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[Near-field microscopy: from the isolated molecule to the living cell].

Near field (or scanning probe) microscopy is a recent technology which, owing to the huge amount of publications, is becoming a reference method in molecular and cellular imaging. These microscopies consist in the scanning of the sample, line by line, with a very tiny tip and thus providing informations on its surface down to the nanometer scale. These methods gather scanning tunelling microscopy (STM), which measures a current between the tip and the specimen support, atomic force microscopy (AFM), which measures the repulsive and attractive forces of the tip in contact or very close to the specimen, and scanning near field optical microscopies (SNOM), for which a glass tip allows to catch light signals. Atomic force microscopy, which allows the observation of specimens in air or physiological conditions environments, is presently dominant in biology, in complementarity with the classical optical and electron microscopies, which by the way, have also shown considerable improvements during the last years. The complementarity of these microscopies is due to their very different basic principles, which provide them various possibilities and limits. The biological applications of STM is limited by the need of conducting samples, but the different models of SNOM, often still in development, allow to consider very interesting applications, particularly for detecting very faint and tiny fluorescence signals. Different examples will be given concerning the visualization by AFM of isolated DNA molecules, naked or associated with proteins, the observation of intact or decondensed chromosomes, as well as living cells. One of the originality of AFM is its capacity to observe objects in a wide range of enlargements, with fields from a few hundred of nanometers to several micrometers.

Animals↗

Nanoscale in vivo evaluation of the stiffness of Drosophila melanogaster integument during development.

A quasistatic nanoindentation technique, enhanced by scanning probe microscopy, was used to measure cuticle stiffness of live Drosophila melanogaster during its larval, pupal, and early adult development in vivo. Stiffness was defined as the reduced elastic modulus (E(r)), which is a material property related to the elastic modulus. E(r) was measured at the local contact while indenting the live sample at a constant loading rate using a spherical tip. E(r) was derived from the resultant force-displacement curves. Insect cuticle exhibits viscoelastic behavior. Constant loading rate quasistatic measurements were used so that the effects of viscosity and contact force adhesion introduced systematic measurement effects. E(r) values were as follows: larvae, mean (SE), 0.39 (0.01) MPa; the puparium without evidence of adult structures 15.43 (1.78) MPa; and the adult, measured in the puparium at the completion of metamorphosis, 4.37 (0.31) MPa. Thus, as expected, the puparium and adult cuticle were very much stiffer than larval cuticle. Results also indicated stiffness variation that related to developmental events. This study has shown that this quasistatic nanoindentation-scanning probe microscopy approach is a suitable method for analyzing live biological samples.

Animals↗

Multifunctional probe array for nano patterning and imaging.

This letter reports the design, fabrication, and testing of a multifunctional scanning probe array for nanoscale imaging and patterning. The probe array consists of multiple cantilever probes, with each probe being able to perform a dedicated function such as scanning probe lithography (e.g., dip pen nanolithography and scanning probe contact printing) or scanning probe microscopy (e.g., atomic force microscopy and lateral force microscopy). The bending states of each probe can be controlled by using an integrated thermal electric actuator so that it is possible to engage any individual probe(s) independently for writing or imaging purposes. The multifunctional probe array is therefore capable of performing a rich variety of operations with minimal chemical crosstalk and high registration accuracy. It will eliminate the need for probe chip exchanges and increase the operational efficiency. The probe tips in a given array may be made of different materials. Further, the tip and cantilever may be made of different materials for a given probe. In this work, we focus on the development of a probe array consisting of dip pen nanolithography probes, scanning probe contact printing probes (of various tip sizes), and scanning probe microscopy probes.

Journal Article↗

Nanoelectrodes integrated in atomic force microscopy cantilevers for imaging of in situ enzyme activity.

