Quantitative dimensional measurements of micro- and nanometre-sized structures are urgently required from science and industry. Due to their very high vertical resolution (down to sub nanometres) and high lateral resolution (<10 nm) scanning probe microscopes (SPMs) are of great interest for such metrological applications. Additionally, SPM methods are able to measure surfaces in a number of modes like contact, intermittent-contact and non-contact mode. The forces between tip and sample are low during the measurement and, even in contact mode, reach only a few nanonewtons. This fact prevents scratching of the measured surface during the SPM scanning procedure even when very sharp tips are used.

Atomic Force Microscopy (AFM) has become a ubiquitous tool for analyzing the topography of a wide variety of materials, especially as nanoscale features become more significant for both understanding as well as determining materials properties [1]. Many AFM variations have also been developed for measuring surface properties beyond straightforward cartography. In many of these cases, the contrast mechanisms are often either extremely complex, or not well understood, even though the principles are simple. For example, Piezo-Force Microscopy (PFM) is relatively easy to understand and use in a standard lab for measuring electromechanical properties of materials, but care must be taken in order to obtain quantitative results as described below.

Scanning Probe Microscopy (SPM) [1] has been an important tool to organize matter on the nanometer scale. It has been proved to be a powerful tool not only for imaging but also for nanofabrication. SPM-based nanofabrication comprises manipulation of atoms or molecules and SPM-based nanolithography. SPM-based nanolithography, referred to as scanning probe lithography (SPL) holds good promise for fabrication of nanometer-scale patterns as an emerging generic lithography technique that employs SPM to directly pattern nanometer-scale features under appropriate conditions. The water meniscus formation between the tip and the flat substrate, due to the water layer present on any surface of a material at ambient conditions, has been studied experimentally and theoretically [2-6] using SPM techniques. The water effect in the imaging process is well understood [2, 4, 6-8]. Dip pen nanolithogaphy (DPN) [9] is one example of a technique that uses the water effect to transfer material from the tip onto sample surface in direct-write fashion with nanoscopic resolution.

Nanoscale resolution in microscopy characterization has become crucial for state-of-the-art science and technology. We have developed a Magneto-optical Scanning Near-Field Optical Microscope (MO-SNOM), and it has demonstrated to be a powerful tool to study local magnetic properties [1,2]. One of the critical steps in producing a reliable instrument and consistent images is the fabrication of the microscope tip. This work presents concepts and results on tip processing by chemical etching on FS-SN-3224 optical fibers from 3M. The quality of the tips produced was tested on magnetic multilayers presenting exchange-bias coupling.

This work proposes a parallel implementation for image restoration, applied to biological images, obtained from an Atomic Force Microscope (AFM) [1]. Depending on the scanning dimensions, any image obtained from this technique can present either poor signal/noise ratios and blurred contents. In order to restore or, at least, decrease the effects of such degradations, image restoration methods are usually employed. The algorithm of restoration is based on the use of the Tikhonov methodology, described elsewhere, known here as the serial approach [2].

One of the pillars of Nanotechnology is the possibility of matter manipulation on nanometer scale. At the basis of such pillar, the importance of Scanning Probe Microscopy (SPM) is twofold: surface visualization and material manipulation at a sub-molecular level. There is a myriad of reports in the literature showing astonishing cases of high resolution imaging and manipulation carried out by different SPM techniques [1]. Within such examples, anodic oxidation nanolithography (AON) appears as one of the most promising tools due to its simplicity and applicability to a large variety of materials [1,2]. Therefore, this work reports on a detailed study of possibilities and limitations of AON towards the fabrication of semiconductor- and/or metal-based devices.

Progress in structural biology very much depends upon the development of new high-resolution techniques and tools. Despite decades of study of viruses, bacteria and bacterial spores and their pressing importance in human medicine and biodefense, many of their structural properties are poorly understood. Thus, characterization and understanding of the architecture of protein surface and internal structures of pathogens is critical to elucidating mechanisms of disease, immune response, physicochemical properties, environmental resistance and development of countermeasures against bioterrorist agents. Furthermore, even though complete genome sequences are available for various pathogens, the structure-function relationships are not understood. Because of their lack of symmetry and heterogeneity, large human pathogens are often refractory to X-ray crystallographic analysis or reconstruction by cryo-electron microscopy (cryo-EM). An alternative high-resolution method to examine native structure of pathogens is atomic force microscopy (AFM), which allows direct visualization of macromolecular assemblies at near-molecular resolution. The capability to image single pathogen surfaces at nanometer scale in vitro would profoundly impact mechanistic and structural studies of Progress in structural biology very much depends upon the development of new high-resolution techniques and tools. Despite decades of study of viruses, bacteria and bacterial spores and their pressing importance in human medicine and biodefense, many of their structural properties are poorly understood. Thus, characterization and understanding of the architecture of protein surface and internal structures of pathogens is critical to elucidating mechanisms of disease, immune response, physicochemical properties, environmental resistance and development of countermeasures against bioterrorist agents. Furthermore, even though complete genome sequences are available for various pathogens, the structure-function relationships are not understood. Because of their lack of symmetry and heterogeneity, large human pathogens are often refractory to X-ray crystallographic analysis or reconstruction by cryo-electron microscopy (cryo-EM). An alternative high-resolution method to examine native structure of pathogens is atomic force microscopy (AFM), which allows direct visualization of macromolecular assemblies at near-molecular resolution. The capability to image single pathogen surfaces at nanometer scale in vitro would profoundly impact mechanistic and structural studies of pathogenesis, immunobiology, specific cellular processes, environmental dynamics and biotransformation.

