Andor Technology iXon Ultra 897 User Manual page 156

TUTORIAL 3
Record a Kinetic Series - From the Acquisition menu on the Main
Window, select Setup Acquisition. In Acquisition Mode select Kinetic
Series, and type in the number of frames to be recorded on the run, e.g. 50.
In the Setup CCD tab, ensure gain is set to zero and set exposure time to
approximately 10-20 ms (the Kinetic Cycle Time will update as exposure
time is changed, as will the indicated Frame Rate in Hz). Now acquire the
signal image – from the Acquisition menu on the Main Window, select
Take Signal (or alternatively click the Take Signal button).
Playback Kinetic Series - Playback of the kinetic series can easily be done
through the Play, Pause and Stop icons at the RHS of the top row of icons.
Furthermore, Playback Autoscale may be selected or deselected as
required. The dynamics of the fluorescent beads moving across the field of
view and in and out of the focal plane should be readily observable.
Use of ROI and Kinetic Plots – Using the scroll bar at the RHS of the Main
Window, select a frame for processing. 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 a fluorescent bead, then
resize until it falls within the signal area (or define it numerically to
measure say 5x5 pixels, then drag it onto the signal).
Click the Add ROI button again and a second ROI will appear of the same
resized dimensions as the first. Drag this over the position of another
fluorescent bead. More ROIs can be added in this fashion. The mean value
of pixels within each ROI will appear for each frame, and can be accessed
for that particular frame by sliding across the scroll bar within the ROI
display window. The mean of the top 10%, 20%, 30% etc. pixels can be
calculated by clicking on the Hot Spot Approximation icon in the ROI
display window. This enables spot intensities to be compared without
taking the immediately surrounding background pixel values into account.
Section 7. 3
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