Imaging ROMK1 inwardly rectifying ATP-sensitive K+ channel protein using atomic force microscopy.

The inwardly rectifying K+ channel ROMK1 has been implicated as being significant in K+ secretion in the distal nephron. ROMK1 has been shown by immunocytochemistry to be expressed in relevant nephron segments. The development of the atomic force microscope has made possible the production of high resolution images of small particles, including a variety of biological macromolecules. Recently, a fusion protein of glutathione S-transferase (GST) and ROMK1 (ROMK1-GST) has been used to produce a polyclonal antibody for immunolocalization of ROMK1. We have used atomic force microscopy to examine ROMK1-GST and the native ROMK1 polypeptide cleaved from GST. Imaging was conducted with the proteins in physiological solutions attached to mica. ROMK1-GST appears in images as a particle composed of two units of similar size. Analyses of images indicate that the two units have volumes of approximately 118 nm3, which is close to the theoretical volume of a globular protein of approximately 65 kDa (the molecular mass of ROMK1-GST). Native GST exists as a dimer, and the images obtained here are consistent with the ROMK1-GST fusion protein's existence as a heterodimer. In experiments on ROMK1 in aqueous solution, single molecules appear to aggregate, but contact to the mica was maintained. Addition of ATP to the solution produced a change in height of the aggregates. This change (which was reversible) suggests that ATP induces a structural change in the ROMK1 protein. The data show that atomic force microscopy is a useful tool for examination of purified protein molecules under near-physiological conditions, and furthermore, that structural alterations in the proteins may be continuously investigated.

Imaging of single molecule diffusion.

In recent years observations at the level of individual atoms and molecules became possible by microscopy and spectroscopy. Imaging of single fluorescence molecules has been achieved but has so far been restricted to molecules in the immobile state. Here we provide methodology for visualization of the motion of individual fluorescent molecules. It is applied to imaging of the diffusional path of single molecules in a phospholipid membrane by using phospholipids carrying one rhodamine dye molecule. For this methodology, fluorescence microscopy was carried to a sensitivity so that single fluorescent molecules illuminated for only 5 ms were resolvable at a signal/noise ratio of 28. Repeated illuminations permitted direct observation of the diffusional motion of individual molecules with a positional accuracy of 30 nm. Such capability has fascinating potentials in bioscience--for example, to correlate biological functions of cell membranes with movements, spatial organization, and stoichiometries of individual components.

Study of interaction between Si(O,C) nanowires and the biological system

Nanomedicine is a new branch of medicine, based on the potentiality and intrinsic properties of nanomaterials. Indeed, the nanomaterials ( i.e. the materials with nano and under micron size) can be suitable to different applications in biomedicine. The nanostructures can be used by taking advantage of their properties (for example superparamagnetic nanoparticles) or functionalized to deliver the drug in a specific target, thanks the ability to cross biological barriers. The size and the shape of 1D-nanostructures (nanotubes and nanowires) have an important role on the cell fate: their morphology plays a key role on the interaction between nanostructure and the biological system. For this reason the 1D nanostructure are interesting for their ability to mime the biological system. An implantable material or device must therefore integrate with the surrounding extracellular matrix (ECM), a complex network of proteins with structural and signaling properties. Innovative techniques allow the generation of complex surface patterns that can resemble the structure of the ECM, such as 1D nanostructures. NWs based on cubic silicon carbide (3C-SiC), either bare (3C-SiC NWs) or surrounded by an amorphous shell (3C-SiC/SiO2 core/shell NWs), and silicon oxycarbide nanowires (SiOxCy NWs) can meet the chemical, mechanical and electrical requirements for tissue engineering and have a strong potential to pave the way for the development of a novel generation of implantable nano-devices. Silicon oxycarbide shows promising physical and chemical properties as elastic modulus, bending strength and hardness, chemical durability superior to conventional silicate glasses in aggressive environments and high temperature stability up to 1300 °C. Moreover, it can easily be engineered through functionalization and decoration with macro-molecules and nanoparticles. Silicon carbide has been extensively studied for applications in harsh conditions, as chemical environment, high electric field and high and low temperature, owing to its high hardness, high thermal conductivity, chemical inertness and high electron mobility. Also, its cubic polytype (3C) is highly biocompatible and hemocompatible, and some prototypes of biomedical applications and biomedical devices have been already realized starting from 3C-SiC thin films. Cubic SiC-based NWs can be used as a biomimetic biomaterial, providing a robust and novel biocompatible biological interface . We cultured in vitro A549 human lung adenocarcinoma epithelial cells and L929 murine fibroblast cells over core/shell SiC/SiO2, SiOxCy and bare 3C-SiC nanowire platforms, and analysed the cytotoxicity, by indirect and direct contact tests, the cell adhesion, and the cell proliferation. These studies showed that all the nanowires are biocompatible according to ISO 10993 standards. We evaluated the blood compatibility through the interaction of the nanowires with platelet rich plasma. The adhesion and activation of platelets on the nanowire bundles, assessed via SEM imaging and soluble P-selectin quantification, indicated that a higher platelet activation is induced by the core/shell structures compared to the bare ones. Further, platelet activation is higher with 3C-SiC/SiO2 NWs and SiOxCyNWs, which therefore appear suitable in view of possible tissue regeneration. On the contrary, bare 3C-SiC NWs show a lower platelet activation and are therefore promising in view of implantable bioelectronics devices, as cardiovascular implantable devices. The NWs properties are suitable to allow the design of a novel subretinal Micro Device (MD). This devices is based on Si NWs and PEDOT:PSS, though the well know principle of the hybrid ordered bulk heterojunction (OBHJ). The aim is to develop a device based on a well-established photovoltaic technology and to adapt this know-how to the prosthetic field. The hybrid OBHJ allows to form a radial p–n junction on a nanowire/organic structure. In addition, the nanowires increase the light absorption by means of light scattering effects: a nanowires based p-n junction increases the light absorption up to the 80%, as previously demonstrated, overcoming the Shockley-Queisser limit of 30 % of a bulk p-n junction. Another interesting employment of these NWs is to design of a SiC based epicardial-interacting patch based on teflon that include SiC nanowires. . Such contact patch can bridge the electric conduction across the cardiac infarct as nanowires can ‘sense’ the direction of the wavefront propagation on the survival cardiac tissue and transmit it to the downstream surivived regions without discontinuity. The SiC NWs are tested in terms of toxicology, biocompatibility and conductance among cardiomyocytes and myofibroblasts.

