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Unbinding forces of weak, noncovalent bonds have been measured by scanning force microscopy (1) or biomembrane force probes (2). Initially, these scanning force microscopy measurements focused on feasibility studies to measure single biomolecular interactions (3-5). Recently, however, a few groups showed that these single molecule experiments give a direct link to bulk experiments where thermodynamic data are experimentally acquired (6-9). In contrast with bulk experiments where averaged properties are measured, a single molecular approach gives access to properties that are hidden in the ensemble. These experiments can give insight into the geometry of the energy landscape of a biomolecular bond (7,9-11). Some experiments even showed that intermediate states during unbinding (unfolding) exist which only can be detected by single molecule experiments (12-14). [ABSTRACT FROM AUTHOR]

With the advent of the atomic force microscopy (AFM), the study of biological samples has become more realistic because, in most cases, samples are not covered or fixed and this makes it possible to observe them while alive (1,2). This advantage of the AFM prompted a new invention: nanobiosensors using the cantilever (probe) of the AFM, which made possible the observation of specific molecules (including medications) as they enter or exit living cells (3,4). [ABSTRACT FROM AUTHOR]

Ordered aggregation of the β-amyloid (Aβ) peptide in the brain as plaques consisting of fibrils is an important characteristic of Alzheimer's disease (AD), a late onset neurodegenerative disease (1). Aβ derives from the endoproteolysis of the amyloid precursor protein (APP), which is a transmembrane protein containing 677-770 amino acids (2-9). The two most common forms of Aβ are the 40 and 42 residues long fragments respectively referred to as Aβ(40) and Aβ(42) (sequence shown in Fig. 1; ref. 10). The insoluble aggregated form of Aβ, which deposits in the extra cellular space in the brain and on the walls of cerebral blood vessels (6), exhibits an enhanced β-sheet conformation as opposed to the partially α-helical soluble form found in body fluids (11,12). Despite the lack of the definitive establishment of the causative role of Aβ in AD, evidence points to its aggregation and deposition in the pathogenesis of AD. [ABSTRACT FROM AUTHOR]

Transcription is a fundamental biochemical process in which an RNA molecule is synthesized according to its corresponding DNA template. [ABSTRACT FROM AUTHOR]

Since the appearance of the first pioneering article in the 1970s, particular efforts have been made to study the effect of exposure to electric and magnetic fields (EMFs) on living matter (1-10). The initial interest in radio frequency and microwaves has shifted to include the nonthermal, and essentially magnetic, effect of extremely low frequencies (ELF, 1-300 Hz), in particular the 50 and 60 Hz of the electric power system (3,4,9). The density of normal ELF fields is usually below 0.1 μT, but values of 0.5 μT may be found in front of television sets and computer monitors (11) as well as at a distance of 50 m from 300-kV high-voltage power lines (12). Values up to two to three orders of magnitude higher can be found near some domestic appliances or in some industrial processes, and pulsed magnetic fields in the range 1-10 mT are used in nuclear magnetic resonance imaging and for the therapy of soft tissue or nonhealing bone fractures (1).The increasing interest in ELF fields has been partially motivated by epidemiological reports of an increase in some types of cancer and leukemia in children (13,14) and workers exposed to high levels of ambient fields. Carcinogenesis is considered to be a multistep process consisting of initiation-promotion-progression stages (15). Although ELF magnetic fields are not considered sufficiently energetic to interact with DNA and initiate a cancerous process, they could, by acting on cellular receptors, affect cell proliferation and modulate the promotion or progression stage. [ABSTRACT FROM AUTHOR]

Before the advent of the atomic force microscope (AFM), scanning electron microscopy (SEM) was used to obtain high-resolution visualizations of the surface of biological samples. Normally, to scan samples of yeast cells, each preparation was coated with a film of evaporated gold approx 20 nm in thickness (1,2). Although necessary for scanning, the application of gold to the sample resulted in distortions in its surface. In addition, the application of a conductive coating to the surface effectively masked all the information that can exist below the gold film. The AFM apparatus permits the observation of samples without the use of this mask (samples are uncoated and nonfixed). If we compare the thickness of the gold coating to the thickness of the yeast cell wall (Saccharomyces cerevisiae cell wall is about 25 nm; ref. 3), we find that they have approximately the same dimensions, which results in loss of resolution from the surface of the cells, including any changes that might occur on the cell wall. With improvements in AFM technology, it became possible to examine the surface of many preparations at much greater resolutions than previously described (4). Recently, it has become possible to observe, with AFM, that the surface of the cell wall of S. cerevisiae contains natural undulations (rugosities) never described when SEM was used (4) and that these cell walls contain pores along the surface that vary from strain to strain (4). With AFM is also possible to observe pores on membrane of others eukaryotic cells (5). [ABSTRACT FROM AUTHOR]

