Andor Technology iXon Ultra 897 User Manual page 157

TUTORIAL 3
The row of mean values can be highlighted for any ROI then plotted vs.
either frame number or time by clicking the appropriate icon in the ROI
display window. The kinetic plot will appear in a new window. In the
present example, the fluctuations in the plot over time represent fluorescent
beads passing through or coming into focus within the field of view.
Furthermore, a smaller number of frames may be selected and plotted in
order to concentrate more closely on the interesting sections of the kinetic
sequence.
Use of Gain to Enhance S:N of Weak Signal - from the Acquisition menu
on the Main Window, select Setup Acquisition. In the Setup CCD tab,
ensure gain is set to zero and set exposure time to approximately 10 ms. In
the Video Mode tab, ensure that Use Settings From Standard Setup is
selected. Click the Video Mode Run, the images of dynamic fluorescent
beads should appear in real time. From the Acquisition menu on the Main
Window, toggle Autoscale Acquisition (or alternatively press F6).
Reduce the aperture of the excitation light, insert neutral density filters and
lower the exposure time (this is easily done through the Exposure Slide
Control Bar), such that the intensity of fluorescence coming from the
beads will be significantly reduced, representing a low light signal (using a
mismatched emission filter can also markedly reduce the signal intensity).
Ideally, the intensity of emission detected from the beads will approach, or
become lost within the readout noise of the camera under this condition of
zero gain setting. (Operating under subdued ambient light conditions will
increase the probability that the background detection limit is indeed
defined by the camera readout noise).
Then increase gain using the Gain Slide Control Bar, visually monitoring
the markedly enhancing effect this has on the S:N ratio. Estimate the
optimum approximate gain setting required to amplify the signal from the
fluorescence beads significantly above the readout noise floor.
If the quantitative level of the noise floor can be seen to increase as gain is
Section 7. 3
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