Formal definitions of nanotechnological devices for drug delivery typically feature the requirements that the device itself or its essential components be man-made, and in the 1-1000 nm range in at least one dimension [1]. The known nanovectors or nanostructures can be filled with drugs for different therapies and for diagnostical aims. Targeting moieties can also be attached to their surface. Polymeric nanovectors are generally made from biodegradable polymers such as polyesters, for example, poly-e-caprolactone (PCL). The drug delivery system known as nanocapsules (NCs) can be defined as a complex nanovector that is composed by a polymeric wall surrounding an oil core, where lipophilic drugs can be encapsulated. The advantages of NCs compared to other nanovectors are the high entrapment efficiencies of lipophilic drugs, low polymer content and low inherent toxicity. On the other hand, because of its complex blend of components NCs suspension allow several forms of nanovectors to be present at the same time, such as nanospheres, liposomes and nanoemulsions [2]. These ‘contaminants’ would be present in accordance with the type of formulation and method of preparation. Atomic force microscopy (AFM) has been used as a method for imaging the surfaces of liposomes [3] and nanospheres [4] allowing information in nanoscaled dimensions. In the present work, the NCs were prepared loading two different drugs, the antifungal albaconazole (ABZ), showing a crystalline drug structure and the antimalarial halofantrine (Hf) free base, having an amorphous form. These drugs possess high lipophilic character, which favours the association of the drug with the oily core, with drug loadings above 94%. Herein we studied the behavior of ABZ-loaded and Hf-loaded NCs through the AFM technique, searching to analyze and understand possible alterations induced by the drug inclusion in these nanostructures.

Some microalga species may accumulate extracellular polysaccharides, such as a gelatinous mass that encloses its cells, and is known as envelopes, sheaths or capsules. These structures are very common among the Desmidiales. However, studies concerning their functions, properties and structures are still incipient. Spondylosium panduriforme is a filamentous desmids, enclosed by a great and continuous capsule with well-defined borders firmly bond to the cell. These characteristics can be observed through light microscopy in Indian ink preparations. The S. panduriforme capsule is taken as an essential part of the cell, and points out the importance of this structure for its proper functioning [1,2].

Biological membranes are constituted of lipids organized as a two dimensional bilayer supporting peripheral and integral proteins, providing a barrier between the inside and the outside of a cell [1]. Similar membranes can be prepared from the lipid mixtures forming liposomes. The liposomes are multi or unilamellar spherical vesicles in which an aqueous volume is enclosed and can be used to encapsulate some drugs [2]. In order to better expose the details of their structure, these membranes are generally deposited on the surface of a flat substrate. These supported planar lipid membranes can also provide a model system for investigating the properties and functions of the complex cell membrane and membrane mediated processes such as recognition events and biological signal transduction. Various methods have been used to create artificial lipid membranes supported on a solid surface, being the most used the Langmuir-Blodgett monolayers formation [3], the vesicle fusion or liposome adsorption [4] and the solution spreading [5].

Polymeric nanoparticles containing an oily core, named nanocapsules (NCs), have been widely studied in the life sciences field due to their therapeutic potentialities of drug targeting in the body accompanied also by its larger stability in the biological fluids compared to other colloidal carriers. Many studies have shown different applications of nanocapsules for therapeutic use concerning their properties [1] of loading poorly water-soluble drugs, protection drugs from inactivation in the gastro-intestinal tract [2], gastric mucosal toxicity protection [3,4], increased drug permeation through mucous epithelium [5,6] and prolongation of drugs in blood circulation for surface modified nanocapsules [7]. The characterization of the nanocapsules is frequently performed by mean size, surface charge of the particles (zeta potential), hydrophobicity, drug loading yield and release kinetic [8]. These features are of great importance for biodistribution profile and interactions with the cells of mononuclear phagocyte system of any injected particles by intravenous route [1]. However, few data on structural organization of the nanocapsules constituents are available in literature and several hypotheses only suggest the presence of an oil "capsular" structure surrounded by a polymeric envelope. Recently, atomic force microscope (AFM) has been used as a method for imaging the surfaces of colloidal systems, such as liposomes [9,10] and nanospheres [11], supplying high resolution information in nanoscaled dimension. In the present work, unloaded nanocapsules were deposited on mica in order to analyze by AFM the diameter, height, particles polydispersion, and topographic characteristics of nanocapsule surface.