Atomic force microscopy (AFM) has been successfully applied not only to the topography of biological specimens but also to the measurement of their local mechanical properties. This technique is very useful for imaging such biological specimens as cells, proteins, and DNA, because no special treatments of samples are required (1,2). The measurement of local mechanical properties in a living cell is achieved with a nanoindentation technique (3). The method has been applied to several kinds of cultured cells, including Madin-Darby canine kidney (MDCK) cells (4), myocytes (5), fibroblasts (6,7), and endothelial cells (8,9). However, morphology and properties are different between cells cultured in vitro on substrate and intact cells in vivo (10). [ABSTRACT FROM AUTHOR]

In the last years the atomic force microscope (AFM; ref. 1) has become a powerful imaging tool for the biologist. The unique features like the possibility to image biological structures in their native environment (i.e., in buffer solution, at room temperature, and under normal pressure), the high lateral and vertical resolution, and the high signal-to-noise ratio of the topographs acquired by AFM make this instrument outstanding. It has made the observation of different single biomolecules at work and the monitoring of biomolecular interactions by time-lapse AFM possible (for recent reviews, see refs. 2-4). [ABSTRACT FROM AUTHOR]

Epithelial sodium channels (ENaC) mediate Na reabsorption across a variety of sodium reabsorbing epithelia, such as the kidney, distal colon, and airway. Normal function of these channels is critical for processes as diverse as blood volume control and airway fluid homeostasis. The molecular cloning of ENaC from a variety of epithelial cells has revealed that they are composed of three homologous subunits, such as α, β, and γ (1). [ABSTRACT FROM AUTHOR]

The steroid hormone aldosterone, which is synthesized in the suprarenal glands and secreted in response to a reduction in circulating blood volume, increases water and sodium reabsorption in the kidney (1,2). Although kidney is the major target organ, various other cell types, including different epithelia, smooth muscle, and endothelium, respond to the hormone (3-6). The acute aldosterone-induced responses of target cells are an intracellular calcium change and an intracellular pH increase (3,7,8) along with activation of plasma membrane Na+/H+ exchange and plasma membrane proton conductance (9,10). Therefore, it is not surprising that researchers have postulated that these acute transmembrane shifts of electrolytes are accompanied by cell swelling (2,11). Cell swelling or shrinkage plays a critical role in endothelial cell (EC) function. ECs tightly coat the luminal surface of blood vessels, playing an important role in the regulation of vascular tone, in vascular remodeling, in the pathogenesis of arteriosclerosis, and in arterial hypertension of humans (12). It has been shown that swelling of EC may disturb cell-to-cell interactions, resulting in an increase of transendothelial permeability, a precursor mechanism in the development of arteriosclerosis (13,14). Moreover, environmental stress (e.g., mechanical forces or hyperosmolarity) induces changes in cell volume and stimulates tissue plasminogen activator synthesis (15-18). Direct evidence for the importance of cell-volume regulation of endothelial cells is the existence of a volume-sensitive protein kinase (19). [ABSTRACT FROM AUTHOR]

The detailed understanding of the nuclear cell functions requires an accurate knowledge of the spatial organization of their structures. For many years the study of human metaphase chromosomes was conducted with light microscopy after staining protocols disturbing the native chromosomal structure. The approach by scanning electron microscopy (SEM) provides higher resolution as compared with light microscopy and permits surface analysis of the chromosomal structure, which cannot be adequately obtained from transmission electron microscopy. Nevertheless, to obtain high resolution in SEM observations, the use of a high electron accelerating voltage (up to 30 kV) is required (1). With these experimental conditions, the sputter coating or a conductive staining of the samples is generally required (1,2). Both procedures allow electron-charging dispersion from the sample but may obscure fine details and produce sample alterations (3). [ABSTRACT FROM AUTHOR]

