Using Imaging Workbench 6 in
FRET Experiments
Fluorescence resonance energy transfer (FRET) is a distance dependent
physical process by which energy is transferred nonradiatively from
an excited molecular fluorophore (the donor) to another fluorophore
(the acceptor) within a distance of 10 – 100 Å by means
of intermolecular long range dipole-dipole coupling. It is a sensitive
technique to investigate a variety of biological phenomena that produce
changes in molecular proximity.
An example experiment, with cells transfected with both CFP- and
YFPtagged proteins, consists of 3 exposures in rapid succession: (1)
expose 200 ms at 440 nm and measure at 480 nm; (2) exposure 200 ms
at 480 nm and measure at 545 nm; and (3) expose 200 ms at 440 and
measure at 545 (the actual FRET measurement).
Thus, for a complete experiment as outlined above, Imaging Workbench 6 controls two wavelength controllers to change excitation wavelengths
and emission filters respectively. However, if a user with CFPand
YFP-tagged cells wants to measure only changes in FRET and not the
absolutely quantified FRET signal, the user needs only a shutter to
control the duration of the excitation light and a filter wheel to
select one of two emission wavelengths; no change of excitation wavelength
is needed. For example, expose 200 ms at 440 nm and measure at 480
nm; then expose again at 440 nm and measure at 545 nm. Imaging Workbench 6 can accurately monitor the change in FRET online by calculating
the ratio of intensities at 545 and 480 nm.
The emission filter wheel can be replaced by the Dual-ViewTM Micro-Imager
(Optical Insights, LLC) that splits the incoming image into side-by-side
images at e.g. 480 and 545 nm that appear on the camera image sensor.
Imagng Workbench then can grab the two images at two respective emission
lights in one exposure.
and
Electrophysio-