Biology Reference
In-Depth Information
the in-focus image, a processed image that is normalised for this intensity variation is
created to give the smooth final image shown in Panel D. Although most image pro-
cessing packages contain a 'flat field correction' function that is designed to take into
account signal variation across an image, we have found that the above approach
gives a more reliable and higher quality corrected image, particularly when imaging
cells on agarose pads.
8.1
Signal comparison
Once the image has been processed, MetaMorph
®
has a very useful function that
allows the intensity of fluorescence from labelled proteins in different strains to
be compared directly. By creating a stack of two or more fields imaged under iden-
tical conditions, the intensity of fluorescence between the fields can be directly com-
pared. This is done by firstly stacking the images to equalise their signal intensities
and then presenting them as a montage.
Figure 4.5
shows the results that equalising
can achieve, using GFP-tagged metabolic enzymes CitC and CitZ from
B. subtilis
.
When comparing the unequalised images in the top panels, the intensity and there-
fore the cellular concentration of the fluorescent protein fusions appear to be similar.
However, by comparing the bottom two panels that were equalised for intensity, it is
CitC
CitZ
Before
After
Max. Pixel
Intensity = 2899
Max. Pixel
Intensity = 1592
FIGURE 4.5
Equalising intensity for protein abundance estimation. The top panels show the processed
images of CitC and CitZ before being equalised. The bottom two panels show the results of
image stacking andmontaging, which allow direct comparisons of fluorescent intensities to be
observed.
