultaneous acquisition of emission
images with the use of a spectral separator. Such equipment is
commercially available (Dual-View from Photometrics or W-View from
Hamamatsu Photonics) and enables imaging with high temporal resolu-
tion. Details of the practical experimental procedures are provided in an
excellent review. 101