Topic : Fluorescence compensation overview

         Flow cytometry relies in many aspects on the ability to detect fluorescent dyes, or "fluorochromes" which are bound in or on particles of interest. These fluorochromes have the universal characteristic that when excited by a light source, normally a laser, they emit energy as light at longer wavelengths. These emissions are rarely spectrally discrete and therefore overlap to some degree, so in order to measure the amount of signal in a given detector, a compensation network is employed to derive the appropriate amount of signal which should appear in each detector.

example of compensation from under to over..          Consider the animated picture to the left. It shows three populations of beads, one unlabelled and one each labelled with FITC and PE. Watch as the red and green coloured populations move from an uncompensated to a correctly compensated, then an over compensated position relative to the X and Y axes.

          So when is a population correctly compensated?
The short answer is when the median of a population positively labelled for Y is the same as the median of a population negatively labelled for Y when displayed relative to the X axis.

         Now let's use the simulator to adjust for spectral overlap on some real data files. The simulator will work with any FCS compliant data file, so you can practice and become comfortable with this concept using any file in the database, however if you'd like to use some data files which contain completely uncompensated data please select the following files:

Unstained:
FITC only:
PE only:
PerCP only:
APC only:

The basic procedure to follow is:

  1. select the file "beads_unstained";
  2. display plots of various fluorescence parameters;
  3. ensure that you have at least displayed plots which show fluorochromes which are spectrally close e.g. FITC & PE;
    Information on this and other combinations of fluorochromes can be viewed at TSRI Java spectrum viewer;
  4. click the quadrant icon in the lower left of the dotplot window;
    Note that quadrant statistics are now available below each plot;
  5. next select and display a positively labelled sample, in this case we recommend the file "beads_FITC";
  6. select the "Adjust" menu and the Compensation item

position the crosshairs such that they surround the unstained population of