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A Software Method for Color Compensation
Publication Name:
Current Protocols in Cytometry
Unit Number:
Unit 10.15
DOI:
10.1002/0471142956.cy1015s23
Online Posting Date:
February, 2003 Abstract
When two or more fluorochromes are measured simultaneously, every detector sees some fluorescence from every fluorochrome. Spectral compensation is the process of removing the undesired overlap of signal. Although very successful for two and three fluorochromes, the general practice of adjusting instrument compensation becomes increasingly inadequate and unforgiving as the number of fluorochromes increases. When data are collected uncompensated, software compensation provides the flexibility of setting correct compensation every time for every sample. Software methods do have problems. The linearization assumptions made by the software algorithms may be more or less in error. Binning effects become more of a problem with increasing numbers of compensated parameters. This explanatory unit also contains protocols that illustrate the process of software compensation utilizing matrix algebra that provides for elements of all possible PMT detection combinations. Although details are limited to four colors, the principles described can be applied to any desired number. When two or more fluorochromes are measured simultaneously, every detector sees some fluorescence from every fluorochrome.
Keywords: flow cytometry; spectral compensation; software compensation; tandem dyes
Table of Contents
- Unit Introduction
- Acquisition of Data from Compensation Standards
- Correction of Compensation for all Nontandem Fluorochromes
- Verification of Compensation
- Generate Compensation Matrices for each Antibody Combination
- Data from Specimens
- Using a Macro
- Literature Cited
- Figures
Figures
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Figure 10.15.1Projection of uncompensated data. For clarity, only three bivariate histograms are shown and parameters without any antibody of that color are designated by the parameter name. Otherwise the antibody name and fluorochrome are used. -
Figure 10.15.2Overcompensation. For clarity, only two bivariate plots are shown and parameters without any antibody of that color are designated by the parameter name. Otherwise, the antibody name and fluorochrome are used. -
Figure 10.15.3Correcting overcompensation. Overcompensation can also result in the apparent loss of expression of an eptitope. This is exacerbated when very bright staining is encountered. -
Figure 10.15.4Equal fluorescence detected by two detectors. -
Figure 10.15.5Setting up the instrument. Human leukocytes were used and a region R1 drawn around lymphocytes, shown in the left view, was used as a gate. Parameters without any antibody of that color are designated by the parameter name. Otherwise, the antibody name and fluorochrome are used. The top row shows unstained cells used for adjusting the high-voltage settings. Row 2 is uncompensated CD45-FITC in FL1, row 3 is uncompensated CD4-PE in FL2, row 4 is uncompensated CD2-PE-Cy5 in FL3, and row 5 is uncompensated CD45-APC in FL4. The software will automatically compensate each parameter. Refer to the software help menu if additional information is required. -
Figure 10.15.6Software compensation. The software has automatically compensated the uncompensated data shown in Figure -
Figure 10.15.7Compensation toolbox. -
Figure 10.15.8Verifying compensation. The compensation matrix single.cmp has been applied and All Trace Lines is selected in the dialog box. In the top row, FL3-%FL2 and FL3-%FL4 may require adjustment, shown as heavy trace lines. The trace lines for the other views, shown as light lines, do not require adjustment and the trace lines A and B are correctly set for CD8-PE-Cy5. These lines are reset here to zero so that basic.cmp can be applied to other tandem fluorochromes for their unique adjustment. A trace line parallel to an axis means there is no software compensation for that parameter combination. -
Figure 10.15.9Unique compensation matrix for each tandem. The listmode file of monocytes (gated by moving R1 from the lymphocyte to the moncyte cluster shown in Figure
Literature Cited
| Literature Cited | |
| Loken, M.R., Parks, D.R., and Herzenberg, L.A. 1977. Two-color immunofluorescence using a fluorescence-activated cell sorter. J. Histochem. Cytochem 25:899-907. | |
| Shapiro, H.M. 1995. Practical Flow Cytometry. 3 | |
| Stewart, C.C. and Mayers, G.L. 2000. Kinetics of antibody binding to cells. In Immunophenotyping, (C.C. Stewart and J. Nicholson, eds.) pp 1-21. J. Wiley & Sons, Inc., New York. | |
| Stewart, C.C. and Stewart, S.J. 1999. Four color compensation. Comm. Clin. Cytometry 38:161-175. | |
| Stewart, C.C. and Stewart, S.J. 2001a. Cell preparation for the identification of leukocytes. In Methods in Cell Biology, vol. 64 (Z. Darzynkiewicz, H. Crissman, and J.P. Robinson, eds.) pp. 218-270. Academic Press, New York. | |
| Stewart, C.C. and Stewart, S.J. 2001b. Multiparameter data acquisition and analysis of leukocytes by flow cytometry. In Methods in Cell Biology, vol. 64 (Z. Darzynkiewicz, H. Crissman, and J.P. Robinson, eds.) pp. 289-312. Academic Press, New York. | |
| van Vugt, M.J., van den Herik-Oudijk, I.E., and van de Winkle, J.G. 1996. Binding of PE-CY5 conjugates to the human high-affinity receptor for IgG (CD64). Blood 88:2358-2361. | |
| This work was supported by a NIH core grant and NYS DOH. The authors thank David Sheedy and Earl Timm Jr. for their editing and clarifications. | |
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