Andor Technology iXon Ultra 897 User Manual page 152

TUTORIAL 2
determine approximately how close the most intense pixels are to the
saturation level. If this signal level is less than the saturation limit of 16384
counts, the exposure time may be proportioned up accordingly to approach
this maximum value. If the signal is already saturating the CCD, the
exposure time will have to be reduced and the above procedure repeated.
Carry out this measurement with the calculated optimum exposure time in
order to ensure that it does not saturate.
You must now calculate the signal to background (S:B) ratio of the lowest
intensity signal at this exposure time, as defined by the individual user or
assay criteria. Background relates to the combined contribution from
sources of 'undesirable' signal and noise, such as readout noise
(remember that the main benefit of EMCCD technology is to amplify the
weakest of signals above the readout noise 'detection limit'), clock
induced charge, darkcurrent and 'leaked' background light level (the
latter three are amplified by EMCCD gain). A suggested way to measure
S:B ratio is to integrate the signal, from say 20 pixels, from within the
signal area (S) and from a neighbouring background region (B). This can be
accomplished within the iXon software as follows:
From the Display menu on the Main Window, select ROI (Region of
Interest). This opens the ROI display window, which lists parameters such
as mean, standard deviation etc. for the ROI and also displays a list of ROI
manipulation buttons along the bottom. Click the Add ROI button (bottom
left), and a ROI will appear on screen. Pan the ROI centrally onto the
image of the LED, then resize until it falls within the signal area (or define
it numerically to measure say 5x5 pixels, then drag it onto the signal).
Take an average reading from within this ROI. Then drag the ROI to a
neighbouring background area and repeat this measurement. The S:B ratio
of the weakest signal can now readily be calculated.
Next, the exposure time (E2) must be determined at which the S:B ratio
from the weakest signal exceeds the assay S:B cut-off limit. Set the
exposure time to a greater value within the Setup CCD sub-window. The
intensity of the highest concentration signal will now saturate the detector
Section 7. 3
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