Analysis of Protein Turnover by Quantitative SNAP-Based Pulse-Chase Imaging

Assessment of protein dynamics in living cells is crucial for understanding their biological properties and functions. The SNAP-tag, a self labeling suicide enzyme, presents a tool with unique features that can be adopted for determining protein dynamics in living cells. Here we present detailed protocols for the use of SNAP in fluorescent pulse-chase and quench-chase-pulse experiments. These time-slicing methods provide powerful tools to assay and quantify the fate and turnover rate of proteins of different ages. We cover advantages and pitfalls of SNAP-tagging in fixed- and live-cell studies and evaluate the recently developed fast-acting SNAPf variant. In addition, to facilitate the analysis of protein turnover datasets, we present an automated algorithm for spot recognition and quantification.

Big cat scan: magnetic resonance imaging of the tiger

In August 2002, we performed MRI scans on a female juvenile Bengal tiger. We present the clinical course, imaging and autopsy findings, and some comparative anatomy of the tiger brain and skull. Magnetic resonance images of a tiger have not previously been published.

Noninvasive localization of petroleum-derived spray oil in plants with chemical shift selective magnetic resonance imaging

Nuclear magnetic resonance (NMR) spectroscopy and magnetic resonance imaging (MRI) were used to detect petroleum-derived spray oils (PDSOs) in citrus seedlings and trees. The NMR spectrum of the phantom containing 10% (v/v) of a nC24 agricultural mineral oil (AMO) showed the resonance of the water protons at delta = 5 ppm, while the resonance of the oil protons at delta = 1.3 to 1.7 ppm. The peak resolution and the chemical shift difference of more than 3.3 ppm between water and oil protons effectively differentiated water and the oil. Chemical shift selective imaging (CSSI) was performed to localize the AMO within the stems of Citrus trifoliata L. seedlings after the application of a 4% (v/v) spray. The chemical shift selective images of the oil were acquired by excitation at delta = 1.5 ppm by averaging over 400 transients in each phase-encoding step. Oil was mainly detected in the outer cortex of stems within 10 d of spray application; some oil was also observed in the inner vascular bundle and pith of the stems at this point. CSSI was also applied to investigate the persistence of oil deposits in sprayed mature Washington navel orange (Citrus x aurantium L.) trees in an orchard. The trees were treated with either fourteen 0.25%, fourteen 0.5%, four 1.75%, or single 7% sprays of a nC23 horticultural mineral oil (HMO) 12 to 16 months before examination of plant tissues by CSSI, and were still showing symptoms of chronic phytotoxicity largely manifested as reduced yield. The oil deposits were detected in stems of sprayed flushes and unsprayed flushes produced 4 to 5 months after the last spray was applied, suggesting a potential movement of the oil via phloem and a correlation of the persistence of oil deposit in plants and the phytotoxicity. The results demonstrate that MRI is an effective method to probe the uptake and localization of PDSOs and other xenobiotics in vivo in plants noninvasively and nondestructively.

