Abstract
The structural diversity of different lipid species within the membrane defines its biophysical properties such as membrane fluidity, phase transition, curvature, charge distribution, and tension. Environment-sensitive probes, which change their spectral properties in response to their surrounding milieu, have greatly contributed to our understanding of such biophysical properties. To realize the full potential of these probes and avoid misinterpretation of their spectral responses, a detailed investigation of their fluorescence characteristics in different environments is necessary. Here, we examined the fluorescence lifetime of two newly developed membrane order probes, NR12S and NR12A, in response to alterations in their environments such as the degree of lipid saturation, cholesterol content, double bond position and configuration, and phospholipid headgroup. As a comparison, we investigated the lifetime sensitivity of the membrane tension probe Flipper in these environments. Applying fluorescence lifetime imaging microscopy (FLIM) in both model membranes and biological membranes, all probes distinguished membrane phases by lifetime but exhibited different lifetime sensitivities to varying membrane biophysical properties (e.g., cholesterol). While the lifetime of Flipper is particularly sensitive to the membrane cholesterol content, the NR12S and NR12A lifetimes are moderately sensitive to both the cholesterol content and lipid acyl chains. Moreover, all of the probes exhibit longer lifetimes at longer emission wavelengths in membranes of any complexity. This emission wavelength dependency results in varying lifetime resolutions at different spectral regions, which are highly relevant for FLIM data acquisition. Our data provide valuable insights on how to perform FLIM with these probes and highlight both their potential and limitations.