Solid lipid nanoparticles (SLN) have generated increasing attention as an alternative carrier system, particularly for lipophilic drugs [1-2]. The system consists of lipid nanoparticles, which are solid at room temperature. The solid matrix offers the possibility to improve the stability against coalescence and the reduced mobility of incorporated drug molecules is a pre requisite for controlled drug release [3]. Dexamethasone acetate was used as a model drug because of its wide application in the pharmaceutical field and lipophilic properties.

Protein adsorption plays a major role in a variety of important technological and biological processes [1-2] and the understanding of the fundamental factors that determine protein adsorption are imperative to the development of biocompatible materials and biotechnological devices [3-4] as for example biosensors [5]. The adsorption of proteins on surfaces is a complex process. Due to the large size and different shapes of these adsorbing particles, the interactions between the adsorbed proteins on the surface can be strongly influentiated by the fact that the particles may undergo conformational changes upon adsorption [6-7]. In a previous work the adsorption behaviour of creatine phosphokinase (CPK) onto hydrophilic (silicon wafers and amino-terminated surfaces) and hydrophobic (Polystyrene, PS, coated wafers) substrates was investigated by means of null-ellipsometry and contact angle measurements [8]. This previous ellipsometric study led to a model, where CPK adsorption takes place in four stages: (i) a diffusive one, where all the arriving biomolecules are immediately adsorbed; (ii) the arriving biomolecules might stick on the latter and afterward diffuse to the free sites on the substrate, followed by conformational changes [6-7], (iii) formation of a monolayer and (iv) continuous and irreversible adsorption. A multilayer system might be formed, as well as aggregation processes might play a role at this stage. In this work Atomic Force Microscopy (AFM) measurements under water were done in order to confirm this four steps model and to observe changes in the film topography and homogeneity along the adsorption process. The thickness of the adsorbed CPK biofilm obtained by ellipsometry was also compared with that obtained by the wet AFM method.

Actinic Kerarosis (AK) are hyperqueratotic cutaneous lesions, in which an abnormal proliferation of keratinocytes of the epidermis is present [1]. Since 1960s, topical 5-Fluorouracil (5-FU), a hydrophilic antimetabolite drug, has been widely used as an effective treatment for many pre-cancerous conditions and certain malignant tumors. However, the severe inflammatory reaction, clinically characterized by erosion and ulceration, which occurs in the skin following topical therapy, is still a major treatment disadvantage. Topical delivery of hydrophilic drugs through intact skin usually creates problems due to the inability of the drugs to penetrate into stratum corneum. Liposomal formulations have demonstrated capacity to increase penetration across and into skin of these drugs when compared to conventional formulations [2]. Besides, when applied on the stripped skin, in absence of barrier for drug permeation, liposomes were found to provide targeted and sustained topical delivery [3]. Thus, we hypothesized that liposomal formulation containing entrapped 5-FU could be an interesting alternative for topical treatment of AK. 5-FU encapsulation efficiency (EE) and retention in liposomes are usually low (EE less than 10%). 5-FU does not associate with the lipid bilayer [4] and EE depends mainly on the internal aqueous volume of vesicles [5]. Therefore, EE has been greater in large unilamellar vesicles (LUV) when compared to multilamellar vesicles (MLV) and the physical state of the bilayer determines the retention capacity of the vesicles [6]. Recently, MLV liposomes containing 5-FU were prepared with high EE, but with an average vesicle diameter of 5 m [7].

Atomic Force Microscopy (AFM) is a recent technique that allows evaluation of features in biological systems that could not be previously observed by other instruments. Red Blood Cells (RBC) have been extensively studied because of their relatively simple membrane structure, convenience of preparation and scanning [1]. As an ancillary way of confirming diagnoses, AFM has mostly been used to determine shape and size of RBCs, which are important indicators of some blood diseases or disordered erythropoiesis [2]. In Myelodysplastic Syndrome (MDS) hematopoiesis is inefficacious with consequent anemia that may evolve to acute leukemia. Genomic alterations lead to structural defects in the biomolecular network that forms the erythrocyte membrane. The deformation capability of the cells and their lifetime in circulation are diminished [5]. AFM allows us to observe in a controlled way the response of these membrane molecular networks under physical and chemical stimuli in many different physiological conditions, such as in air and liquids [6]. Using this technique, many new characteristics have been found in erythrocyte membranes that are still of undetermined significance [3,4]. The aim of our work is to compare membrane morphology of two groups of blood donors, that is, healthy subjects and patients with MDS. The images yielded by AFM confirm the structure of the erythrocytes and reveal interesting submicron features on the cell, suggesting a way to distinguish between RBCs from healthy donors and RBCs from patients with MDS.

