Probing single molecule orientations in model lipid membranes with near-field scanning optical microscopy

Abstract

Single molecule near-field fluorescence measurements are utilized to characterize the molecular level structure in Langmuir-Blodgett monolayers of L-␣-dipalmitoylphosphatidylcholine ͑DPPC͒. Monolayers incorporating 3ϫ10 Ϫ4 mol % of the fluorescent lipid analog N-͑6-tetramethylrhodaminethiocarbamoyl͒-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt ͑TRITC-DHPE͒ are transferred onto a freshly cleaved mica surface at low ( ϭ8 mN/m) and high ( ϭ30 mN/m) surface pressures. The near-field fluorescence images exhibit shapes in the single molecule images that are indicative of the lipid analog probe orientation within the films. Modeling the fluorescence patterns yields the single molecule tilt angle distribution in the monolayers which indicates that the majority of the molecules are aligned with their absorption dipole moment pointed approximately normal to the membrane plane. Histograms of the data indicate that the average orientation of the absorption dipole moment is 2.2°( ϭ4.8°) in monolayers transferred at ϭ8 mN/m and 2.4°( ϭ5.0°) for monolayers transferred at ϭ30 mN/m. There is no statistical difference in the mean tilt angle or distribution for the two monolayer conditions studied. The insensitivity of tilt angle to film surface pressure may arise from small chromophore doped domains of trapped liquid-expanded lipid phase remaining at high surface pressure. There is no evidence in the near-field fluorescence images for probe molecules oriented with their dipole moment aligned parallel with the membrane plane. We do, however, find a small but significant population of probe molecules ͑ϳ13%͒ with tilt angles greater than 16°. Comparison of the simultaneously collected near-field fluorescence and force images suggests that these large angle orientations are not the result of significant defects in the films. Instead, this small population may represent a secondary insertion geometry for the probe molecule into the lipid monolayer.

Probing confined fields with single molecules and vice versa

“…Vice versa, if the electric field distributions are precisely known a wealth of information can be inferred from microscopy images. We show that it is possible to determine precisely the orientation of molecules from their image pattern (Betzig & Chichester, 1993;Veerman et al, 1999;Hollars & Dunn, 2000). A fundamental requirement for the determination of arbitrary molecular dipole orientations is that the three independent components of the optical excitation field are of comparable magnitude.…”

Sample heating in near-field scanning optical microscopy

“…The NSOM microscope is a homebuilt design, the details of which can be found elsewhere. 15 Figure 2 shows the bulk emission spectra of the perylene-NA polymer revealing two peaks located at ϳ475 and ϳ510 nm. Upon heating of the polymer, the ratio between the 475 and 510 nm peaks increases, providing a convenient marker of temperature that is not sensitive to intensity or concentration changes.…”

Interaction of an Asymmetric Scanning Near Field Optical Microscopy Probe With Fluorescent Molecules

“…These molecules are usually imaged as two lobes with aperture probes, because the longitudinal component of the electric field close to the aperture is strong only on the aperture edge at two diametrically opposite positions aligned along the direction of the input linear polarization. From a practical standpoint, better imaging of molecules with a mainly longitudinal dipole moment (like terylene molecules in p-terphenyl [33] or TRITC-DHPE molecules in DPPC monolayers [24]) could be achieved. Moreover the determination of the orientation of molecules with the same polar angle and different azimuthal orientation becomes easier.…”

“…The interaction of single fluorescent molecules with aperture probes has been thoroughly analyzed both experimentally and theoretically [24][25][26]. The interest in such a topic stems not only from its importance as a characterization technique, but also because single molecule detection is a hot issue in biological as well as in material science [1,27].…”

“…SNOM looks particularly suitable for this purpose due to the strong localization of the field emitted by the probe; moreover, due to the presence of transverse and longitudinal field components close to the probe apex, it has been used for the determination of the three-dimensional (3D) orientation of single fluorophores by controlling the polarization of the excitation light [29]. Aperture SNOM probes have been employed for fluorescence studies on biological molecules and polymers [24,[28][29][30][31].…”