Biomedical Engineering Reference
In-Depth Information
Fig. 12 Intensity line diagram of intensity values for replicated spots (sciANA 4.1, Scienion AG,
Berlin, Germany)
Currently, determining the spot locations within each image requires operator
assistance, but this process could become fully automated with the inclusion of
reference spots. The inclusion of spots for correct orientation and positive and
negative controls enable the automated alignment of the grid, the automated control
of array quality and the control of the array evaluation. Having suitable reference
spots at hand automatic grid finding algorithms can be adjusted to a wide range of
image formats and grid types as displayed in Fig.
11
. Shown is a software screenshot
after automatic grid finding (sciANA 4.1, Scienion AG, Berlin, Germany).
Additionally analysis software with automatic grid finding algorithms is a valuable
tool for quality control in the protein array production process.
5.2 Determination of Spot Intensity
Signal intensities are generally quantified by subtracting the foreground intensities
with background intensities. The intensity of a specific spot can be estimated by
integrating over a fixed spot area or by using flexible radii for each spot. For the
determination of the spot intensity the local background intensity has to be mea-
sured and used for intensity correction and noise reduction. The background
intensity can be estimated either by a number of empty spots within the image or
as background signals from regions immediately surrounding the spot of interest.
In protein microarrays the local background regions are often distorted by local
artefacts or strong positive signals in the neighbourhood resulting in over shining
