Lesson 3: Compensation – Art or Science

You will need to download the following data files (zip archive) in order to complete this exercise. This exercise is again based on Weasel. If you choose to use another software package, you will need to be sure that it can perform post-acquisition compensation.

Spectral Overlap

Research BCRF Flow Cytometry Spectragraph Overlap

Consider the basic detection unit on any flow cytometer. The unit consists of a photodectector (photomultiplier tube or photodiode) coupled with a bandpass filter to allow only select wavelengths the detector. Now consider the emission properties of a typical fluorochrome. Though excited by a discreet wavelength from the excitation source (laser), the fluorochrome will not emit a discreet wavelength of light, but rather a spectrum, often a very broad spectrum. If two fluorochromes are used in an experiment with overlapping emission spectra, it is possible for the light emitted from both flourchromes to be detected, to some degree, in the same detector. The image below shows the emission spectra of the Fitc fluorochrome and the PE fluorochrome. The colored lines indicate the bandpass of the filters for the Fitc detector (530/30) and the PE detector (585/42). These are the specific values for the filters used in the FACSVantage SE cell sorter, but are typical values for any flow cytometer. Look closely at the image and notice that there is very little spectral overlap detectable within the Fitc Filter, but there is a significant amount of Fitc emission falling within the PE bandpass. The result of this will be an artificially elevated PE signal (false positives, inaccurate percentages). In the case of clinical diagnosis, this could prove disastrous if not corrected.

Research BCRF Flow Cytometry Spectral Overlap graph



The method for correcting for this spectral overlap is known as compensation and is really nothing more than a mathematics exercise. Since we can’t separate out the Fitc-originated photons within the 585nm bandpass from the PE-originated ones, we need to estimate the Fitc contribution and subtract it out from the gross detected signal. This is done by subtracting a percentage of the Fitc Signal detected in the Fitc Channel from the signal detected in the PE Channel (PE – %Fitc). To see how this works, open the Weasel application and create the following six plots using file COMPENSATION.001:

  • Fitc vs PE
  • Fitc vs PerCP
  • Fitc vs APC
  • PE vs PerCP
  • PE vs APC
  • PerCP vs APC

Place quad stats on each plot. COMPENSATION.001 is a sample of unstained polystyrene beads. It is used to adjust the detector sensitivity on the cytometer and to allow for the placement of the quad stats.

Once the stats are in place, change each plot to COMPENSATION.002. This file is a sample of negative and fitc-stained beads. Examine the dot plots carefully. What do you notice? Channels other than Fitc are showing positive events. As these are beads, we can say without doubt that these are false positives and need to be corrected for via compensation.

Right-Click in the Fitc vs PE plot and choose Compensate. The Set Spillover (Compensation) dialog opens. Enter a value for the PE -%Fitc correction (see image), and click Apply to All.

Research BCRF Flow Cytometry Compensation data

How does the image change? Continue to adjust the value until there are no more false PE positives (be careful not to overcompensate). The proper compensation level is when the mean PE value of the Fitc-negative population is equal to the Mean PE value of Fitc-Positive population. This can be done by “eyeballing” it (that would be the art) or by creating a stats window and adjusting the compensation value until the mean values for the two quadrants are equal (that would be the science). The method you choose depends on your preferences and how many colors are being used for the analysis (the more colors, the more difficult eyeballing becomes).

Research BRCF Flow Cytometry Training Compensation Scatter Plot
Research BRCF Flow Cytometry Training Compensation Scatter Plot


Wrapping Up

In order to asses your understanding of the material thus far, please email the answers to the following exercise to the address below. After receiving this I will provide you with access to the next module.

Complete the analysis of the data files. Apply compensation as needed to all channels. The COMPENSATION.001-.006 are Blank, Fitc only, PE only, PerCP only, APC only, and all beads respectively.

  1. What would be the benefit of reducing the bandwidth of the bandpass filters? the drawback?
  2. Attach a copy of your completed compensation exercise (COMPENSATION.006 – Fitc vs PE, Fitc vs PerCP, and PerCP vs APC)
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