Glycan distribution and density in native skin's stratum corneum.

BACKGROUND: The glycosylation of proteins on the surface of corneocytes is believed to play an important role in cellular adhesion in the stratum corneum (SC) of human skin. Mapping with accuracy the localization of glycans on the surface of corneocytes through traditional methods of immunohistochemistry and electron microscopy remains a challenging task as both approaches lack enough resolution or need to be performed in high vacuum conditions. MATERIALS AND METHODS: We used an advanced mode of atomic force microscope (AFM), with simultaneous topography and recognition imaging to investigate the distribution of glycans on native (no chemical preparation) stripped samples of human SC. The AFM cantilever tips were functionalized with anti-heparan sulfate antibody and the lectin wheat germ agglutinin (WGA) which binds specifically to N-acetyl glucosamine and sialic acid. RESULTS: From the recognition imaging, we observed the presence of the sulfated glycosaminoglycan, heparan sulfate, and the glycans recognized by WGA on the surface of SC corneocytes in their native state. These glycans were found associated with bead-like domains which represent corneodesmosomes in the SC layers. Glycan density was calculated to be ~1200 molecules/µm2 in lower layers of SC compared to an important decrease, (~106 molecules/µm2 ) closer to the surface due probably to corneodesmosome degradation. CONCLUSION: Glycan spatial distribution and degradation is first observed on the surface of SC in native conditions and at high resolution. The method used can be extended to precisely localize the presence of other macromolecules on the surface of skin or other tissues where the maintenance of its native state is required.

A single-molecule approach to explore binding, uptake and transport of cancer cell targeting nanotubes.

In the past decade carbon nanotubes (CNTs) have been widely studied as a potential drug-delivery system, especially with functionality for cellular targeting. Yet, little is known about the actual process of docking to cell receptors and transport dynamics after internalization. Here we performed single-particle studies of folic acid (FA) mediated CNT binding to human carcinoma cells and their transport inside the cytosol. In particular, we employed molecular recognition force spectroscopy, an atomic force microscopy based method, to visualize and quantify docking of FA functionalized CNTs to FA binding receptors in terms of binding probability and binding force. We then traced individual fluorescently labeled, FA functionalized CNTs after specific uptake, and created a dynamic 'roadmap' that clearly showed trajectories of directed diffusion and areas of nanotube confinement in the cytosol. Our results demonstrate the potential of a single-molecule approach for investigation of drug-delivery vehicles and their targeting capacity.

Continuous capacitance-voltage spectroscopy mapping for scanning microwave microscopy.

A new method, scanning sawtooth capacitance spectroscopy (SSCS), is proposed to measure a map of capacitance/voltage curves (C-V) by applying a low frequency voltage sawtooth signal (20-100 Hz) to the AFM tip while scanning. For this a scanning microwave microscope (SMM) is used to acquire calibrated capacitance data in the high frequency range of 1-20 GHz. While the capacitance is acquired pixel by pixel, the applied voltage signal is recorded as well, and each pixel of the capacitance is assigned the corresponding voltage value. Assuming the voltage variable is smooth over time, adjacent pixels within a scan line will have similar voltage values and a small sequence of neighboring pixels can be combined into a virtual C-V spectroscopy curve. With standard SMM operation parameters roughly 26,000 C-V curves can be acquired within few minutes data acquisition time. The method is demonstrated for n-type and p-type silicon semiconductor samples and can be applied to other samples including new materials and bio-membranes.

Improved localization of cellular membrane receptors using combined fluorescence microscopy and simultaneous topography and recognition imaging.

The combination of fluorescence microscopy and atomic force microscopy has a great potential in single-molecule-detection applications, overcoming many of the limitations coming from each individual technique. Here we present a new platform of combined fluorescence and simultaneous topography and recognition imaging (TREC) for improved localization of cellular receptors. Green fluorescent protein (GFP) labeled human sodium-glucose cotransporter (hSGLT1) expressed Chinese Hamster Ovary (CHO) cells and endothelial cells (MyEnd) from mouse myocardium stained with phalloidin-rhodamine were used as cell systems to study AFM topography and fluorescence microscopy on the same surface area. Topographical AFM images revealed membrane features such as lamellipodia, cytoskeleton fibers, F-actin filaments and small globular structures with heights ranging from 20 to 30 nm. Combined fluorescence and TREC imaging was applied to detect density, distribution and localization of YFP-labeled CD1d molecules on alpha-galactosylceramide (alphaGalCer)-loaded THP1 cells. While the expression level, distribution and localization of CD1d molecules on THP1 cells were detected with fluorescence microscopy, the nanoscale distribution of binding sites was investigated with molecular recognition imaging by using a chemically modified AFM tip. Using TREC on the inverted light microscope, the recognition sites of cell receptors were detected in recognition images with domain sizes ranging from approximately 25 to approximately 160 nm, with the smaller domains corresponding to a single CD1d molecule.

Improving the contrast of topographical AFM images by a simple averaging filter.

New image-processing methods were applied to atomic force microscopy images in order to visualize small details on the surface of virus particles and living cells. Polynomial line flattening and plane fitting of topographical images were performed as first step of the image processing. In a second step, a sliding window approach was used for low-pass filtering and data smoothing. The size of the filtering window was adjusted to the size of the small details of interest. Subtraction of the smoothed data from the original data resulted in images with enhanced contrast. Topographical features which are usually not visible can be easily discerned in the processed images. The method developed in this study rendered possible the detection of small patterns on viral particles as well as thin cytoskeleton fibers of living cells. It is shown that the sliding window approach gives better results than Fourier-filtering. Our method can be generally applied to increase the contrast of topographical images, especially when small features are to be highlighted on relatively high objects.

Single molecule recognition between cytochrome C 551 and gold-immobilized azurin by force spectroscopy.

Recent developments in single molecule force spectroscopy have allowed investigating the interaction between two redox partners, Azurin and Cytochrome C 551. Azurin has been directly chemisorbed on a gold electrode whereas cytochrome c has been linked to the atomic force microscopy tip by means of a heterobifunctional flexible cross-linker. When recording force-distance cycles, molecular recognition events could be observed, displaying unbinding forces of approximately 95 pN for an applied loading rate of 10 nN/s. The specificity of molecular recognition was confirmed by the significant decrease of unbinding probability observed in control block experiments performed adding free azurin solution in the fluid cell. In addition, the complex dissociation kinetics has been here investigated by monitoring the unbinding forces as a function of the loading rate: the thermal off-rate was estimated to be approximately 14 s(-1), much higher than values commonly estimated for complexes more stable than electron transfer complexes. Results here discussed represent the first studies on molecular recognition between two redox partners by atomic force microscopy.