Biomedical Engineering Reference
In-Depth Information
ensure a unique assignment of every microbead to a specific population (Fig.
4
).
The probability of misclassification using this method is well below 1%.
Finally, the fluorescence intensity of the labeled analyte captured on the surface
of the microbead is determined by taking an image of the same scene with a filter
set corresponding to the wavelength of the ''ligand channel''. The initial inte-
gration time of routine images is typically 500 ms. In contrast to the encoding
dyes, the ligand dye is located on the surface of microbeads only, so it creates a
halo around the microbead. The intensity of this halo, resulting from binding
analyte, is quantified on the edge of every microbead (Fig.
5
).
To increase the dynamic range of a given assay, the intensity of every halo is
checked. If its value exceeds a predefined limit, overexposure is assumed. If one or
more microbead halos are overexposed, an additional image is captured with the
integration time halved. This procedure is repeated until no halo is overexposed.
Considering the integration time used, the absolute intensity value of the halo is
recalculated and finally referenced to the intensity of one or both encoding
channels. Referencing compensates completely for different intensities of fluo-
rescence extinction and other long-term drifts of device-specific parameters. This
allows comparison of numeric results measured with different devices at different
time points or in different experiments. To get comparable results from different
microbead populations, despite different concentrations of encoding dye, intensity
differences in the encoding channel(s) are compensated by a population-specific
correction factor (Eq.
2
).
:
X
all beads population
i
¼
Correction
Population
Greyvalue hal
ð Þ
Greyvalue encoding1
i
refMFI
Population
Beadcount
Population
ð
Þ
i
i
ð
2
Þ
The process is repeated at different positions in a well until a predefined
minimum number of microbeads is identified. The measurement value for an
analyte is calculated by the quantile-filtered mean value of all microbeads of the
corresponding microbead population (refMFI). A user-defined quantile filter
(default 3%) can be applied to handle outliers. Microbead measurement leads to a
refMFI ranging from 0.003 to 300 (± 10%). In this dynamic range (100 dB), the
measurement value is linear (deviation less than 3 dB).
3 Design Principles of VideoScan Microbead Assays
3.1 Immobilization of Microbeads
An optional step during VideoScan microbead array fabrication is microbead
immobilization. The assay workflow of sequential handling steps is simplified
considerably and refocusing is reduced to a minimum. Most importantly, reactions