Pulmonary surfactant (PS) is a lipid protein complex secreted at the terminal airways of the lung. The material is secreted as lipid rich multilamellate bodies, which transforms into lipid—protein tubules, planar bilayers, and monomolecular films at the alveolar air—aqueous interface (1,2). The films reduce the surface tension of the interface and prevents lung collapse during end expiration (3). PS layers also act as a protective barrier against inhaled particles and bacteria and keeps the upper airways or bronchioles open during respiration (3). Dysfunction of PS has been implicated in various lung diseases, such as asthma, acute respiratory distress syndrome, cystic fibrosis, and pneumonia (4). The composition of PS is conserved in most air-breathing species; however, its high content of saturated phosphatidylcholine (PC) and phosphatidylglycerol (PG) is unique compared with other secretory materials and cell membranes, which lack these phospholipids (1,5). Specifically, PS contains significant amounts of dipalmitoylphosphatidylcholine (DPPC), palmitoyl-oleyl-PC (POPC) and PG (POPG), cholesterol, and small amounts (10%) of surfactant proteins SP-A, SP-B, SP-C, and SP-D (1). It is not clear to date how this lipid—protein complex functions by forming alveolar films or barrier in situ because such fragile and dynamic films are difficult to preserve for traditional electron microscopy (2,3). In vitro studies have focused on model lipid—protein films of PS and also by extracting the material out of lungs and studying interfacial properties of surface tension of such material using Langmuir and other surface balances (6-8). [ABSTRACT FROM AUTHOR]

Scanning probe microscopy (SPM) is a rather new family of surface studies methods, having broad applications to biomedical science. The main advantage of SPM over conventional microscopic techniques (i.e., scanning and transmission electron microscopy) is its ability to study living objects in their natural environment and, therefore, the ability to observe dynamical processes occurring to them with subnanometer resolution. Here, we will discuss one of its family members, atomic force microscopy (AFM), which has yielded fascinating results for the past decade. [ABSTRACT FROM AUTHOR]

In 1986, Binnig et al. (1) revolutionized microscopy through the invention of the atomic force microscope (AFM). Subsequently, commercial instruments of this new imaging technique began to appear in the 5 yr after its discovery. In the early 1990s, Henderson et al. (2) and Radmacher et al. (3) both illustrated the potential of the instrument to image biological preparations in real time under nearby physiological conditions with nanometer resolution. From that point onwards the AFM has attracted biologists and the number of publications describing biological applications of AFM have grown rapidly (4). [ABSTRACT FROM AUTHOR]

The cell nucleus is an organelle where molecules involved in gene expression are highly compartmentalized (1) in very dynamic (2) territories. This current notion of functional organization has been possible because of many studies of this organelle that included its cell and molecular organization, and where microscopy played an important role. Light and electron microscopy in conjunction with molecular approaches, such as the construction of both antibodies and nucleic acid probes, as well as molecular construction of genes with green fluorescent protein and fluorescence recovery after photobleaching technology, in addition to high-resolution in situ hybridization and immunocy-tochemistry now offer a more complete knowledge. [ABSTRACT FROM AUTHOR]

Bacteria are typically smaller than eukaryotic cells. The average diameter of Staphylococcus aureus is 1±0.5 μm, whereas Escherichia coli is on average 0.5×1.5 μm. The bacterial cell is also characterized by the presence of a complex external rigid structure called cell wall, which protects the internal protoplast and gives also the cellular shape, that generally falls into one of the, three basic morphologic categories, spherical (cocci), rod-shaped (bacilli), and spiral. Some bacteria show an atypical bacterial shape. [ABSTRACT FROM AUTHOR]

At the present time there exists a great deal of interest in the application of scanning probe microscopy methods to the imaging of cellular systems (1,2). It would now not be an exaggeration to state that atomic force microscopy (AFM), in particular, represents perhaps the most powerful means of structural/functional analysis at the level of a single live cell. In recent years this technology has been applied, amongst many other systems, to studies of bacterial flagella (3), erythrocytes (4), human platelets (5), endothelial cells (6), skin fibroblasts (7), plant cuticles (8), and cardiac myocytes (9). In resolution terms, perhaps the most impressive work is that of Engel et al. (10). Although not on whole cells (thereby greatly simplifying the experiment), molecular-level images of isolated cellular gap junctions, which play an important role in intracellular communication and signal transduction, have been obtained. More recently, experimental protocols have advanced to the point where it is possible to monitor, simultaneously, both cellular topography and ion channel flux (11). The ability to characterize functionally active cellular systems at a nanometre resolution under controlled fluid conditions has also been used in the monitoring of time-dependent cellular change (12,13). [ABSTRACT FROM AUTHOR]

