Biomedical Engineering Reference
In-Depth Information
Fig. 4 Representative encoding of 18 microbead populations. Microbead populations are
encoded by a combination of two fluorescent dyes at varying intensity levels which are processed
as gray values. The decadal logarithm of the gray value ratio defines the PopID. Additionally, the
microbead size can be used as an encoding parameter. Due to variations in impregnated dye
quantity and microbead diameter, a negligible fraction is excluded from analysis (outer range of
ellipsoids)
Fig. 5 Microbead with red
halo of ligand dye (analyte).
The intensity of the ligand
dye is quantified on the edge
of the microbeads
bubbles, etc.; centre coordinates and diameters of the remaining microbeads can be
easily obtained from the circular area. Because of the edge-based detection
method, the diameter calculation is independent from the intensity. Then a second
image is taken at the same position with a filter set matched to the second encoding
dye (second encoding channel). Gray values of the first and second encoding
channel are determined in the centre of every microbead detected. This so-called
PopID of every microbead is calculated by the decadic logarithm of the ratio of
these two gray values (Eq. 1 ).
ð
Þ
Grayvalue encoding2
PopID ¼ log 10
ð 1 Þ
Grayvalue encoding1
ð
Þ
If the two encoding dyes are graded in a ratio from 1:10 to 10:1 for every
microbead, a PopID from -1 to +1 can be calculated. The diameter and PopID
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