Two-photon (2P) fluorescence lifetime imaging microscopy (FLIM) is a powerful, quantitative, non-invasive technique used for a wide range of applications in chemistry, biology, biophysics, drug screening, and medicine. Unlike fluorescence-intensity based imaging microscopy, 2P-FLIM provides spatio-temporal mapping of the excited-state fluorescence lifetime per pixel that is sensitive to changes in both molecular structure and surrounding microenvironment of living cells and tissues under different physiological conditions. For practical reasons, a compromise may be made during 2P-FLIM imaging of live cells to reduce cellular phototoxicity, which may undermine some quantitative aspects of this powerful technique. Here, we carried out 2P-FLIM on cleaved and intact mCerulean3-linker-mCitrine construct (or RD) as a model system with two oppositely charged alpha helices in the linker region. We hypothesize that as the ionic strength increases; the Debye ionic screening of the electrostatic interactions in the linker region will decrease the energy transfer efficiency due to the enhanced donor-acceptor distance. To test this hypothesis, we quantified sensitivity of RD using Förster resonance energy transfer (FRET) from the donor (mCerulean3) to the acceptor (mCitrine) as a function of potassium chloride concentration (0–500 mM). The results are compared with single-point, time-resolved 2P-fluorescence measurements, as a benchmark, quantitative approach, on cleaved and intact RD. Our results demonstrate the sensitivity of 2P-FLIM to the signal-to-noise ratio, magic-angle detection, measured vs. computer-generated instrument response function, and fitting modes for protein-protein interaction studies. These studies highlight the need for careful experimental design and FLIM data analysis for reliable FRET analysis and protein-protein interactions studies.
Hanauska et al. (Sun,) studied this question.