Molecular Devices SpectraMax Paradigm User Manual page 42

SpectraMax Paradigm Multi-Mode Detection Platform User Guide
When neglecting the time delay t
accumulated signal A can be approximated with the following equation:
A / A
In the previous equation, M is the size of the time window (or integration time) divided by
the exponential decay time constant (or the fluorescence lifetime of the label).
M = (integration time) / (fluorescence lifetime)
For example, using Europium, which has a fluorescence lifetime of 700 µs, and the suggested
integration time per cycle of 1.890 ms (or 1890 µs), M = 1890 / 700 = 2.7. Inserting this value
of M into the first equation yields A / A
To optimize the integration time per cycle (pulse), the integration time should be set such
that the value of M produces the desired signal. For example, to get more than 86% signal,
select an integration time such that M is greater than 2.0. Using the previous Europium
example and solving for the integration time, the integration time can be set to M (2.0) times
the fluorescence lifetime (700 µs), or 1400 µs (1.4 ms).
Table 2-2: Achievable accumulated signal percentage compared to M
M
A / A
max
M can be technically limited by the time between pulses. Further gain in signal above some
value of M can be negligible to improve results.
When performing a dual-label Europium-Samarium measurement, there are more timing
parameters. There is some residual cross-talk of the Samarium signal captured in the
Europium emission channel. Samarium has a much shorter fluorescence lifetime, so to
reduce the cross-talk of Samarium in the Europium channel, Europium is measured in a time
window shifted away from the time window for Samarium. This lets the Europium be
quantified without cross contamination from the Samarium. The known Europium
concentration can then be used to remove the Europium cross-contamination in the
Samarium channel.
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= (1 – exp(–M)) x 100%
max
0.25 0.50
[%] 22 39
compared to the integration time window t
2
= 93%.
max
0.75 1.00 1.25
53 63 71
, the
3
1.50 2.00 3.00
78 86 95
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