Hydration Effects on Membrane Structure Probed by Single Molecule Orientations

Abstract

Single molecule fluorescence measurements are used to probe the structural changes in glass-supported DPPC bilayers as a function of relative humidity (RH). Defocused polarized total internal reflection fluorescence microscopy is employed to determine the three-dimensional orientation of the fluorescent lipid analogue BODIPY-PC, doped into DPPC membranes in trace amounts. Supported DPPC bilayers formed using vesicle fusion and Langmuir-Blodgett/Langmuir-Schäfer (LB/LS) transfer are compared and show similar trends as a function of relative humidity. Population histograms of the emission dipole tilt angle reveal bimodal distributions as observed previously for BODIPY-PC in DPPC. These distributions are dominated by large populations of BODIPY-PC molecules with emission dipoles oriented parallel (≥81°) and normal (≤10°) to the membrane plane, with less than 25% oriented at intermediate tilts. As the relative humidity is increased from 13% to 95%, the population of molecules oriented normal to the surface decreases with a concomitant increase in those oriented parallel to the surface. The close agreement in trends observed for bilayers formed from vesicle fusion and LB/LS transfer supports the assignment of an equivalent surface pressure of 23 mN/m for bilayers formed from vesicle fusion. At each RH condition, a small population of BODIPY-PC dye molecules are laterally mobile in both bilayer preparations. This population exponentially increases with RH but never exceeds 6% of the total population. Interestingly, even under conditions where there is little lateral diffusion, fluctuations in the single molecule orientations can be observed which suggests there is appreciable freedom in the acyl chain region. Dynamic measurements of single molecule orientation changes, therefore, provide a new view into membrane properties at the single molecule level.

Single molecule probes of membrane structure: Orientation of BODIPY probes in DPPC as a function of probe structure

“…26–28 These studies have been valuable for understanding how monolayer and bilayer systems may be influenced by factors such as surface pressure, humidity, and composition. To expand the capabilities of this approach and fully explore how probe orientations reflect membrane properties, here we analyze the single molecule orientations of a series of BODIPY-linked fluorescent lipid analogs doped into DPPC monolayers.…”

“…Recently, we have shown that single molecule fluorescence measurements can characterize the orientation of individual fluorescent lipid probes doped into lipid membranes. [26][27][28][29][30] Using polarized total internal fluorescence microscopy (P-TIRFM), the three-dimensional orientation of fluorescent lipid analogs doped into films at trace levels can be characterized by emission pattern mapping. Using an acyl chain linked BODIPY-C 4 C 9 -PC probe, we have shown that these measurements are sensitive to membrane structure at the single molecule level.…”

Interaction of Cholesterol in Ternary Lipid Mixtures Investigated Using Single-Molecule Fluorescence

“…Single-molecule fluorescence measurements carried out using defocused polarized total internal reflection fluorescence microscopy (PTIRF) lead to distinct shapes in the single-molecule emission images that reflect the three-dimensional orientation of each fluorophore in the image. We have previously used singlemolecule orientation measurements to characterize the evolution in lipid monolayers with surface pressure, 32−34 relative humidity, 37 and the presence of additives such as GM1 and cholesterol. 35,36 Here we extend those studies to compare the tilt angle of BODIPY-cholesterol doped into DPPC/DOPC/Chol and SM/DOPC/Chol monolayers as a function of cholesterol.…”

“…This approach enables quantitative analysis of fluorophore orientations which we have used to characterize changes due to surface pressure, 32−34 presence of additives, 35,36 and ambient conditions. 37 Here we use fluorescence microscopy and single-molecule fluorescence measurements of BODIPY-cholesterol to probe its interactions with lipid monolayers formed using the Langmuir− Blodgett (LB) technique. BODIPY-cholesterol is studied in ternary mixed monolayers of DPPC/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Chol and SM/DOPC/Chol, which are often used models to study cholesterol mixing.…”

Resolving the Effects of Nanoscale Membrane Curvature on Lipid Mobility

“…Optical techniques enable the observation of living samples (i.e., aqueous, 0 to 45 °C, and non-ionizing radiation). Interference contrast microscopy and polarized total internal reflection fluorescence microscopy (TIRFM) have enabled the detection of membrane curvature in diverse samples [22][23][24][25][26][27][28][29][30][31]. However, these optical techniques are traditionally limited by diffraction to a spatial resolution of >200 nm.…”