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Phasor ratio analysis is a simple way to quantify the respective contribution of two species to a fluorescence signal.
It involves defining two "reference" phasor points (check the Phasor Graph manual page to learn how to define phasor references) and computing the fractional distance of an unknown phasor to these two references.
A phasor ratio equal to 0 corresponds to a sample identical to Reference 1 and a phasor ratio equal to 1 corresponds to a sample identical to Reference 2. More details on the definition of the phasor ratio can be found in the following reference:
Colyer et al., Phasor imaging with a widefield photon-counting detector, Journal of Biomedical Optics 17 (2012) 016008. DOI: 10.1117/1.JBO.17.1.016008
In favorable cases, the standard phasor ratio f1 can be related to the corresponding amplitude fraction a1 of a two-component fluorescence decay fit by the following formula:
where τ1 and τ2 are the reference lifetimes used for the phasor ratio calculation and are assumed to correspond to the fitted lifetime in the 2-component fluorescence decay fitting approach.
The Settings>>Phasor Graph panel has a checkbox (Compute Amplitude Ratio) which can be used to output a1 instead of f1 when computing the phasor ratio.
1. Computation of a single phasor ratio
An example of a typical situation is shown in the figure below.
In this example, two reference phasors have been defined (green and red dot), corresponding to the intersections of the line connecting the first two cursors with the universal semicircle (UC).
The new (calibrated) phasor (blue dot) is located in between these two references.
To compute its phasor ratio, right-click on (or close to) the phasor and select Compute Phasor Ratio:
This opens a Dialog window in which the user is invited to associate an horizontal coordinate (Sample Abscissa) to the computed phasor ratio:
This value is user-defined and could for instance represent the known fraction of one component of a mixture.
The corresponding point is then added to the last plot in the Phasor Ratio Graph of the Phasor Ratio Analysis panel.
Note that it is possible to create new plots in the Phasor Ratio Graph by right-clicking in it and select New Plot. This will create a new empty plot in the graph, to which subsequent phasor ratios will be added.
This feature is useful to group phasor ratios from different data points into different groups (or plots).
2. Computation of a series of phasor ratios in one step
Instead of manually computing the phasor ratios of individual data points, it is possible to select a Phasor Plot in the Phasor Graph and use the right-click menu item Compute Time-Series Phasor Ratios. The abscissa of successive points in the corresponding phasor ratio plot will be 0, 1, etc. and can easily be modified in the Plot Editor accessible via the context menu.
Another way to compute phasor ratios is by using the Time Series Analysis script (see the corresponding manual page for details). When phasor references have been defined, this script automatically computes the phasor ratio (and lifetime) of each data point, and add them to a new plot in the respective graphs.
3. Fitting a phasor ratio series to a line
The Linear Fit right-click shortcut menu can be called by clicking on the desired plot's legend. This will fit a line to the series of data point in this graph and reports the Slope and Intercept parameters of the fit in the Notebook (as well as the Residue and number of data points n used in the fit). Obviously, more than one point is needed for this to be meaningful.
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