Channel Calibration

Axis scales for histograms and 2D plots traditionally have been displayed on arbitrary scales: either a channel number or a logarithmic intensity scale. For some applications, this is inadequate since the investigator needs to know the absolute number of antigen molecules or binding sites on the surface of the cell. Several companies have developed beads of different intensities for which the precise numbers of dye molecules are known. By correlating the intensity level in terms of channel number to the number of dye molecules, it is possible to specify the results in terms of molecules of antigen, antibody, or whatever you wish.

This type of calibration has  been very labor intensive: using your flow cytometry software to obtain a set of statistics and plugging those statistics into a spreadsheet to obtain a calibration curve.  The results were obtained as standard channel values from the flow cytometry software and then transformed using the spreadsheet into number of binding sites. FCS Express automates the entire process for you. Once you have set up a channel calibration, all plots will automatically display the calibrated axes and statistics will be displayed as calibrated values as demonstrated in the clip below.  The channel calibration is applied and removed to illustrate the changing median fluorescent intensity (MFI) for the gated population defined by the marker.



Uncalibrated vs. Calibrated

In the example below, two plots are displaying the same data file.  The left plot is uncalibrated, while the right plot is calibrated.  Although both have identical marker positions to gate the same events for the sample, the median fluorescence intenstity (MFI) statistic differs between the two.  The left plot is reporting a relative measurement, while the right plot is reporting an absolute measurement based upon calibration created using multipeak data and related values in the channel calibration tool.  It also is notable the plots' scales differ along the x-axis for this fluorochrome due to the calibration being applied or not.

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Manual and Tutorials

Channel Calibration in the FCS Express Manual

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