Even at realistic photon-limited conditions (~5–10 photons/pixel):
• Phasor analysis remains robust and resilient to noise, although harder to quantify in commercial software.
• Fast FLIM gives clean kinetics but depends on setup
• Fitting yields the most amount of information in ideal situations

We also compared Fast FLIM, Fitting and Phasor estimators during osmotic shocks, a direct test of membrane tension.
All track the expected lifetime shifts, but with distinct sensitivities and noise behaviors.

We quantified the shot-noise constant (c), the factor translating photon number (N) to lifetime standard deviation (σ): σ ∝ c N⁻⁰·⁵.
📊 Shot-noise constant results: Leica yields consistent photon efficiency; PicoQuant varies by estimator.

We show the behavior of tau average and tau 1 across a range of packing standards (GUV with known lipid compositions), showing that tau average has less variability than tau 1.

Flipper-TR is now a standard probe for lipid packing and membrane tension, but everyone analyses it differently.
We compared:
• Fast FLIM (mean photon arrival)
• Bi-exponential reconvolution & tail fits
• Phasor analysis
…across photon counts, repetition rates (20–80 MHz), and different set-ups.

We first review what we know about the photophysics of the Flipper-TR probe and how this lipid packing probe can be used as a tension reporter