For investigation of laterally resolved information on biological activity, techniques for simultaneously obtaining complementary information correlated in time and space are required. In this context, recent developments in scanning probe microscopy are aimed at information on the sample topography and simultaneously on the physical and chemical properties at the nanometer scale. With the integration of submicro- and nanoelectrodes into atomic force microscopy (AFM) probes using microfabrication techniques, an elegant approach combining scanning electrochemical microscopy with AFM is demonstrated. This instrumentation enables simultaneous imaging of topography and obtainment of laterally resolved electrochemical information in AFM tapping mode. Hence, topographical and electrochemical information on soft surfaces (e.g., biological species) and polymers can be obtained. The functionality of tip-integrated electrodes is demonstrated by simultaneous electrochemical and topographical studies of an enzyme-modified micropattern.

Enzymes↗

Near-field optics: from subwavelength illumination to nanometric shadowing.

Near-field optics uniquely addresses problems of x, y and z resolution by spatially confining the effect of a light source to nanometric domains. The problems in using far-field optics (conventional optical imaging through a lens) to achieve nanometric spatial resolution are formidable. Near-field optics serves a bridging role in biology between optical imaging and scanned probe microscopy. The integration of near-field and scanned probe imaging with far-field optics thus holds promise for solving the so-called inverse problem of optical imaging.

Delivery of Health Care, Integrated↗

Metallic ReO3 nanoparticles.

ReO3 nanoparticles in the diameter range of 8.5-32.5 nm have been prepared by decomposition of the Re2O7-dioxane complex under solvothermal conditions and characterized by X-ray diffraction, electron microscopy, optical spectroscopy, and scanning probe microscopy. The nanoparticles have the cubic (Pm3m {221} space group) structure with the lattice parameter increasing with decreasing size. The particles are metallic and show a plasmon band around 520 nm, which becomes blue-shifted with a decrease in size. The metallicity of the nanoparticles is also confirmed by tunneling conductance measurements. The nanoparticles show paramagnetic or diamagnetic behavior depending on the size, with evidence for superparamagnetism at low temperatures when the size is small.

Journal Article↗

Scanning probe technology in metalloprotein and biomolecular electronics.

The interfacing of man-made electronic components with specifically-folded biomacromolecules lies central not only to the development of sensory interfaces and potential new molecular-scale devices, but also enables us to analyse processes of great biological importance in a refined and controllable manner. Recent advances in both available technology, most notably optical and scanning probes in nature, and our understanding of suitable methodologies, have led us to the point where the characteristics of single biological molecules can be interrogated with good levels of reproducibility. We review here the application of scanning probe microscopy to the analysis of and experimentation on biological redox systems. Within this paper the tunnel transport characteristics, as assayed by both scanning tunnelling microscopy (STM) and conducting probe atomic force microscopy (AFM), of single metalloproteins are discussed. In a specific case study the electron transfer characteristics of the blue copper metalloprotein, azurin, are reported. The modulation of these properties under the influence of calibratable compressional force has also been examined in some detail. Work such as this enables one to reproducibly establish the conductance, barrier height, environmental sensitivity and electromechanical properties of these molecules.

Journal Article↗

Scanning electrochemical microscopy as a probe of Ag+ binding kinetics at Langmuir phospholipid monolayers.

A new method has been developed for measuring local adsorption rates of metal ions at interfaces based on scanning electrochemical microscopy (SECM). The technique is illustrated with the example of Ag+ binding at Langmuir phospholipid monolayers formed at the water/air interface. Specifically, an inverted 25 microm diameter silver disc ultramicroelectrode (UME) was positioned in the subphase of a Langmuir trough, close to a dipalmitoyl phosphatidic acid (DPPA) monolayer, and used to generate Ag+ via Ag electro-oxidation. The method involved measuring the transient current-time response at the UME when the electrode was switched to a potential to electrogenerate Ag+. Since the Ag+/Ag couple is reversible, the response is highly sensitive to local mass transfer of Ag+ away from the electrode, which, in turn, is governed by the interaction of Ag+ with the monolayer. The methodology has been used to determine the influence of surface pressure on the adsorption of Ag+ ions at a phospholipid (dipalmitoyl phosphatidic acid) Langmuir monolayer. It is shown that the capacity for metal ion adsorption at the monolayer increased as the density of surface adsorption sites increased (by increasing the surface pressure). A model for mass transport and adsorption in this geometry has been developed to explain and characterise the adsorption process.

