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62 BD FACSAria II User’s Guide

Breakoff Window

The upper camera transmits an image of the drop breakoff to the Breakoff

window, where video image processing software converts the visual

characteristics of the image into numerical properties. The drop breakoff

is analyzed for two key features: Drop 1 and Gap.

Drop 1 is defined as the number of pixels from the top of the image to the

center of gravity of the first broken-off drop. A thin gray line on the image

is used to identify this drop. The number shown to the right of the Drop 1

field at the bottom of the window is the actual pixel location of the gray

line. The number entered into the Drop 1 field is the user-defined Drop 1

target.

The Gap is defined as the number of pixels from the first discontinuity in

the stream to the next stream object, generally the first broken-off drop. A

gray line of varying thickness represents the Gap. The number shown to

the right of the Gap field is the pixel width of the gray line. The number

entered into the Gap field is a user-defined target.

When the Sweet Spot is turned on, the drop drive amplitude (Ampl) is

automatically adjusted to approximately match the target Drop 1 and Gap

values. The amplitude is initially adjusted in larger increments until Drop

1 is achieved. The amplitude is then adjusted in smaller increments until

the cytometer attains the target Gap. The Sweet Spot feature will make

adjustments as necessary to maintain the required breakoff conditions

throughout the day.

The Sweet Spot performs two other functions during sorting. When

sorting, if the Drop 1 or Gap are out of range, sorting is paused until the

values are back within range. This ensures that sorting occurs only under

the proper breakoff conditions. If a more severe problem such as a clog is

detected by the Sweet Spot, the stream is shut off and sorting is stopped:

the deflection plates shut off, the aspirator drawer closes, and the sample

tube is unloaded.

Pre-programmed values can be downloaded to the Breakoff window by

choosing one of the nozzle sizes (70, 85, 100, 130 micron) from the Sort

Sort Setup menu.

Drop 1

Gap

target

value

actual

value

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Aria 8.0 Betriebsanweisung Seite 62