Spectral Phasor

Phasor approach is powerful technique for visualization and analysis of lifetime and spectral images. The spectra are mapped into a 2D plot according to their emission maximum and spectral widths. Unmixing, segmentation and visualisation becomes easier.


Spectral Phasor Example 1

This example shows the method for segmentation based on reciprocal transformation and unmixing of three components. To start download the image from the link below :
This is an image recorded with a spectrograph with 128 spectral channels.This is a cell stained with DAPI (DNA), Bodipy (tubulin) and Texas Red (actin). The description about the sample can be found here.
From Image menu choose Stacks> Z Project...
Then from Projection Type choose Average Intensity.
The Image should looks like this:

Now select the stack image again and from Plugins menu select Spectral phasor.

In Threshold text box, as the name imply you can enter the value for thresholding the pixels to remove them from analysis.
By entering large values we make sure that we have removed the background signal.
Background is the value to be subtracted from the pixels value.
Now press the OK button.

Three distinct areas are observed in the phasor plot.The central cloud corresponds to the background signal and the rest are originated from the cell. Basic segmentation based on phasor cloud position can be performed. First we start from the central cloud.
From ImageJ toolbar select the “Oval” and select a region of interest on the phasor plot as shown in the figure below.
Then fromPlugins menu select the Phasor To Image. The corresponding pixels and the spectrum will be shown.This confirm the position dependency of phasor plot on the spectrum.

Next choose the region as shown in the figure; This belongs to signal from DAPI which originates from nucleus :


Farzad Fereidouni, Arjen N. Bader, and Hans C. Gerritsen,
“Spectral phasor analysis allows rapid and reliable unmixing of fluorescence microscopy spectral images," Opt. Express 20, 12729-12741 (2012)



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