Compensation (cytometry)

Compensation (cytometry) is a critical process in Flow Cytometry that corrects for the overlap of fluorescence signals. This overlap, also known as spectral overlap, occurs when the emission spectra of two or more fluorochromes used in a flow cytometry experiment overlap, causing a false positive signal. Compensation is the mathematical process used to correct for this spectral overlap.

Overview

In flow cytometry, cells or particles are labeled with fluorescent markers, known as fluorochromes. Each fluorochrome emits light at a specific wavelength when excited by a laser. However, the emission spectra of different fluorochromes can overlap, leading to a phenomenon known as spectral overlap or spillover. This can result in a false positive signal, where a cell or particle appears to be labeled with a fluorochrome when it is not.

Compensation is the process used to correct for spectral overlap. It involves the use of single-stained controls, which are samples that are stained with only one fluorochrome. The signal from these controls is used to calculate the amount of spectral overlap between different fluorochromes, and this information is then used to adjust the signals from the experimental samples.

Process

The process of compensation involves several steps:

1. Preparation of single-stained controls: These are samples that are stained with only one fluorochrome. They are used to calculate the amount of spectral overlap between different fluorochromes.
2. Measurement of spectral overlap: The single-stained controls are run through the flow cytometer, and the amount of spectral overlap is measured.
3. Calculation of compensation values: The amount of spectral overlap is used to calculate the compensation values. These are the values that will be used to adjust the signals from the experimental samples.
4. Application of compensation values: The compensation values are applied to the signals from the experimental samples, correcting for the spectral overlap.

Importance

Compensation is a critical step in flow cytometry experiments. Without proper compensation, the results of a flow cytometry experiment can be misleading, leading to incorrect conclusions. Therefore, it is essential to perform compensation correctly and to check the compensation settings regularly.

See also

• Flow Cytometry
• Fluorescence
• Fluorochrome

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