Biology Reference
In-Depth Information
images are captured far away from the optic axis. Factors defined for offsets
and thresholding in the pre-bleach images are applied to all images in the time-
lapse sequence. For long sequences, estimates for the rate of fade of the images
are required. These can be obtained by integration of the CFP intensity over
the whole cell and application of a second factor to both images to
compensate for this. We have found it advantageous to match the rates of fade
of YFP and CFP by careful adjustment of the laser excitation intensities.
FLAP: Some points to consider
Besides chromatic aberration, two possible causes of non-uniformity of
prebleach FLAP images are (1) the two fluorophores are not co-localized and
(2) FRET is occurring. We have never experienced the first case but there is
often evidence for FRET in dense regions of F-actin. As discussed previously,
CFP and YFP constitute an effective FRET pair (Pollok and Heim, 1999) and
one effect of FRET occurring is to reduce the donor (CFP) signal when
exciting the CFP channel. The CFP detector does not register the increased
YFP emission caused by FRET and no FRET occurs when the YFP channel
is excited. The net result is that the FLAP image shows less intensity in regions
of dense actin though the effect is seldom more than one or two grey levels and
can be ignored. Moreover, the effect is reduced in regions where the YFP has
been photobleached and so the strength of the FLAP signal is hardly affected
by FRET. It is likely that the FRET eciency is always going to be low in the
system described above since the statistical probability of two labelled actin
molecules being within 10 nm of one another is not high.
Theoretically, FLAP could be simplified if the actin molecule carried both
fluorophores, one on the N-terminus and the other on the C-terminus. In this
case, co-localization of CFP and YFP would be perfect, not a statistical
probability, eliminating probabilistic noise in the FLAP signal. Early
indications suggest that the additional noise due to statistical co-localization
is small compared to instrumental noise.
Significant levels of FRET would be expected in this simplified FLAP since
the N- and C-termini of actin are adjacent to one another and this could act to
enhance the FLAP signal. CFP emission would be reduced except in cases
where CFP was fused to the same molecule as a bleached YFP (remember that
the unbleached YFP signal would be unaltered). This would increase the
FLAP signal. However, complications arise since incorporation of G-actin
into F-actin results in a marked conformational change. This would be
expected to lead to repositioning of the CFP-YFP pair with respect to one
another, a consequent change in FRET e
ciency and therefore an altered
difference image. The FLAP signal would thus represent two variables:
number of bleached molecules and polymerization state of the actin. The
