Pyrene as a membrane depth gauge: Wavelength selective fluorescence approach to monitor pyrene localizations in the membrane

We take the advantage of pyrene's unique spectral properties as a reliable polarity indicator to monitor pyrene localizations in the membrane depth by using wavelength selective fluorescence approach. We show that fine structure of pyrene fluorescence emission spectra and excimerization rate in model and native phospholipid membranes depend on the excitation wavelength. This phenomenon is not observed in neat solvents. In membranes, the dependence on the excitation wavelength reflects selective excitation of pyrene molecules located close to the membrane–water polar interface, or deep in the hydrophobic core of the membrane, verified with the aid of pyrene derivatives of fatty acids of various lengths.

► Py value and excimerization of pyrene in membranes vary with excitation wavelength.
► Excitation spectra for the emission components at 373, 383 and 470 nm are shifted.
► The wavelength dependence phenomenon is not observed in neat solvents.
► Varying excitation selects pyrene located at different depths in the membrane.
► This conclusion is verified using pyrene derivatives of fatty acids.