Adsorption↗

Characterization of a protein-based adhesive elastomer secreted by the Australian frog Notaden bennetti.

When provoked, Notaden bennetti frogs secrete an exudate which rapidly forms a tacky elastic solid ("frog glue"). This protein-based material acts as a promiscuous pressure-sensitive adhesive that functions even in wet conditions. We conducted macroscopic tests in air to assess the tensile strength of moist glue (up to 78 +/- 8 kPa) and the shear strength of dry glue (1.7 +/- 0.3 MPa). We also performed nanomechanical measurements in water to determine the adhesion (1.9-7.2 nN or greater), resilience (43-56%), and elastic modulus (170-1035 kPa) of solid glue collected in different ways. Dry glue contains little carbohydrate and consists mainly of protein. The protein complement is rich in Gly (15.8 mol %), Pro (8.8 mol %), and Glu/Gln (14.1 mol %); it also contains some 4-hydroxyproline (4.6 mol %) but no 5-hydroxylysine or 3,4-dihydroxyphenylalanine (L-Dopa). Denaturing gel electrophoresis of the glue reveals a characteristic pattern of proteins spanning 13-400 kDa. The largest protein (Nb-1R, apparent molecular mass 350-500 kDa) is also the most abundant, and this protein appears to be the key structural component. The solid glue can be dissolved in dilute acids; raising the ionic strength causes the glue components to self-assemble spontaneously into a solid which resembles the starting material. We describe scattering studies on dissolved and solid glue and provide microscopy images of glue surfaces and sections, revealing a porous interior that is consistent with the high water content (85-90 wt %) of moist glue. In addition to compositional similarities with other biological adhesives and well-known elastomeric proteins, the circular dichroism spectrum of dissolved glue is almost identical to that for soluble elastin and electron and scanning probe microscopy images invite comparison with silk fibroins. Covalent cross-linking does not seem to be necessary for the glue to set.

Adhesiveness↗

Application of scanning force and near field microscopies to the characterization of minimally adhesive polymer surfaces.

This mini-review reports efforts to develop new scanning probe microscopies to characterize the function and aging of textured, minimally adhesive polymer surfaces used for antifouling applications in the marine environment. Novel atomic force and infrared near field microscopy techniques have been used to characterize the polymer surface adhesion and structural properties. These techniques may find promise for characterizing the deposition of the extracellular matrix of organisms as well as aging of the polymer coating itself. The reported work is part of a larger effort to reduce biofouling on ships' hulls through the development and use of improved coating materials.

Adhesiveness↗

The scanning probe microscope.

Scanning probe microscopy has evolved into a powerful tool since its inception in 1982. The scanning probe microscope has found applications in metrology, spectroscopy, and lithography. We will review the background of the technology, discuss the different types of scanning probe microscopes including the scanning tunneling microscope and the scanning force microscope, and present many of the applications for the instrument.

DNA↗

Examination of dentin surface using AFM (our experience).

Atomic force microscopy (AFM) as one the technique of Scanning Probe Microscopy is useful for imaging of surface structure. This method can yield three-dimensional high-resolution topographic images of sample surfaces by using a scanning technique for conductors and insulators on atomic scale. It is based upon mapping of atomic-forces on a surface of an investigated sample. The method is useful not only in physics and chemistry; it can be also applied in biological fields. Special construction of AFM scanner enables to follow biological samples in liquid environments. Artifacts caused by dehydration of samples are removed this way. Dentin of human teeth is a vital hydrated tissue. It is strongly sensitive to dehydration and drying that are commonly used in preparation of samples in examinations by Scanning Electron Microscopy (SEM). We describe our experience in examination of dentin surfaces of extracted human third molars using contact method of AFM under moist conditions.

Dentin↗

High resolution scanning force microscopy of cardiac myocytes.

The advent of scanning probe microscopy has introduced a powerful new method of probing the structural features of biological specimens. In this study, high resolution atomic force microscopy micrographs of single, isolated, cardiac myocytes are presented. Significantly, our images show not only the features to be expected of the external sarcolemma, but also resolve sub-surface features, including the striated pattern of the contractile proteins and their associated sarcoplasmic reticulum and mitochondria.

