Probing the Orientational Distribution of Dyes in Membranes through Multiphoton Microscopy

Abstract

Numerous dyes are available or under development for probing the structural and functional properties of biological membranes. Exogenous chromophores adopt a range of orientations when bound to membranes, which have a drastic effect on their biophysical behavior. Here, we present a method that employs optical anisotropy data from three polarization-imaging techniques to establish the distribution of orientations adopted by molecules in monolayers and bilayers. The resulting probability density functions, which contain the preferred molecular tilt μ and distribution breadth γ, are more informative than an average tilt angle [φ]. We describe a methodology for the extraction of anisotropy data through an image-processing technology that decreases the error in polarization measurements by about a factor of four. We use this technique to compare di-4-ANEPPS and di-8-ANEPPS, both dipolar dyes, using data from polarized 1-photon, 2-photon fluorescence and second-harmonic generation imaging. We find that di-8-ANEPPS has a lower tilt but the same distributional width. We find the distribution of tilts taken by di-4-ANEPPS in two phospholipid membrane models: giant unilamellar vesicles and water-in-oil droplet monolayers. Both models result in similar distribution functions with average tilts of 52° and 47°, respectively.

The orientation of a membrane probe from structural analysis by enhanced Raman scattering

“…They found a polar angle of 52° for di-4-ANEPPS. 13 These measurement results are rather widely varied, but likely for good reasons. One is the longer acyl chains of di-8 compared to di-4.…”

“…28 The orientation reported here from SABERS for di-4-ANEPPS is at a large tilt angle (63°) and closest to the result by nonlinear microscopy (52°) on GUV's. 13 Note that the much smaller angle (14°) from linear dichroism is calibrated to measurements on retinol, which is presumed to be perfectly normal based on measurements on the retinol group in bacteriorhodopsin. 29 It is possible that free retinol in a membrane is at a tilted angle (like all other optical probes studied to date), which would increase the estimated angles of di-4-ANEPPS and di-8-ANEPPS by that method.…”

“…The probe's position and orientation in the membrane, the exact meaning of a probe's signal, how that signal might be affected by membrane phase and composition, and how the probe may alter the inherent membrane properties are important open questions. [10][11][12][13][14] Here we focus on a class of "voltage sensitive dyes" which report on the electric fields inside membranes through changes in their fluorescent properties. 15,16 These dyes are based on aminostyryl pyridinium chromophores with alkane chains at one end and hydrophilic groups at the other end to orient the probe within the membrane.…”

“…[21][22][23][24][25] The molecular orientation of di-8-ANEPPS and di-4-ANEPPS have been studied with polarized fluorescence and second harmonic generation (SHG), as well as linear dichroism on aligned lipid membranes. 13,[26][27][28][29] These measurements give a range of conflicting answer for the probe tilt from the membrane normal. Here we apply a method we recently introduced called structural analysis by enhanced Raman scattering (SABERS) to study the probe orientation.…”

Fluorescence lifetime imaging (FLIM): Basic concepts and some recent developments

“…in the membrane (Benninger et al, 2005b;Reeve et al, 2012), muscle fibres (Brack et al, 2004) or in DNA (Mojzisova et al, 2009). In these cases, neither the depolarization due to Brownian rotational motion nor homo-FRET is measured, but rather the angle between the electric vector of the light exciting the sample, and the transition dipole moment of the static fluorophore, thus yielding its orientation.…”