Differences between FLIM phasor analyses for data collected with the Becker and Hickl SPC830 card and with the FLIMbox card.

The phasor approach to FLIM (Fluorescence Lifetime Imaging Microscopy) is becoming popular due to the powerful fit free analysis and the visualization of the decay at each point in images of cells and tissues. However, although several implementation of the method are offered by manufactures of FLIM accessories for microscopes, the details of the conversion of the decay to phasors at each point in an image requires some consideration. Here, we show that if the decay is not properly acquired, the apparently simple phasor transformation can provide incorrect phasor plots and the results may be misinterpreted. In particular, we show the disagreement in experimental data acquired on the same samples using the two cards (FLIMbox, frequency domain and Becker & Hickl BH 830, time domain) and the effect produced by using the BH 830 card with different settings. This difference in data acquisition translates to the assignment of phasor components calculated using different acquisition parameters. This effect is already present in the original data that are not acquired with the proper parameters for the phasor conversion. We also show that the difference in the resolution of components already exists in the data acquired in the time domain when used with settings that do not allow acquisition of the fluorescence decay on a sufficient large time scale. RESEARCH HIGHLIGHTS: This paper is intended to made researchers aware of some simple requirements for the conversion of time-domain data (typically TCSPC) to phasors. The use of phasors for FLIM analysis has seen a surge of popularity. Since the phasor approach is a fit free method and has a powerful visualization of the data, it appears very simple to use. This paper shows that when the original data in the time domain is not acquired with the proper time range to cover the lifetimes in a sample, the conversion to phasors can produce very erroneous results. These results are appearing more frequently in the literature since many of the manufacturers of FLIM accessories for microscopes are now offering the phasor analysis in their software. Here, we show that the phasor transformation per se cannot correct for the problems with data acquisition and that one is misled to think that the "phasor approach" is a universal fix for the lack of the proper time range for data acquisition.