Phasor Analysis for FLIM (Fluorescence Lifetime Imaging Microscopy)

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.

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.

2. Multi-exponential lifetimes are found inside the universal circle and are a linear combination of their single exponential lifetime components.

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 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).