Ideally, when one uses a dye in an experiment, its emission spectrum will be narrow enough that fluorescence from that dye is only detected by a single detector in the instrument. In practice, however, because of the broad emission spectra of available fluorochromes, the dye you are using will likely emit significant amounts of fluorescence in several different detectors. In other words, the light reaching a given detector consists of the signals from multiple fluorochromes.

 

Compensation is the process of transforming the data such that the values from a single detector come from an individual dye. In order to do this, a percentage of the overlapping emission is subtracted from the target emission. Traditionally, this compensation was performed by the instrument during acquisition. However, modern instruments are capable of storing the data in uncompensated form and compensation can be applied by the analysis software.

 

 

Example of the calculation: Compensated Parameter 2 Fluorescence = Observed Parameter 2 Fluorescence minus 5% of Observed Parameter 1 Fluorescence.

 

 

Compensation involves creating two matrices. The spillover matrix represents the percentage of the signal from a given channel that spills into adjacent channels. The compensation matrix, used to correct for the spillover, is the inverse of the spillover matrix.

 

FCS Express uses the following terminology when dealing with compensation. The target parameter is the parameter that is detecting signal (potentially from multiple sources). The source parameter is the primary parameter that you want the signal to be in, but that dye is also bleeding (potentially) into multiple targets. In other words, we are subtracting the percentage of the source that is “bleeding” into the target.

 

In the example given above, Parameter 2 is the target and Parameter 1 is the source. A family of sources and targets is called a compensation definition. A compensation definition describes all the ways that fluorescence from different channels affect each other under a given set of conditions and is equivalent to a single compensation matrix. Typically, the instrument user will set the gains for all the channels at the beginning of the experiment and use these settings for the duration of the experiment. Thus, the compensation definition would apply for the entire experiment.

 

FCS Express can store multiple compensation definitions in a single layout. This is useful when one wants to analyze data acquired on different days in the same layout. It is likely that the gains will be different on different days and will require a different compensation definition for each day. Each overlay on a plot can use a different compensation definition.

 

In addition, the compensation definition can be assigned to a data file. This means that when the .fcs file is loaded into a plot, it will automatically use the proper compensation. In this way, you do not have to remember the appropriate compensation definition for each of your data files.