BD FACSymphony A1 Flow, User Manual

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Reflective mirrors
Reflective mirrors reflect all light in their performance band. They are intended to pass light to the next optic
with minimal loss regardless of wavelength. These are used when a detector position needs to be bypassed due
to space constraints.
Compensation theory
Fluorochromes emit light over a range of wavelengths. Optical filters are used to limit the range of frequencies
measured by a given detector. However, when two or more fluorochromes are used, the overlap in wavelength
ranges often makes it impossible for optical filters to isolate light from a given fluorochrome. As a result, light
emitted from one fluorochrome appears in a detector intended for another. This is referred to as spillover.
Spillover can be corrected mathematically by using a method called compensation.
In the following example, FITC emission appears primarily in the FITC detector, but some of its fluorescence
spills over into the PE detector. The spillover must be corrected or compensated for. Alternatively, the spillover
can be minimized by discrete excitation of fluorochromes. In the following example, excitation with a yellow-
green configuration will help minimize spillover.
This spillover can be seen in a dot plot of FITC vs PE. The FITC spillover in the PE detector must be corrected as
demonstrated in the two figures that follow.
Using the Compensation tab of the Cytometer window in BD FACSDiva™ software, you can adjust the
PE-%FITC spectral overlap value. Compensation is optimal when the positive and negative FITC populations
have the same medians in the PE parameter statistics.
Chapter 7 Technical overview 83