FlowJo allows you to calibrate any collected parameter. This gives you the ability to express any parameter's values (graphical display, any statistical calculations) in the units of your choice. Most commonly, this is used to convert the scaling into absolute number of molecules (given a standard that converts between the fluorescence intensity collected on your instrument and absolute numbers of fluorophores).
A Calibration Standard is FlowJo's internal representation of the conversion of fluorescence to absolute units.
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When you select Calibration under the Tools menu, you will be presented with the Define New Calibration Standard dialog window (shown right). It asks you to define which parameter you want to calibrate. Once created, the calibrated parameter becomes available from the Graph Window and is saved with the Workspace.
If you want to apply a Calibration Standard to another sample or group (after defining it), click on that sample or group and then select the calibrated parameter from the Tools menu.
For detailed instructions on creating a new Calibration Standard, click on this link.
For an example of bead calibration, click here. This uses an 8-bead set, and shows you how FlowJo creates different kinds of regressions depending on what options you choose.
For an example of CD4 calibration, click here. This uses CD4 expression as a reference standard, and then shows you how graphs and statistics are computed based on the calibrated parameter.
More on Calibration:
Calibrate your samples to a reference when you have collected data on a standard sample which you can use to convert one unit of measure to another (for example, a commercial bead set where each bead has a defined absolute intensity mapped to a known unit value; or, a sample stained with an antibody that has a known binding capacity to the cells). When you use a reference, FlowJo automatically finds all of the peaks in the fluorescence, and asks you to enter any or all of the calibration values for those peaks. FlowJo then performs a linear (or logarithmic) least squares regression to determine the best calibration value, and creates the calibrated parameter.
When you graph your data, select the calibrated parameter on the axis of choice--the scale values on the axis are now drawn in your calibrated units! For example, if you calibrate to absolute number of molecules per cell, then you can display these numbers on the axis. In addition, any statistics you calculate (medians, CVs, etc.) will be expressed in units of absolute molecules. Finally, you can even enter a gate based on those units: you could, for example, specify a gate on all cells with more than 5,000 molecules of an antigen.
A Calibrated Parameter is one type of Derived Parameter. For more information on derived parameters, click here.
Download a Calibration Workspace with Demo Data to try out this platform.
