Molecular Devices SpectraMax Paradigm User Manual page 141

The algebraic solutions to (Equation 1) and (Equation 2) are:
Equation 3:
Equation 4:
The unknown coefficients for the overlap (CT) are determined by measuring and inserting the
red only and green only controls into (Equation 1) and (Equation 2), respectively, yielding
Equation 5:
Equation 6:
and
Equation 7:
Equation 8:
where Red
control inducing the green emission spectrum only, R
through the red filter, R
green control as measured through the red filter, and G
through the green filter.
Paired equations (Equation 5) and (Equation 6), and (Equation 7) and (Equation 8) can be
resolved into
Equation 9:
Equation 10:
Because (Equation 9) and (Equation 10) are ratiometric, the concentration of the red and
green only controls can be undefined, though shall be considerable to yield accurate
measurement values.
By inserting the solutions for the coefficients CT enumerated above in (Equation 9) and
(Equation 10) into (Equation 3) and (Equation 4) above, it can be confirmed that the
resulting equations comply with those provided by Promega, after normalization with the
filter efficiencies E
are stored in the cartridge, thus requiring a different measurement to get R and G (without
emission filter) unnecessary.
All the equations in this section make no assumption on the nature of the signal, thus could
be similarly applied to other dual label detection with overlapping spectra, for example in an
absorbance or a fluorescence reading.
5014038 E
Red = (L - [L x CT ]) / (1 - CT
rf gf RG
Green = (L - [L x CT ]) / (1 - CT
gf rf GR
R = Red x CT + 0 x CT
rf Control GR
R = Red x CT + 0 x CT
gf Control GR
G = 0 x CT + Green
rf GR Control
G = 0 x CT + Green
gf GR Control
is the control inducing the red emission spectrum only, Green
Control
is the red control as measured through the green filter, G
gf
R
gf
CT =
GR R
rf
G
rf
CT =
RG G
gf
= (R / R) and E
rf rf
x CT )
GR RG
x CT )
RG GR
RG
RG
x CT
RG
x CT
RG
rf
= (G / G), respectively. The values for filter efficiencies
gf gf
Chapter 5: Available Detection Cartridges
Control
is the red control as measured
is the green control as measured
gf
is the
is the
rf
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