![Phasor Analysis: Separation performed using FLIM phasors](/fileadmin/academy/2019/Phasor_Analysis_for_FLIM/Phasor_Analysis_Separation_with_FLIM_phasors.png)
How is the phasor plot generated?
When the data is acquired with Time Resolved Single Photon Counting (TCSPC) systems, the phasor FLIM distributions are derived from a Fourier transform (Digman et al., 2008). Each pixel in the image corresponds to a point in the phasor plot.
![Phasor Analysis: Relation between FLIM image and phasor plot](/fileadmin/academy/2019/Phasor_Analysis_for_FLIM/Phasor_Analysis_Relation_FLIM_image_and_phasor_plot.png)
![Phasor Analysis: Phasor plot with universal circle](/fileadmin/_processed_/a/9/csm_Phasor_Analysis_Phasor_plot_with_universal_circle_bbc119d45a.png)
Rules: the algebra of phasors
There are only a few rules in the phasor approach and they enable a straightforward interpretation of the lifetime distribution. Three of the most important ones are shown below (E. Gratton, 2018).
1. Single exponential lifetimes lie on the universal circle line.
![Phasor Analysis: Universal circle; Monoexponential lifetime](/fileadmin/academy/2019/Phasor_Analysis_for_FLIM/Phasor_Analysis_Universal_circle_Monoexponential_lifetime.png)
2. Multi-exponential lifetimes are found inside the universal circle and are a linear combination of their single exponential lifetime components.
![Phasor Analysis: Universal circle; Multiexponential lifetime](/fileadmin/academy/2019/Phasor_Analysis_for_FLIM/Phasor_Analysis_Universal_circle_Multiexponential_lifetime.png)
3. The ratio of the linear combination for a multi-exponential lifetime (experimentally measured) determines the fraction of the components (f1 and f2) from their two single exponential lifetimes (τ1 and τ2).
![Phasor Analysis: Universal circle; Experimental point](/fileadmin/academy/2019/Phasor_Analysis_for_FLIM/Phasor_Analysis_Universal_circle_Experimental_point.png)
Advantages of using phasors
Phasor FLIM is a very powerful analysis tool for molecular species separation and FRET analysis, in particular when the donor has a multi-exponential lifetime, something which is typical of CFP [cyan fluorescent protein] (Caiolfa et al., 2007). Moreover, phasor FLIM combined with STED, allows you to use less STED power to reach the same resolution (Lanzanò et al., 2015).
![Phasor Analysis: Gattabeads imaged with STED and FLIM](/fileadmin/_processed_/4/a/csm_Phasor_Analysis_Gattabeads_imaged_with_STED_and_FLIM_e66c2de1e0.png)
Left: Gattabeads imaged with SP8 STED system having FLIM capability. Right: By highlighting the long lifetime pixels in the phasor plot, the resolution of the STED image is improved.