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A software method for color compensation.

Carleton C Stewart¹, Sigrid J Stewart

¹Roswell Park Cancer Institute, Buffalo, New York, USA.

Current Protocols in Cytometry

|September 5, 2008

Summary

This summary is machine-generated.

Spectral compensation is crucial for accurate multi-fluorochrome analysis. Software compensation offers flexibility over instrument compensation, especially with increasing fluorochromes, but has potential algorithm and binning issues.

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Area of Science:

Immunology
Cell Biology
Biotechnology

Background:

Simultaneous measurement of multiple fluorochromes in flow cytometry leads to signal overlap.
Spectral compensation corrects for this undesired signal overlap.
Instrument-based compensation becomes inadequate with increasing numbers of fluorochromes.

Purpose of the Study:

To explore the advantages and limitations of software-based spectral compensation.
To provide protocols for software compensation using matrix algebra.
To demonstrate principles applicable to any number of fluorochromes.

Main Methods:

Data collected uncompensated.
Software compensation algorithms applied.
Matrix algebra utilized for compensation calculations.
Protocols illustrated for up to four colors.

Main Results:

Software compensation provides flexibility for accurate correction with multiple fluorochromes.
Potential inaccuracies arise from software linearization assumptions.
Binning effects can be problematic with more compensated parameters.

Conclusions:

Software compensation is a flexible alternative to instrument compensation for multi-fluorochrome analysis.
Understanding algorithm limitations and binning effects is essential for accurate results.
The principles of software compensation are scalable to higher numbers of fluorochromes.