5. Set an initial laser delay value only for the laser you are optimizing.
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If you are optimizing the violet laser, set its delay to –35µs.
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If you are optimizing the UV laser, set its delay to –65µs.
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If you are optimizing the red laser, set its delay to 35µs.
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If you are optimizing the blue laser, set its delay to 0µs.
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If you are optimizing the yellow-green laser, set its delay to 65µs.
6. While observing the positive events on the histogram, adjust the laser delay in 1-µs increments. You might
need to adjust the delay above or below the initial setting.
Choose the setting that moves the events farthest to the right (highest fluorescence intensity).
7. Draw an interval gate on the histogram for the positive events.
8. Create a statistics view to display the mean fluorescence intensity (MFI) of the gated population.
9. While observing the MFI for the gated population, adjust the laser delay in 0.1-µs increments within a range
of 2.0µs of the setting obtained in While observing the positive events on the histogram, adjust the laser
delay in 1-µs increments. You might need to adjust the delay above or below the initial setting. (page 96).
Preserve the setting that maximizes the fluorescence intensity.
10. Reset the Window Extension to 10µs.
96 BD FACSymphony™ A5 SE User's Guide
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