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Fluorescence Compensation

The inherent overlap of emission spectra from antibody fluorescent labels makes compensation
necessary. This is of particular importance when you attempt to make simultaneous immunofluorescence measurements from several cell-bound antibodies(2, 3, and 4 color analysis of
samples). Figure 3.1 and 3.2 help illustrate the phenomena of fluorescence spectral overlap as
it applies to flow cytometry.

Figure 3.1 The Emission spectra shown are for equal concentrations ( mg antibody/ml) of mouse anti-human
IgG directly conjugated with fluorescein (FITC), phycoerythrin (PE), and tandem conjugates of phycoerythrin
with Texas red (PE-TR or 613) and Cy5 (PE-Cy5 or Tricolor), with excitation at 490nm. Boxes demarcate the
passband of the green (525nm), yellow (575nm), orange(620nm), and red (660nm long pass) filters used in
front of the fluorescence detectors. (From Practical Flow Cytometry Third Edition, Howard M. Shapiro p.164).

Compensation is the process by which the fluorescence spillover originating from a fluorochrome other than the one specified for a particular PMT detector is subtracted as a percentage of the signal from other PMTs. For example, spectral overlap between FITC and
PE produces light which is detected by both the FL1 and FL2 detectors. The amount of FITC
fluorescence being detected by the FL2 detector (i.e. PE detector) can regarded as excess
fluorescence and should therefore be compensated out. Figure 3.1, show the emission spectra of the fluorochromes commonly used in our XL analysers . You can see the portion of fluorescence emission from each dye which leaks to other detectors. This phenomena is
referred to as SPECTRAL OVERLAP.

By looking at the emission spectra of FITC and PE together with the optical specifications for their
respective detectors you can see how more of the FITC spills into PE than PE spills into FITC. This
amount of spectral overlap is corrected by subtracting a percentage from the total FITC pulse
generated by the FL1 detector. The FITC spillover detected by the FL2 detector can be viewed as
interference. By subtracting a percentage of the total FITC pulse from the total pulse generated
by the FL2 detector (PE + FITC interference) you can obtain a PE only pulse.

Figure 3.2 A close up view of FITC and PE Fluorescence overlap ( From Operation Principles, Beckman Coulter
Corp.)

Figure 3.2 above helps illustrate why you can expect a significantly greater amount of FITC spectral spill-over into the FL2 detector used for PE fluorescence. Based on the emission spectra of
FITC it can be predicted that the percentage subtraction of FL2 FL1 is going to be significantly
higher than the FL1 - FL2. For example, if you set the FL2-FL1 subtraction at 40%, this amount
would be subtracted from the pulses corresponding to the total light detected on FL2.
The best way (and probably most accurate method ) for determining the fluorescence
compensation requires running samples that are individually stained with the antibody-fluorochrome components of your multi-color samples.
Setting fluorescence Compensation. The Percentage (%) compensation/subtraction is adjusted
while observing a display of data being run on a 2 color histogram(2 parameter histogram displayed by your Protocol). In the example below a CD8 FITC single stain is displayed. You make
the compensation adjustments while the sample is running.
The illustration below shows a CD8 FITC single stain displayed on a 2 parameter histogram consisting of FL1-FITC on the x axis and FL2-PE on y axis. In this example the FL2 parameter is not
compensated or it is under compensated (since the sample was only stained with a FITC
conjugated antibody, there should not be any FL2 positive cells).

Compensation is correct when an


imaginary line drawn across the top
of the negatives and positives is
level.Notice how the Quatstats
Regions are being used in this
example. They are quite useful markers for determining if your
compensation settings are correct.

The color compensation box shows an


entry of 12.0% in the FL2 - FL1 slot.
This entry has brought the CD8-FITC
population out of the FL2 parameter.
Also, the mean channel for the Y axis
in quadrant 3 and for are very similar.

You can use the statistics generated by the Quatstat regions to help determine if your
compensation settings are correct. Set the position of Quadstat regions so that you separate
your FITC positives from your negatives. Look at the Mean peak position corresponding to the
appropriate region for the parameter you are compensating. They should be the same.
After you have determined the correct compensation setting FL2 (PE) using your FITC stained
sample repeat the same procedure using your PE stained sample. This is illustrated below. In
this instance you will be using the FL1-%FL2 setting running a PE(FL2 parameter) only stained
sample.

Summary of Guidelines to Follow with Multi-Color Flow Cytometry


1.

As a general rule use 1ugram of your antibody per 106 cells.

2.

Use a negative control to set your High Voltages (HV) per detector. As a rule of
thumb set the High Voltages so that your negatives are in the 1st decade of the
parameters being collected. Lower high voltage settings will also result in lower com
pensation settings.

3.

Run individually stained samples to adjust your fluorescence compensation.

4.

By examining figure 3.1 you can make a number of predictions concerning spectral
overlap.

5.

Compensation settings stored in your Protocol will apply only to samples stained with the
same antibodies/same fluorochromes panels. For example, if you calculated your
compensations for a CD4-FITC/CD8-PE/TCR-TC multicolor stain these settings would
not necessarily be correct if you change to a CD8-FITC/CD4-PE/TCR-TC.

6.

In practice, however, compensation settings dont usually vary too much, specially within a
particular cell type.

7.

The goal is to achieve optimum compensation %s settings. This is done by adjusting


your high voltage settings for your PMTs using negative controls, followed by your
individual stained samples for setting your compensations. Once these settings have
been determined, it should not be necessary to make re-adjustments.