Multi-focal lipid membrane characterization by combination of DAS-deconvolution and anisotropy: Novel insight of fluorescence analysis

Abstract

Three analogue 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 peaks (λ) acquired from time-resolved emission spectrum, one can effectively determine the local lipid environment using the analytical approach refer to as τ and λ plots. Herein, 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 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 (∼ 3 ns). However, it was impossible to emulate the long lifetime values (4 ns ∼) observed in lipid membranes containing 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) 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 isotropic environment (r∞ < 0.15) and a highly ordered region enough to define as 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. Updated analytical model was created to visualize multiple fluorescence components of each probe in 6 types of lipid bilayers, confirming the different distributions between these probes. Our results well-illustrated the multiplicity of lipid environments modeled with solvent and ordered environments in each lipid bilayer systems.