Animals↗

Atomic force microscopy with carbon nanotube probe resolves the subunit organization of protein complexes.

Among many scanning probe microscopies, atomic force microscopy (AFM) is a useful technique to analyse the structure of biological materials because of its applicability to non-conductors in physiological conditions with high resolution. However, the resolution has been limited to an inherent property of the technique; tip effect associated with a large radius of the scanning probe. To overcome this problem, we developed a carbon nanotube probe by attaching a carbon nanotube to a conventional scanning probe under a well-controlled process. Because of the constant and small radius of the tip (2.5-10 nm) and the high aspect ratio (1:100) of the carbon nanotube, the lateral resolution has been much improved judging from the apparent widths of DNA and nucleosomes. The carbon nanotube probes also possessed a higher durability than the conventional probes. We further evaluated the quality of carbon nanotube probes by three parameters to find out the best condition for AFM imaging: the angle to the tip axis; the length; and the tight fixation to the conventional tip. These carbon nanotube probes, with high vertical resolution, enabled us to clearly visualize the subunit organization of multi-subunit proteins and to propose structural models for proliferating cell nuclear antigen and replication factor C. This success in the application of carbon nanotube probes provides the current AFM technology with an additional power for the analyses of the detailed structure of biological materials and the relationship between the structure and function of proteins.

Carbon↗

Solution structure and direct imaging of fibronectin adsorption to solid surfaces by scanning force microscopy and cryo-electron microscopy.

In this study, we present the scanning force and electron microscopic visualization of single molecules of fibronectin either frozen hydrated or adsorbed onto metallic and polymeric surfaces with different solid surface tensions. The surfaces were characterized by dynamic contact angle measurements, X-ray photo emission spectroscopy (XPS or ESCA) and scanning force microscopy. The proteins were prepared by fast protein liquid chromatography (FPLC) and characterized by gel electrophoresis. Protein films on surfaces were investigated by surface plasmon resonance spectroscopy and directly imaged by scanning force microscopy. The spreading of the adsorbed fibronectin revealed dependence on the chemical composition and the solid surface tension. Structure of fibronectin in solution as well as on solid interface appeared as an extended straight strand as obtained by imaging with electron and scanning probe microscopies. Imaging of DNA was performed by scanning force microscopy to test the accuracy and reproducibility of our measurements. The measured contour lengths were accurate and the larger widths were caused by convolution of the tip shape and sample. Frictional forces during the scan have been of significant contribution in the imaging mechanism. Moreover, this work demonstrated that scanning force microscopy can be used for mapping the orientation and organization of protein film adsorbed onto various surfaces at the nanoscale.

Adsorption↗

Immobilization of fine particles on lactose carrier by precision coating and its effect on the performance of dry powder formulations.

The feasibility of immobilization of fine particles on a lactose carrier by precision coating and producing carrier particles of different surface roughness from the same core was explored. A relationship between the resultant surface roughness of the carrier and the in vitro deposition pattern of salbutamol sulfate was established. Lactose carrier particles in the precision coating chamber were spray coated with liquid suspensions consisting of micronized lactose dispersed in isopropyl alcohol (IPA) and/or water mixtures. The surface-modified lactose particles were fractionated and then characterized by laser diffraction for size, image analysis for shape, and scanning probe microscopy for surface roughness. The in vitro deposition pattern of salbutamol sulfate from the drug-lactose mixtures was determined with the twin-stage glass impinger and expressed as the fine particle fraction and dispersibility of the drug. Immobilization of fine particles on carrier particles was feasible by the precision coating process as shown by the scanning probe topographs and the roughness values of the carrier particles. Generally, more discrete fine particles were deposited on the carrier surface and a higher surface roughness was seen when the spray suspension consisting of a higher proportion of IPA was used. A significant correlation was found between the fine particle fraction of salbutamol sulfate with the roughness of lactose. This relationship established between the in vitro drug deposition pattern and the microscopic surface roughness of the carrier would be helpful in the optimization of drug delivery to targeted areas in the lungs.

Albuterol↗