Fluorescence correlation spectroscopy with sub-diffraction-limited resolution using near-field optical probes

We report fluorescence correlation spectroscopy (FCS) measurements using near-field scanning optical microscopy (NSOM) probes to produce a sub-diffraction-limited observation area. An order of magnitude reduction in the area compared to confocal FCS has been achieved. We also demonstrate a simple means to model the autocorrelation decay due to diffusion within the excitation profile at the NSOM probe aperture. The use of probes with smaller apertures is expected to provide an additional order of magnitude reduction in the observation area, thus enabling the study of cellular membranes with higher concentrations of fluorophores than is currently possible with diffraction-limited techniques

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

Fine DNA structure revealed by constant height frequency modulation AFM imaging

Few examples of sub nanometer resolution on biomolecules can be found in literature. In particular, AFM experiments on DNA mostly fail to show the double helical groove periodicity, and so far such a feat was only achieved in liquid. Here we describe a method that produces highly resolved images of DNA where contrast can be assigned to submolecular features such as DNA helix's grooves. Two types of AFM experiments are presented: (1) We started by imaging DNA with Frequency Modulation (FM) AFM, where a shift in resonance frequency is used as a feedback signal and maintained constant during imaging. With FM-AFM we performed experiments in the non-contact (NC) mode, with z-feedback on, where only gentle (few tens of pN) non compressive force is applied between the tip and the sample. (2) Then we switched off the z-feedback and scanned the DNA sample at constant height measuring frequency modulation in order to acquire frequency shift maps of the sample. This was done at several separations including the distances where the frequency feedback is not stable due to the non-monotonicity of the frequency shift curve. At this distance sub-molecular resolution was reached and a calculation of the tip-sample van der Wools interaction at constant height, as well as the periodicity of the observed features that ranges from 3 to 5 nm, suggest that these correspond to the DNA helix grooves. This is the first experimental study where submolecular features of single DNA molecules are observed in dry environment. The knowledge of DNA structure when dried and deposited on flat substrates is important for proper understanding of DNA's electronic behavior and its eventual nanotechnology applications. (C) 2013 Elsevier Ltd. All rights reserved

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

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 &lt;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&ndash;tube junctions within the network

Damping Behavior of Bent Fiber NSOM Probes in Water

The damping behavior of bent fiber near-field scanning optical microscopy (NSOM) probes operating in tapping mode oscillation is investigated in air and water. We show that the significantdrop in probe quality factor Q, which occurs at the air-water interface, is due to meniscus damping. As the probe is immersed in water viscous damping adds to the meniscus damping. Damping effects which lead to a progressive drop in the peak tapping mode resonance frequency are accounted for by additional torsional modes of probe vibration. Understanding the damping processes should lead to the design of high sensitivity NSOM probes for scanning soft biological samples under liquid.Peer reviewed: YesNRC publication: N