Issue 27, 2024

Sensing cholesterol-induced rigidity in model membranes with time-resolved fluorescence spectroscopy and microscopy

Abstract

Here, we report the characterization of cholesterol levels on membrane fluidity with a twisted intramolecular charge transfer (TICT) membrane dye, namely DI-8-ANEPPS, using fluorescence lifetime techniques such as time-correlated single photon counting (TCSPC) and fluorescence lifetime imaging microscopy (FLIM). The characterized liposomes comprised a 3 : 1 ratio of POPC and POPG, respectively, 1% DI-8-ANEPPS, and increasing cholesterol levels from 0% to 50%. Fluorescence lifetime characterization revealed that increasing the cholesterol levels from 0% to 50% increases the fluorescence lifetime of DI-8-ANEPPS from 2.36 ns to 3.65 ns, a 55% increment. Such lengthening in the fluorescence lifetime is concomitant with reduced Stokes shifts and higher quantum yield, revealing that localized excitation (LE) dominates over TICT states with increased cholesterol levels. Fluorescence anisotropy measurements revealed a less isotropic environment in the membrane upon increasing cholesterol levels, suggesting a shift from liquid-disorder (Lα) to liquid-order (LO) upon adding cholesterol. Local electrostatic and dipole characterization experiments revealed that changes in the zeta-potential (ζ-potential) and transmembrane dipole potential (Ψd) induced by changes in cholesterol levels or the POPC : POPG ratio play a minimal role in the fluorescence lifetime outcome of DI-8-ANEPPS. Instead, these results indicate that the cholesterol's effect in restricting the degree of movement of DI-8-ANEPPS dominates its photophysics over the cholesterol effect on the local dipole strength. We envision that time-resolved spectroscopy and microscopy, coupled with TICT dyes, could be a convenient tool in exploring the complex interplay between membrane lipids, sterols, and proteins and provide novel insights into membrane fluidity, organization, and function.

Graphical abstract: Sensing cholesterol-induced rigidity in model membranes with time-resolved fluorescence spectroscopy and microscopy

Supplementary files

Article information

Article type
Communication
Submitted
22 Apr 2024
Accepted
15 Jun 2024
First published
17 Jun 2024

J. Mater. Chem. B, 2024,12, 6570-6576

Sensing cholesterol-induced rigidity in model membranes with time-resolved fluorescence spectroscopy and microscopy

B. Biswas, D. Shah, S. J. Cox-Vázquez and R. J. Vázquez, J. Mater. Chem. B, 2024, 12, 6570 DOI: 10.1039/D4TB00872C

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