Measuring the Diameter of Single-Wall Carbon Nanotubes Using AFM.

In this work, we identify two issues that can significantly affect the accuracy of AFM measurements of the diameter of single-wall carbon nanotubes (SWCNTs) and propose a protocol that reduces errors associated with these issues. Measurements of the nanotube height under different applied forces demonstrate that even moderate forces significantly compress several different types of SWCNTs, leading to errors in measured diameters that must be minimized and/or corrected. Substrate and nanotube roughness also make major contributions to the uncertainty associated with the extraction of diameters from measured images. An analysis method has been developed that reduces the uncertainties associated with this extraction to <0.1 nm. This method is then applied to measure the diameter distribution of individual highly semiconducting enriched nanotubes in networks prepared from polyfluorene/SWCNT dispersions. Good agreement is obtained between diameter distributions for the same sample measured with two different commercial AFM instruments, indicating the reproducibility of the method. The reduced uncertainty in diameter measurements based on this method facilitates: (1) determination of the thickness of the polymer layer wrapping the nanotubes and (2) measurement of nanotube compression at tube-tube junctions within the network.

Analysis Method for Quantifying the Morphology of Nanotube Networks.

While atomic force microscopy (AFM) is a powerful technique for imaging assemblies and networks of nanoscale materials, approaches for quantitative assessment of the morphology of these materials are lacking. Here we present a volume-based approach for analyzing AFM images of assemblies of nano-objects that enables the extraction of relevant parameters describing their morphology. Random networks of single-walled carbon nanotubes (SWCNTs) deposited via solution-phase processing are used as an example to develop the method and demonstrate its utility. AFM imaging shows that the morphology of these networks depends on details of processing and is influenced by choice of substrate, substrate cleaning method, and postdeposition rinsing protocols. A method is outlined to analyze these images and extract relevant parameters describing the network morphology such as the density of SWCNTs and the degree to which tubes are bundled. Because this volume-based approach depends on accurate measurements of the height of individual tubes and their networks, a procedure for obtaining reliable height measurements is also discussed. Obtaining quantitative parameters that describe the network morphology allows going beyond qualitative descriptions of images and will facilitate optimizing network preparation methods based on measurable criteria and correlating performance with morphology.

Real-time monitoring of protein conformational changes using a nano-mechanical sensor.

Proteins can switch between different conformations in response to stimuli, such as pH or temperature variations, or to the binding of ligands. Such plasticity and its kinetics can have a crucial functional role, and their characterization has taken center stage in protein research. As an example, Topoisomerases are particularly interesting enzymes capable of managing tangled and supercoiled double-stranded DNA, thus facilitating many physiological processes. In this work, we describe the use of a cantilever-based nanomotion sensor to characterize the dynamics of human topoisomerase II (Topo II) enzymes and their response to different kinds of ligands, such as ATP, which enhance the conformational dynamics. The sensitivity and time resolution of this sensor allow determining quantitatively the correlation between the ATP concentration and the rate of Topo II conformational changes. Furthermore, we show how to rationalize the experimental results in a comprehensive model that takes into account both the physics of the cantilever and the dynamics of the ATPase cycle of the enzyme, shedding light on the kinetics of the process. Finally, we study the effect of aclarubicin, an anticancer drug, demonstrating that it affects directly the Topo II molecule inhibiting its conformational changes. These results pave the way to a new way of studying the intrinsic dynamics of proteins and of protein complexes allowing new applications ranging from fundamental proteomics to drug discovery and development and possibly to clinical practice.

FM-AFM constant height imaging and force curves: high resolution study of DNA-tip interactions.

Interaction of the atomic force microscopy (AFM) tip with the sample can be invasive for soft samples. Frequency Modulation (FM) AFM is gentler because it allows scanning in the non-contact regime where only attractive forces exist between the tip and the sample, and there is no sample compression. Recently, FM-AFM was used to resolve the atomic structure of single molecules of pentacene and of carbon nanotubes. We are testing similar FM-AFM-based approaches to study biological samples. We present FM-AFM experiments on dsDNA deposited on 3-aminopropyltriethoxysilane modified mica in ultra high vacuum. With flexible samples such as DNA, the substrate flatness is a sub-molecular resolution limiting factor. Non-contact topographic images of DNA show variations that have the periodicity of the right handed helix of B-form DNA - this is an unexpected result as dehydrated DNA is thought to assume the A-form structure. Frequency shift maps at constant height allow working in the non-monotonic frequency shift range, show a rich contrast that changes significantly with the tip-sample separation, and show 0.2 to 0.4 nm size details on DNA. Frequency shift versus distance curves acquired on DNA molecules and converted in force curves show that for small molecules (height < 2.5 nm), there is a contribution to the interaction force from the substrate when the tip is on top of the molecules. Our data shine a new light on dehydrated and adsorbed DNA behavior. They show a longer tip-sample interaction distance. These experiments may have an impact on nanotechnological DNA applications in non-physiological environments such as DNA based nanoelectronics and nanotemplating.

Nanoscale imaging of epidermal growth factor receptor clustering: effects of inhibitors.

