Multifocal lipid membrane characterization by combination of DAS-deconvolution and anisotropy

Three analog solvatochromic probes, Laurdan, Prodan, and Acdan, are extensively used in the study of biological sciences. Their locations in lipid membranes vary greatly in depth, and their fluorescence responds to their surrounding environment based on their corresponding locations in the membrane. Utilizing the fluorescence lifetimes (τ) and emission peak positions (λ) acquired from the time-resolved emission spectrum, one can effectively determine the local lipid environment using the analytical approach, referred to as τ and λ plots. Herein, a τ and λ plot was created using the aforementioned probes to expand the analytical field according to their location. Furthermore, the solvent modeling method in the τ and λ plot was upgraded to artificially emulate the complex environment in lipid membranes by utilizing liquid paraffin and glycerol to assess the contribution of viscosity to each fluorescence distribution. According to the results from a series of solvent mixtures, the effect of solvent viscosity on lifetime values was confirmed in the short lifetime region (τ < 3 ns). However, it was impossible to emulate the longer than 4 ns lifetime values observed in lipid membranes containing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine in the range of viscosity applied in this study. From the insight of the limiting anisotropy (r_∞), the τ and λ plot was divided into a solvent-like region with an isotropic environment (r_∞ < 0.15) and a region highly ordered enough to define it as an anisotropic environment (0.15 < r_∞) at τ = 4 ns. Also, the membrane-specific distribution was illustrated as 4 ns < τ and λ < 460 nm from this work. An updated analytical model was created to visualize multiple fluorescence components of each probe in six types of lipid bilayers, confirming the different distributions between these probes. Our results well illustrate the multiplicity of lipid environments modeled with solvent and ordered environments in each lipid bilayer system.