spechronImage Analysis tools For SHARING

Time Gated Phasor Example 1

To start download the image from the link below :

After selecting the Time gatedphasor from plugins menu, a dialogue window will appear.
In “Threshold” text box as the name imply you can enter the value for thresholding the pixels to remove them from analysis.
In the “Total window” you can enter the total detection time which by default is set to 8ns which is the detection window for LIMO.
“Background” is the value to be subtracted from the pixels value.
“Phasor Dimension” is to choose the initial dimension for the phasor plot. Smaller plot you choose, denser points you get and more contrast in color density.
To start with the plugin we need to do some calibrations. It is for taking into account the instrument response effect. This can be done by any sample with known single exponential decay. You can Enter the lifetime of your reference sample in“reference Lifetime” textbox.
in the drop down list “Pixel by Pixel” box allows you to estimate the average lifetime in each pixel and the "Average Lifetimne” is to find the average lifetime of the whole image. The Global two components option allows you to estimate the lifetimes and fractional intensities of a system with two components. For motre information see here
For calibration open the Green.tif image and then click the Timegatedphasor from Plugins menu. Choose the “Calibration” and in the Reference lifetime text box enter “4.66” and then press the OK button.





















Now run the Timegatedphasor from plugins menu and this time choose Pixel by pixel and press OK. The lifetime image and the phasor plot will be shown. The circular shape of the phasor cloud denotes the mono-exponential behavior for the measured decay. Moving the mouse pointer over the image will show the lifetime on the ImageJ toolbar in ns.





























Now open the “Alexa 488-555.tif” file and select the Timegatedphasor from plugins menu.In Threshold textbox enter 200 and choose Pixel by Pixel and press OK.Again the Phasor plot and the lifetime image will shown. You can choose from Look up table menu different color schemes to better visualize the result.



























You can also do some basic segmentation based on reciprocal transformation. Choose the OvalOval from ImageJ toolbar and make a region of interest as shown in figure below. From PlugIn menu choose Phasor to Image to map the pixels which are correlated with the region of interest that you made.



























The selected region of interest corresponds to pixels with longer lifetimes.