Compensation

The compensation dialog

Compensation is a way to deal with spectral overlap. As fluorescence emmission spectra can cover a wide range of wave lengths, the spectra of different fluorochromes can overlap. This means that multiple fluorochromes may be detected by the same filter/detector. An example: fluorochrome FA is detected mainly in detector DA, but also to some lower extent in detector DB - it 'spills over' into detector DB. To eliminate this overlap, a specific percentage of the value detected in DA is subtracted from the value in DB. This percentage is the compensation value. The collection of compensation values for a given set of fluorescence channels is called compensation matrix.

However, fluorescence spill-overs can criss-cross: fluorochrome FA is expected to be seen in detector DA and spills into detector DB, and fluorochrome FB is expected in detector DB and spills into DA. The compensation matrix needs to be adjusted for this effect. Using single-color controls, the spill-over of a fluorochrome into the various detectors can be determined. The collection of all spill-overs is the spill-over matrix. The compensation matrix is calculated from the spill-over matrix. For a better explanation, see here: http://www.bioinformin.net/cytometry/compensation.php

Some FCS files contain spill-over matrizes internally. FCSalyzer can read some of these matrizes, which are then automatically loaded and applied to FCS files.
Note: up to 0.9.18, FCSalyzer treated spill-over matrizes wrongly as compensation matrixes. Since 0.9.19, FCSalyzer corrects this bug and calculates compensation matrizes from spill-over matrizes and uses these compensation matrizes for data calculation.

To adjust the spill-over matrix for FCS files, select the "File - Format Datafile" menu. This will open a dialog:

• In the upper left panel, the spill-over matrizes of the current FCX document are shown
  • "New compensation" lets you create a new compensation
  • "Del compensation" deletes a compensation
• In the upper right panel, the currently selected spill-over matrix is shown.
• In the bottom panel, you will see a list of all open FCS files.

How to create a spill-over matrix

1. Click "New compensation". This will create a new spill-over matrix, where each spill-over value is 0.0%. The name of this matrix will be "new Compensation [##]", where ## is the parameter count of the spill-over matrix. That count is taken from the currently selected FCX file in the bottom panel. If no FCS file is selected, the file on top of the list is used.
2. select this "new Compensation [##]" in the top left panel.
3. now the spill-over values of this matrix are shown in the top right panel. The parameters are simply labeled "Parameter".
4. select a relevant FCS file in the bottom panel to apply its paramter labels to the spill-over matrix shown in the top right panel
5. now adjust the spill-over matrix in the top right panel. You can change the name of the matrix and also each spill-over value.
   • The matrix is defined by the "Parameter" and "Spillover % into channel", as well as the spill-over value. The spill-over value is determined using single-stained controls. For those, the spill-over is the percent required to reduce the fluorescence in the'unwanted' detectors to background levels. Example: the "Parameter" is FITC, the 'unwanted' detector is PE. An single stained control with only FITC fluorochromes has a mean FITC value of 1000 and a PE value of 200 - but the background level of PE should be 100 (based on the no-staining control). Hence, the spill-over from FITC into the PE detector is 100, and this the spill-over is 10%. This '10' would be entered in the spill-over matrix.
   • The spill-over value can simply be typed into the field, or selected by moving the slider. If you need a higher spill-over value as provided by the slider, type that value into the value field.
6. to apply a spill-over matrix to a FCS file, select the matrix in the drop-down box to the left of the FCS file names in the bottom panel.
7. FCSalyzer will calculate a compensation matrix based on the spill-over matrix and use that compensation matrix on the FACS data

Keep in mind:

• Regions will not be adjusted when a spill-over matrix is changed. Re-check your regions after editing spill-over matrizes!
• One FCS data file has only one spill-over matrix. If you change the matrix, all plots and statistics for that FCS data file will change.
• Compensation does not only change the display of the data, but also the event values! Statistical data will be changed!
• Automatic compensation is not available. You have to do this the hard way manually. But it is enough to create one compensation and then apply it to all FCS data files (with the same number of parameters)