The development of some solid tumors is associated with overexpression of the epidermal growth factor receptor (EGFR) and often correlates with poor prognosis. Near field scanning optical microscopy, a technique with subdiffraction-limited optical resolution, was used to examine the influence of two inhibitors (the chimeric 225 antibody and tyrosine phosphorylation inhibitor AG1478) on the nanoscale clustering of EGFR in HeLa cells. The EGFR is organized in small clusters, average diameter of 150 nm, on the plasma membrane for both control and EGF-treated cells. The numbers of receptors in individual clusters vary from as few as one or two proteins to greater than 100. Both inhibitors yield an increased cluster density and an increase in the fraction of clusters with smaller diameters and fewer receptors. Exposure to AG1478 also decreases the fraction of EGFR that colocalizes with both rafts and caveolae. EGF stimulation results in a significant loss of the full-length EGFR from the plasma membrane with the concomitant appearance of low molecular mass proteolytic products. By contrast, AG1478 reduces the level of EGFR degradation. Changes in receptor clustering provide one mechanism for regulating EGFR signaling and are relevant to the design of strategies for therapeutic interventions based on modulating EGFR signaling.

Nanoscale organization of beta2-adrenergic receptor-Venus fusion protein domains on the surface of mammalian cells.

Adrenergic receptors are a key component of nanoscale multiprotein complexes that are responsible for controlling the beat rate in a mammalian heart. We demonstrate the ability of near-field scanning optical microscopy (NSOM) to visualize beta(2)-adrenergic receptors (beta(2)AR) fused to the GFP analogue Venus at the nanoscale on HEK293 cells. The expression of the beta(2)AR-Venus fusion protein was tightly controlled using a tetracycline-induced promoter. Both the size and density of the observed nanoscale domains are dependent on the level of induction and thus the level of protein expression. At concentrations between 100 and 700 ng/ml of inducer doxycycline, the size of domains containing the beta(2)AR-Venus fusion protein appears to remain roughly constant, but the number of domains per cell increase. At 700 ng/ml doxycycline the functional receptors are organized into domains with an average diameter of 150 nm with a density similar to that observed for the native protein on primary murine cells. By contrast, larger micron-sized domains of beta(2)AR are observed in the membrane of the HEK293 cells that stably overexpress beta(2)AR-GFP and beta(2)AR-eYFP. We conclude that precise chemical control of gene expression is highly advantageous for the use beta(2)AR-Venus fusion proteins as models for beta(2)AR function. These observations are critical for designing future cell models and assays based on beta(2)AR, since the receptor biology is consistent with a relatively low density of nanoscale receptor domains.

Chemical mapping of ceramide distribution in sphingomyelin-rich domains in monolayers.

The incorporation of ceramide in phase-separated monolayers of ternary lipid mixtures has been studied by a combination of atomic force microscopy (AFM), fluorescence, and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Replacement of a fraction of the sphingomyelin by ceramide in DOPC/SM/cholesterol monolayers leads to changes in the SM-cholesterol-rich liquid-ordered domains. AFM shows the formation of heterogeneous domains with small raised islands that are assigned to a ceramide-rich gel phase. ToF-SIMS provides conclusive evidence for the localization of SM and ceramide in ordered domains and shows that ceramide is heterogeneously distributed in small islands throughout the domains. The results indicate the utility of combining AFM and ToF-SIMS for understanding compositions of phase-separated membranes.

Scanning near-field optical microscopy.

An average human eye can see details down to 0,07 mm in size. The ability to see smaller details of the matter is correlated with the development of the science and the comprehension of the nature. Today's science needs eyes for the nano-world. Examples are easily found in biology and medical sciences. There is a great need to determine shape, size, chemical composition, molecular structure and dynamic properties of nano-structures. To do this, microscopes with high spatial, spectral and temporal resolution are required. Scanning Near-field Optical Microscopy (SNOM) is a new step in the evolution of microscopy. The conventional, lens-based microscopes have their resolution limited by diffraction. SNOM is not subject to this limitation and can offer up to 70 times better resolution.

Transition from nanodomains to microdomains induced by exposure of lipid monolayers to air.

The morphology of monolayers prepared from ternary lipid mixtures that have coexisting fluid phases has been examined by atomic force microscopy for samples transferred to mica before and after exposure to air. Mixtures of 1,2-dioleoyl-sn-glycero-3-phosphocholine and cholesterol with either egg sphingomyelin or 1,2-dipalmitoyl-sn-glycero-3-phosphocholine were studied at several surface pressures. Both lipid mixtures have a combination of small islands and large microdomains at low surface pressure (5-10 mN/m) for monolayers deposited in either air or nitrogen. By contrast, monolayers have small interconnected nanodomains when deposited under nitrogen at 30 mN/m but mixtures of large microdomains and small nanodomains when transferred after exposure to air. These results are consistent with an earlier report that concluded that the formation of large domains at high surface pressures (>30 mN/m) for monolayers exposed to air is caused by lipid oxidation. However, the higher spatial resolution available with atomic force microscopy indicates that exposure of the monolayers to air leads to an increase in the size of preexisting nanodomains, rather than a change in the miscibility pressure. Examination of changes in surface morphology as a function of surface pressure demonstrate a gradual evolution in size and surface coverage for both nano- and microdomains, before formation of a network of interconnected nanodomains. Similar studies for binary mixtures in the absence of cholesterol indicate that lipid oxidation results in analogous changes in domain size for monolayers with coexisting gel and fluid phases. These results illustrate the importance of using techniques capable of probing the nanoscale organization of membranes.

Very high resolution chemical imaging with Infrared Scanning Near-field Optical Microscopy (IR-SNOM).

In this paper we present chemically highly resolved images obtained with Scanning Near-field Optical Microscopy (SNOM) coupled with an Infrared (IR) Free Electron Laser (FEL) at Vanderbilt University, Nashville, USA. Main principles governing SNOM imaging as well as essential components of the experimental setup are described. Chemically resolved images showing the distribution of different phases within the boron-nitride films are presented. Universal character of the experiment and its huge potential applications in biophysics and medical sciences domain are illustrated with highly resolved SNOM images of pancreatic cells.