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.

journal_name

Biophys J

journal_title

Biophysical journal

authors

Reeve JE,Corbett AD,Boczarow I,Wilson T,Bayley H,Anderson HL

doi

10.1016/j.bpj.2012.08.003

subject

Has Abstract

pub_date

2012-09-05 00:00:00

pages

907-17

issue

5

eissn

0006-3495

issn

1542-0086

pii

S0006-3495(12)00861-2

journal_volume

103

pub_type

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