101 Flow Cytometry Compensation

What is Compensation?

Flow cytometry is a powerful technique used to measure the characteristics of cells and particles in suspension.
One of the most important aspects of flow cytometry is the ability to accurately measure multiple fluorescence signals on a single cell or particle.
This can be challenging due to the overlap in fluorescence emission that can occur whenever multiple fluorochromes are being used.
This process can be difficult and time consuming particularly in today’s multi-colour environments.

Why is Compensation Important?

If you have two or more fluorochromes you will often have some amount of overlap, or spillover, which increases in complexity as more fluorochromes are added.
Compensation is required as the fluorochrome from a target channel is also picked up by other detectors in the system. For example, an amount of the green fluorochrome signal may be detected in the detectors intended for yellow or red fluorescence.
Fluorescence compensation is a technique used to correct this overlap, allowing for more accurate measurements.
Compensation is essentially the process of mathematically correcting for the overlap. It involves calculating the overlap between detectors for each fluorochrome then adjusting the signal from any affected detectors to account for the rogue signal from another dye.
VenturiOne software includes a simple method for automatically calculating compensation coefficients for all your fluorochrome combinations. Compensation settings can be saved and reloaded at any point.

What ways can we compensate flow cytometry data?

Which method is used depends on the type of flow cytometer and the type of data being collected. It is important to choose the right compensation method, as incorrect compensation can lead to inaccurate measurements.

Manual Correction of Compensation

Manual adjustment of compensation coefficients, where compensation is corrected by eye, which can be done if you already know the samples well and know where the populations should be. This subjective method is far from ideal as results obtained will vary between operators.
As the numbers of fluorochromes increase this method becomes even less suitable.

Calculated Compensation Matrix

To accurately calculate compensation, you will need to run control samples, where you stain cells with a known fluorescent dye or beads with a singular dye and see how they perform.
You can then measure the signal from each fluorochrome in each detector to calculate interference and know what compensation is required.
In this way the single colour controls are used to build up a compensation matrix which details all the compensation coefficients required.
When using flow cytometry data analysis software, these numbers will be calculated for you.
In VenturiOne, we have tools to do both manual compensation and build a compensation matrix, using our compensation wizard tool.

Compensation in VenturiOne

The Compensation tab contains tools for viewing and modifying the compensation applied to the currently open FCS file and for computing compensation values.
VenturiOne allows you to obtain the correct compensation for a file in a number of ways.
Compensation values can be loaded from the FCS file header values (if the values were stored with the file). By default, the software will automatically load compensation values from the file header.
You can also calculate the compensation within the VenturiOne software using the Compensation Wizard and save these calculated values for future use.
The status of the compensation settings of the current file is displayed in the status bar.
Using the Hyperlog display in VenturiOne allows you to see the full range of data clearly and identify under or over-compensated data.
Hyperlog sliders enable the easy optimisation of the Hyperlog plots.
The quadrant tool also allows you to see where populations should be which allows you to optimally set manual compensation values.

Try a 30 day free trial of VenturiOne today, and see how its compensation tools can help you.








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