Biology Reference
In-Depth Information
A
B
1
B
2
Dichroic
mirror
Filter wheels
Focusing
lens
CCD
Camera
Figure 5.11 Examples of the setup optimized for ratiometric FRET measurements. In (A),
images are acquired sequentially. A fast filter wheel allows fast switching between both
acquisitions (10-30 ms). In (B), images are acquired simultaneously. A dichroic mirror is
used to separate the emission from donor and acceptor molecules. A focusing lens can
then be used for directing the light of both channels on each half of the same camera
(B1), or two different cameras can be used for light collection (B2).
a slower but more precise configuration for measuring sensitized emission.
It consists inmeasuring for each pixel of an image the overall emission spectrum
and is not limited to only two bandwidths using filters (see
Fig. 5.12
for more
details about the setup).
Gathering fluorescence emission spectra then allows donor and acceptor
emission spectra to be separated according to the distinct shape of both spec-
tra after spectral unmixing. The fluorescence signal is then analyzed in the
same way as traditional ratiometric images are. It, however, allows for dis-
tinguishing the real FRET signal from other elements that may alter this
measurement, such as autofluorescence or the presence of multiple
fluorophores in the sample, and it can then be used in more complex bio-
logical environments.
4.1.3 Lifetime-based approaches
Fluorescence lifetime is inherently quantitative and is most of the time
independent of the concentration of the fluorophore. Furthermore, FRET-
FLIMexperiments needmeasurements of the donor fluorescence lifetime only
(cf. Eqs.
5.16, 5.18, and 5.29
), which makes it extremely valuable for
simultaneous multibiosensor measurements. Lifetime measurement,
however, requires dedicated and more sophisticated instruments than for
ratiometric imaging. The measurement can be done in either the time
domain (TD FLIM) or the frequency domain (FD FLIM) (
Fig. 5.13
). In
Search WWH ::
Custom Search