Biology Reference
In-Depth Information
Figure 7.5 A typical FLAP experiment following actin dynamics. (A) A pre-bleach
fibroblast showing (i) CFP-actin expression, (iii) YFP-actin expression and (ii) integrated
intensity traces (cyan line is CFP, yellow line is YFP) through the cell encompassing the
proposed bleach region. The red line corresponds to the difference in intensities of the YFP
and CFP. Since this is a pre-bleach image the difference signal will be zero with noise
artefacts superimposed over it. (B) The same cell after bleaching (the bleach region is
defined by the white circle). Note the red difference signal now registering a significant
difference in integrated CFP and YFP intensities. Descriptions for parts (i-iii) are as in (A)
in this legend. (C) The same as (B) except that (ii) the integrated difference signal has been
replaced with a pseudocolour representation (Hall LUT) of the difference signal for the
whole cell. Red regions correspond to a high FLAP signal (large difference between CFP
and YFP) whereas blue regions correspond to low FLAP or difference signals. (The reader
is referred to the colour montage.) (A colour reproduction of this figure can be found in the
colour plate section)
immediately afterwards. Data from a typical FLAP experiment are shown in
Figure 7.5 (from Dunn et al., 2002).
Simple image processing is required to produce the FLAP signal. Using
Mathematica 4.2, the pre-bleach CFP and YFP images are smoothed using a
363 pixel median filter followed by calculation of the offsets required to bring
both image backgrounds to a mean intensity of zero. User-defined algorithms
are then applied to calculate a variable that when applied to one image
produces a precise match of the integrated intensities of the two images. To
calculate the FLAP image, the YFP image is simply subtracted from the CFP
reference image (Figure 7.5). In the case of the pre-bleach images, the FLAP
image should have a uniform intensity of zero, although in practice the images
are noisy when viewed with a pseudocolour look-up table (Hall LUT). This
noise can be reduced satisfactorily with the minimum of thresholding. Non-
uniformity of the pre-bleach images, particularly around the cell edges,
indicates a problem often due to chromatic aberration of the objective when
