Compensation is not dependent on intensity, BUT the accuracy at which you determine compensation is dependent on intensity. The brighter the control the more accurately compensation is determined.

1. Compensation Issue:
Bright vs. Dim Controls
Rich Hastings
KUMC Flow Cytometry Core Lab
3901 Rainbow Boulevard
Kansas City, KS 66160
913-588-0627
rhastings@kumc.edu
http://www.kumc.edu/flow/

2. Compensation
Compensation/Spectral Overlap
 Most fluorochromes and dyes excited by laser light have long emission curves.
 Flow cytometers have filter sets optimized for specific wavelengths of light.
 Unfortunately, overlapping emission wavelengths from one fluorochrome may
spillover into the filter of another.
 This spillover can be corrected mathematically by a process called compensation.
 Once fluorescence compensation is set for a sample, the compensation values
remain applicable for successive dim or bright samples, because compensation
subtracts a percentage of the fluorescence intensity.

3. Compensation
 Misconceptions:
 A compensation value > 100% is a problem, no it’s not. It is what it is!
 Compensation is not dependent on brightness, compensation is dependent on
the emission spectra of a fluorochrome.*
 Same experiment on same instrument, does compensation becomes
unnecessary?
1. How long were your samples exposed to light or fixative?
2. How careful were you in the set up of your stained cells and the
instrument? Does your instrument drift?
3. Check your compensation matrices over time and apply different
compensation values to different experiment days. Trust but verify!
 Beads and cells cannot be both used to generate compensation values. Sure
they can!
 Compensation introduces errors. Not true, but the data may spread .
Slide adapted from Mario Roederer (Current Protocols in Cytometry)

4. Compensation
 Compensation is and is not dependent on brightness. Why the
Ambivalence?
 Possible to get the proper compensation value using a dim compensation
control.
 Compensation is just much more accurate using bright controls.
 Digital Compensation is dependent on complex matrix algebra.
 This matrix algebra is the simultaneous solution of the equations for the
contributions of the spectral overlaps of each of the colors into every detector.
(BD)
 First Error: Photoelectron counting statistics dictate that the variance of a dim
or “negative” signal will be higher than that of a positive signal. (Howard Shapiro)
 Second Error: The loss of information when a signal is converted to an integer
is called digital error. (Bruce Bagwell)

5. Compensation
Bagwell CB, Adams
EG. Ann NY Acad Sci.
1993;677:167-184.

6. Compensation
Bruce Bagwell Verity Software House

7. Compensation
How does this effect me and my experiment?

8. Compensation
 Experiment: eBioscience OneComp eBeads Unlabeled Beads

9. Compensation
 Experiment: eBioscience OneComp eBeads APC Beads
Bright APC
Comp Control
Dim APC
Comp Control

10. Compensation

11. Compensation
 Experiment: eBioscience OneComp eBeads APC-H7 Beads

12. Compensation
 Experiment: eBioscience OneComp eBeads APC-H7 Beads
Compensation Matrix with Bright CD309 APCCompensation Matrix with Dim CD15 APC

13. Compensation
Compensation Matrix with Bright CD309 APC
APC-Cy7 - %APC = 46.65
CD309 APC Perfectly
Compensated
CD15 APC
Perfectly
Compensated

14. Compensation
Compensation Matrix with Dim CD15 APC
APC-Cy7 - %APC = 85.38
CD309 APC Over-
Compensated
CD15 APC
Perfectly
Compensated

15. Compensation
 I clearly manipulated this experiment  very bright and very dim APC
controls.
 Very possible to get accurate compensation with a dim control, just more
likely with a bright control.
 If your dim control is your only compensation control for a specific color
and is the only version of that color in your experiment  proper
compensation.
 Instrument counting errors and the conversion to digital may create errors
in compensation.
 Know your data, what should the data look like?

16. Compensation
http://ucflow.blogspot.com/Ryan Duggan, University of Chicago