Ultrathin films of polysaccharides provide convenient conditions for the immobilization of biomolecules, promoting the development of biosensors supported on renewable sources [1-3]. Specific interactions such as protein-substrate, antigen-antibody and lectin-carbohydrate can be used with advantage for the creation of biotechnological devices.

Lipases (glycerol ester hydrolases, E.C. 3.1.1.3) are enzymes of great industrial interest due to their ability to catalyze a broad range of hydrolytic and synthetic reactions. They find applications in the synthesis of compounds used in clinical, nutritional, environmental, pharmaceutical and chemical fields. For example, lipases are used to catalyze key intermediate steps in the synthesis of biologically active compounds such as Naproxen, Ibuprofen and Atenolol [1]. Depending on the application, lipases may need to be purified and characterized biochemically before they can be used. However, the purification of microbial lipases is often made difficult by the presence of high molecular weight aggregates. These aggregates can form due to the presence, in the fermentation medium, of lipids used to induce the production of the enzyme by the microorganism or simply due to hydrophobic interactions amongst the enzyme molecules themselves [2]. In previous work, we characterized a new lipase produced by Bacillus megaterium CCOC P2637. The enzyme eluted in the void volume during gel filtration chromatography, indicating that it was present in the form of a high molecular weight aggregate. This aggregate was dispersed when a gradient of 60% (v/v) isopropanol was used, but formed again when the enzyme was injected in a gel filtration column for further purification, even when the elution buffer contained 20% (v/v) isopropanol. Further, when the enzyme was diluted in buffer (phosphate pH 7.0 20 mM) containing 30% isopropanol, its specific activity was double the activity obtained by diluting in buffer without isopropanol [3].

Pollens appear like a fine to coarse powder that is liberated by the microsporangia of Gimnosperms and Angiosperms. The pollen grain wall, the sporoderm, envelopes the microgametophytes (male gametophytes), which produce the male gametes of seed plants. Pollen grains are interesting from the material science point of view since the native polymer, the sporopollenin, found in the sporoderm outer layer (exine), is one of the toughest known materials which is degraded by oxidation but is resistant to reduction. This property permits the sporopollenin persistence as an unaltered polymer in sediments of great age, e.g the Ordovician period, 400 million years ago. Sporopollenin is a mixture of fatty acids, phenyl-derivatives as p-coumaric acid, and carotenes [1]. Its nanostructure is not yet completed revealed. Therefore, more studies must be performed. A number of models have been proposed for the sporopollenin nanostructure of spores and pollen grains [2]. Rowley et al. [3-4] interpret exine structure as being formed by helical subunits, based on transmission and scanning electron microscope (TEM and SEM) studies. The atomic force microscopy (AFM) is the ideal method to study the sporopollenin nanostructure [5] since the arrangement of components is not visualized easily through other microscope techniques (e.g. TEM and SEM). In the present work, we used AFM to study the sporopollenin nanostructure of the Ilex paraguariensis A.St.Hil. exine, an Angiosperm (Aquifoliaceae).

Diamond-like carbon (DLC) films have been intensively studied with a view to improving orthopaedic implants. Studies have indicated smoothness of the surface, low friction, high wear resistance, corrosion resistance and biocompatibility [1-4]. DLC coatings can be deposited using various techniques, such as plasma assisted chemical vapour deposition (PACVD), magnetron sputtering, laser ablation, and others [5]. However it has proved difficult to obtain films which exhibit good adhesion. The plasma immersion process, unlike the conventional techniques, allows the deposition of DLC on three-dimensional workpieces, even without moving the sample, without an intermediate layer, and with high adhesion [6], an important aspect for orthopaedic articulations. In our previous work, DLC coatings were deposited on silicon and Ti-13Nb-13Zr alloy substrates using the plasma immersion process for the characterization of microstructure, mechanical properties and corrosion behaviour [7-9]. Hardness, measured by a nanoindenter, ranged from 16.4-17.6 GPa, the pull test results indicate the good adhesion of DLC coatings to Ti-13Nb-13Zr, and electrochemical assays (polarization test and electrochemical impedance spectroscopy) indicate that DLC coatings produced by plasma immersion can improve the corrosion resistance [9].