Data Processing Algorithms
274 0112-0109 H
In the table below, the results of the experiment are presented without
Spatial Uniformity Correction, Negative Control Correction, Positive
Control Scaling, or Subtract Bias Value.
An inspection of the fluorescence counts taken at Sample 1 (time = 0)
shows some inter-well variability (range 8000–10000). Variability can
be the result of differences in cell density or well positions (so-called
‘edge effects’ where cells on the outside of the plate are less dense
than their internal neighbors), dye loading, non-uniform illumination,
buffer variations or any other effect which is constant throughout the
experimental run. These variations can complicate data interpretation.
Crosstalk Correction
The crosstalk correction algorithm compensates for light from one well
affecting the read out from a neighboring well.
Determining Crosstalk Correction
If the middle well in the drawing below was empty and the eight wells
surrounding it would have signal, the middle well would show up as
having a certain value based on the amount of light crossing over from
each of it’s neighbors. The percentage of crosstalk from wells directly
next to (B) the well of interest is assumed to be different from the
percentage of the crosstalk from the wells diagonal (A) from the well of
interest, therefore these two values are entered for the crosstalk
correction. In this calculation, only the immediate neighboring wells are
assumed to impact the middle well.
When the crosstalk correction is applied, the Horizontal/Vertical factor
and the Diagonal Factor are applied in a matrix, which automatically
subtracts the percentage of each of the neighboring well’s value from
the middle well’s value. If the crosstalk correction option is selected it
will be the first correction applied to the data. Crosstalk correction is
Sample Time
Well
A1
– Ctrl
A2
– Ctrl
3
– Ctrl
A4
Exp
A5
Exp
A6
Exp
A7
+ Ctrl
A8
+ Ctrl
A9
+ Ctrl
1 0 8000 8500 9500 8200 9200 8800 1000 9500 8700
2 5 8200 8600 9400 8300 9000 9000 9500 9700 9000
3 10 8400 8500 9200 8500 9000 9100 9700 9700 9400
4 20 8600 8400 9200 45000 50000 42000 57000 52000 54000
5 25 8600 8800 9200 37000 42000 35000 53000 50000 50000
6 30 8800 8500 9500 30000 25000 29000 50000 51000 50000
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