Biology Reference
In-Depth Information
8
IMAGE PROCESSING
Image processing is one of the most important steps in localisation studies, ensuring
the generation of high-quality images suitable for publication. After a focussed
image is taken, the same field should be moved completely out of focus to allow
a background image to be captured. This image can now be used to subtract any
background fluorescence and autofluorescence that may be present, as well as being
able to take into account micro-heterogeneities that can be present within the agarose
pad. Using an out-of-focus image also takes into account the inherent variation in
intensity across the field, which will vary from microscope to microscope.
Figure 4.4
shows an in-focus image of RNAP-mCherry from
B
.
subtilis
(Panel A)
and an out-of-focus image (Panel B). A linescan from the top left of this image to
the bottom right is represented graphically in Panel C. It shows that the intensity
of the fluorescence varies across the screen. This variation is also present in the
focussed image shown in Panel A. By subtracting the out-of-focus image from
A
B
C
444
D
424
404
384
364
344
0
200
400
600 800
Distance (pixels)
1000
1200
1400
1600
FIGURE 4.4
Image processing using background subtraction. The raw image is shown in (A). An out-
of-focus image is shown in (B). A graphical representation of the linescan taken through B
(diagonal line from top left to bottom right) is shown in (C), while the processed image is
shown in (D).