Synthesis of gadolinium-labeled shell-crosslinked nanoparticles for magnetic resonance imaging applications

Shell-crosslinked knedel-like nanoparticles (SCKs; knedel is a Polish term for dumplings) were derivatized with gadolinium Shell chelates and studied as robust magnetic-resonance-imaging-active structures with hydrodynamic diameters of 40 +/- 3 nm. SCKs possessing an amphiphilic core-shell morphology were produced from the aqueous assembly of diblock copolymers of poly(acrylic acid) (PAA) and poly(methyl acrylate) (PMA), PAA(52)-b-PMA(128), and subsequent covalent crosslinking by amidation upon reaction with 2,2'-(ethylenedioxy)bis(ethylamine) throughout the shell layer. The properties of these materials, including non-toxicity towards mammalian cells, non-immunogenicity within mice, and capability for polyvalent targeting, make them ideal candidates for utilization within biological systems. The synthesis of SCKs derivatized with Gd-III and designed for potential use as a unique nanometer-scale contrast agent for MRI applications is described herein. Utilization of an amino-functionalized diethylenetriaminepentaacetic acid-Gd analogue allowed for direct covalent conjugation throughout the hydrophilic shell layer of the SCKs and served to increase the rotational correlation lifetime of the Gd. In addition, the highly hydrated nature of the shell layer in which the Gd was located allowed for rapid water exchange; thus, the resulting material demonstrated large ionic relaxivities (39 s(-1) mM(-1)) in an applied magnetic field of 0.47 T at 40 degrees C and, as a result of the large loading capacity of the material, also demonstrated high molecular relaxivities (20 000 s(-1) mM(-1)).

Optical scatter imaging using digital Fourier microscopy

An approach reported recently by Alexandrov et al (2005 Int. J Imag. Syst. Technol. 14 253-8) on optical scatter imaging, termed digital Fourier microscopy (DFM), represents an adaptation of digital Fourier holography to selective imaging of biological matter. The holographic mode of the recording of the sample optical scatter enables reconstruction of the sample image. The form-factor of the sample constituents provides a basis for discrimination of these constituents implemented via flexible digital Fourier filtering at the post-processing stage. As in dark-field microscopy, the DFM image contrast appears to improve due to the suppressed optical scatter from extended sample structures. In this paper, we present the theoretical and experimental study of DFM using a biological phantom that contains polymorphic scatterers.

Fluorescence in situ hybridization and spectral imaging of coral-associated bacterial communities

Microbial communities play important roles in the functioning of coral reef communities. However, extensive autofluorescence of coral tissues and endosymbionts limits the application of standard fluorescence in situ hybridization (FISH) techniques for the identification of the coral-associated bacterial communities. This study overcomes these limitations by combining FISH and spectral imaging.

Quantitative analysis of DNA-protein interactions using double-labeled native gel electrophoresis and fluorescence-based imaging

We have developed a sensitive, non-radioactive method to assess the interaction of transcription factors/DNA-binding proteins with DNA. We have modified the traditional radiolabeled DNA gel mobility shift assay to incorporate a DNA probe end-labeled with a Texas-red fluorophore and a DNA-binding protein tagged with the green fluorescent protein to monitor precisely DNA-protein complexation by native gel electrophoresis. We have applied this method to the DNA-binding proteins telomere release factor-1 and the sex-determining region-Y, demonstrating that the method is sensitive (able to detect 100 fmol of fluorescently labeled DNA), permits direct visualization of both the DNA probe and the DNA-binding protein, and enables quantitative analysis of DNA and protein complexation, and thereby an estimation of the stoichiometry of protein-DNA binding.

Imaging and sizing of diamond nanoparticles

Typical disturbances of biological environment such as background scatter and refractive index variations have little effect on the size-dependent scattering property of highly refractive nanocrystals, which are potentially attractive optical labels. We report on what is to our knowledge the first investigation of these scattering optical labels, and their sizing, in particular, by imaging at subvideo frame rates and analyzing samples of diamond nanocrystals deposited on a glass substrate in air and in a matrix of weakly scattering polymer. The brightness of a diffraction-limited spot appears to serve as a reliable measure of the particle size in the Rayleigh scattering limit. (c) 2006 Optical Society of America.