Christopher W. Hollars

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

Single molecule near-field fluorescence measurements are utilized to characterize the molecular level structure in Langmuir–Blodgett monolayers of L-α-dipalmitoylphosphatidylcholine (DPPC). Monolayers incorporating 3×10−4 mol % of the fluorescent lipid analog N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn- glycero-3-phosphoethanolamine, triethylammonium salt (TRITC–DHPE) are transferred onto a freshly cleaved mica surface at low (π=8 mN/m) and high (π=30 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° (σ=4.8°) ...

Evaluation of thermal evaporation conditions used in coating aluminum on near-field fiber-optic probes

The effects that the thermal evaporation conditions have on the roughness of aluminum-coated near-field fiber-optic probes were investigated using the high-resolution capabilities of atomic force microscopy. The coating conditions studied include the effects of background gas composition, base vacuum pressure, and aluminum evaporation rate. The effects of aging on the aluminum-coated tips were also evaluated. The results from topography measurements of the resulting aluminum film indicated that the most dramatic improvements in the tip coatings can be achieved using high aluminum evaporation rates at base vacuum pressures below 10−5 Torr. These results agree with other studies on thin aluminum films and reflect a decrease in oxide formation. For demanding applications of near-field microscopy requiring maximal resolution, the results presented here indicate that it may also be necessary to reduce oxygen and/or water from the vacuum chamber prior to coating.

Progress toward imaging biological samples with NSOM

Advancements in near-field scanning optical microscopy (NSOM) tip design as well as an interferometric feedback mechanism are presented for the common goal of imaging living biological samples under physiological conditions. The ability of a cantilevered tip to track the subtle topography changes of a fragile lipid film in an aqueous environment is demonstrated. In order to further the imaging capabilities, the probes have been chemically etched to reduce the spring constants of the tips, thereby lowering the forces imparted on the sample. An optical feedback mechanism used as an alternative to the conventional force feedback is also described. Utilizing this optical feedback mechanism, images have been obtained of fixed cells underwater. Finally, progress towards modifying the NSOM tip for chemical sensor applications is discussed in the context of eventually measuring ion fluxes through single protein channels. Together these advancements demonstrate the potential of NSOM for studying live cells.

Probing biological systems with near-field optics

The imaging characteristics of cantilevered NSOM probes operating in a tapping-mode feedback arrangement are discussed and compared to conventional tips employing the shear-force feedback method. Images form a wide range of samples are presented to demonstrate the surface tracking capabilities over both high and low topology samples, in addition to the low fluorescence detection limits possible utilizing the new tips. The results show that the cantilevered tip operating in a tapping-mode arrangement offers enhanced force imaging of the sample topology without compromising the low detection limits or high spatial resolution of the NSOM fluorescence images. The examples discussed here indicate that the new design will be particularly useful for applications involving biological samples that frequently exhibit complex surface topologies.

Probing single-molecule orientations in confined sample regions with near-field scanning optical microscopy

Near-field scanning optical microscopy (NSOM) is utilized to study the 3D orientation of fluorescent probe molecules doped into thin polymer films and lipid monolayers. A simplified model of the fields near the NSOM tip aperture is used to simulate the single molecule near-field fluorescence features and extract the orientation of the probe molecules in the films. These measurements are particular useful for semi-ordered systems such as lipid monolayers that are often utilized as models of biological membranes. These single molecule measurements can reveal new features and provide details previously hidden in bulk methods that average over large ensembles of molecules.