Traditional dot plots are replaced with spectrum plots when the 'Spectral' box is checked. To switch to spectral compensation, simply check the 'Spectral' box as shown in Figure 2.ДЇigure 2: Spectral compensation in FlowJo. A normal compensation matrix will be calculated from these selections.

Similar to standard compensation, for each compensation control a gated population should be specified as either positive (events stained with the fluorochrome) or negative (unstained control, or internal negative). To select samples for use within the wizard, you can group samples then select the group, highlight samples in the samples pane before selecting the wizard, or drag and drop them from the workspace into the left side pane of the compensation wizard window. Spectral compensation is accessed from the same compensation wizard that calculates traditional compensation matrices, as shown in Figure 1.ДЇigure 1: Initiating spectral compensation through the compensation wizard Standard compensation is no different, except that the number of detectors equals the number of fluorochromes. Using these exemplars, unmixing can determine what portion of collected 'mixed' data was contributed by each fluorochrome. As with standard compensation, single stained compensation controls are utilized as exemplars of each individual fluorochrome spectrum, along with an unstained control or internal negative population for background autofluorescence. The basic premise is that the emission spectrum of each fluorochrome is measured in multiple detectors, using data from all available detectors on the acquisition cytometer, producing as complete a picture of the emission spectrum as possible. Spectral compensation can be calculated and applied to any cytometric data set that contains more detectors than fluorescent probes.

Spectral compensation is a process in which fluorescence emissions are collected holistically as spectra compensation is performed by unmixing the spectrum of each fluorescent probe from the total signal using linear least squares to solve an over-determined system of equations.

Flowjo 10 compensation