Andor Technology iXon Ultra 897 User Manual page 153

TUTORIAL 2
(for sensors without anti-blooming, the weakest signal should be
sufficiently spaced within the image from the stronger signals, such that
charge does not 'spill over' into a measured region). Measure the S:B ratio
of the weakest signal using ROI, as in the above step, and repeat this
procedure until an optimal exposure time (E2) has been determined which
meets the S:B requirements of the assay.
Division of E2 by E1 (and round upwards) should determine approximately
the number of accumulations needed, using exposure time E1, to approach
the S:B threshold required for the weakest signal to be considered
quantitatively valid. In practice, more accumulations may be necessary due
to extra spurious noise sources such as clock induced charge (CIC), which
will accumulate with each readout and contribute to the overall
background. Simple trial and error above this calculated number should
determine the optimal number of accumulations required. Importantly,
irrespective of the accumulation number used, if E1 is used as the exposure
time, the strongest signal should not result in saturation of any of the pixels
within the image.
This procedure can be carried out at a variety of gain settings in order to
find the setting which affords the required S:B level for the lowest intensity
signal, at the minimum overall accumulation time. Primarily, EMCCD gain
should be applied in order to overcome the readout noise of the detector,
which in a well optimised assay (in which the detector performance defines
the detection limit and not extraneous background sources), defines the
assay detection limit, i.e. the 'concentration' yielding a signal equal to the
readout noise. The EMCCD can effectively reduce the readout noise to sub-
1 electron rms, therefore significantly reducing the detection limit at a
given exposure time. Alternatively gain may be employed to significantly
reduce exposure times, therefore increasing the throughput rate of the assay
procedure.
Bear in mind also that gain performance varies with temperature (see
tutorial one), so ensure that temperature remains stable, as is the case if the
temperature indicator at the bottom right of the Main Window turns blue.
The temperature can be accessed through the command line within the
Section 7. 3
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