Fig. 1. Schematic diagram of fluorescence lifetime measurement and phasor analysis (PA):(a) Frequency domain method; (b) lifetime phasor of single-exponential decay; (c) lifetime phasor of bi-exponential decay; (d) time-correlated single photon counting (TCSPC) method.

Fig. 2. Schematic diagram of phasor-FLIM application:(a) Fluorescence intensity image with untreated lifetime information; (b) lifetime phasor plot obtained by PA analysis; (c) direct analysis of lifetime phasors; (d) phasor-mapped FLIM image based on phasor clustering analysis and pseudo-color assignment.

Fig. 3. Application classification diagram of phasor-FLIM.

Fig. 4. Phasor-FLIM was used to analyze the change of NADH/NAD+ ratio under the stimulation of hypoxia and mitochondrial toxic drug potassium cyanide, for studying the change of metabolic state of cells^[27].

Fig. 5. Phasor-FLIM was used in quantitative FRET efficiency detection of a RhoA-kRas single chain biosensor, studying interaction between proteins^[28].

Fig. 6. Phasor-FLIM was used to analyze the changes of pH value of cells in normal state (at rest) and under oxidative stress^[32].

Fig. 7. Phasor-FLIM clustering analysis and pseudo-color assignment was used to enhance visualization of pathological features of basal cell carcinoma (BCC) sections stained with H&E, assisting pathological diagnosis^[36].

Fig. 8. Phasor-FLIM cluster analysis was used to filter out the photons in the annular depletion region in stimulated radiation depletion (STED) imaging, improving the resolution of super-resolution imaging^[39].