Atomic force microscopy (AFM) is a valuable technique for the study of demineralization and the effects of other solutions and environments on the structure of human dentin because high-resolution studies of changes in structure and dimensions are possible in nearly any environment over time (1). Because dentin forms the bulk of the tooth and is subject to a variety of alterations as a result of disease, age, and treatment (1), such studies are useful for understanding the basic structure—property relations of dentin and for evaluation of various conservative and restorative dental treatments. Initial studies of demineralization and dehydration of dentin were reported in 1993 (2,3) in which relative changes in dimensions were used to evaluate these processes. It soon became apparent that a stable reference layer on the surface or embedded in the specimen would be most desirable for evaluation of time-dependent changes and to take advantage of the high-resolution capabilities of the AFM. In 1995, the use of paint-on varnish or photoresist (4) and evaporated gold layers (5) were developed that allowed evaluation of demineralization, either continuously or sequentially, for a variety of demineralizing agents. An additional technique of interest is the use of AFM-based nanoindention for measurements of site-specific hardness and elastic modulus of dentin. Such measurements can frequently be performed as a portion of the same study in which demineralization experiments are conducted. [ABSTRACT FROM AUTHOR]

A large body of recent literature describes the use of atomic force microscopy (AFM; ref. 1) for the study of living cells. These experimental findings clearly indicate that AFM is a very valuable tool for the 3D imaging of flat biological samples strongly adhering to a substrate, with a lateral resolution in between the resolutions of optical and electron microscopy. Moreover, a very relevant feature of AFM is its capability of analyzing local mechanical properties of living cells. [ABSTRACT FROM AUTHOR]

In its most established mode of operation, named constant force contact mode, atomic force microscopy (AFM) has been applied to image the 2D and 3D architecture of surfaces. Any deflection of the tip as a result of surface topography is recorded. The microscope reconstructs an image of the surface from the x, y, and z scan data to develop a 3D illustration of any surface at the micro- and nanometer level. The production of high-resolution images of a wide variety of biological samples at near-native conditions and the possibility to measure very low local forces is proving to be a powerful tool for cell analysis (1,2). In contrast with electron microscopy observations in particular, AFM improves biological studies involving imaging by also monitoring dynamic processes. However, the investigation of soft biomaterials with this special method is still challenging. This chapter reviews practical details of imaging two cell lines: human chondrocytes and human osteosarcoma. However, characteristics described are not unique to this type of cell. Principally, all types of adherently growing cells can be investigated with the techniques described here. Force curve analysis, as a backdrop for the understanding of the received images (1), will be introduced in detail in Subheading 3.4. Further sections explore how AFM can be used as a helpful tool in observations of the cell surface and the physical interactions that occur there, like adhesion or friction, and their influence on the active cell. In Subheading 7. common artifacts and troubles are described, along with the practical instructions. [ABSTRACT FROM AUTHOR]

Atomic force microscopy (AFM) is an ideal technique for noninvasive examination of hair surfaces (1-11), providing a wealth of structural information not always apparent from electron microscopy. The fine cuticular structure of human head hair is of interest to those engaged in the fields of dermatology (12-14), cosmetics (15-17), and forensic science (18-20). In the former, the morphology of hair can be affected by an underlying inherited or congenital metabolic disorder, such as maple syrup urine disease (21) or monilethrix (22), respectively. The cosmetics industry is interested in the effects of haircare formulations, such as conditioning and bleaching agents, on hair cuticle surfaces (23). There is now increasing legislation on cosmetic manufacturers to be able to substantiate claims made concerning their products. [ABSTRACT FROM AUTHOR]

If a sample has a comparatively even surface and is fixed on a sample stage, atomic force microscopy (AFM) will give a clear image of the surface structure at subnanometer level (1,2). Because a sperm head is flat and can be attached on the slide glass firmly after it is fixed, we consider that AFM is the competent tool for the study of the sperm surface structure. [ABSTRACT FROM AUTHOR]

The cornea is the transparent avascular part of the anterior segment of the eye and consists of a stratified nonkeratinizing squamous epithelium, a stromal dense connective tissue layer, and an endothelium facing the anterior chamber. The cornea contributes largely to the intraocular refraction of the light. Damage can impair its tissue transparency and lead to loss of vision. Significant diseases, such as corneal dystrophies, keratoconus, and refractive errors, are related to the structure and integrity of the cornea. [ABSTRACT FROM AUTHOR]

The force-sensing members of the large family of scanning probe microscopies have become important tools during the past decade for visualizing, characterizing, and manipulating objects and processes on the meso- and nanoscale level. The atomic force microscope (AFM), in particular, has had an impact in the life sciences. In cell science, the pioneering work with AFM was conducted in the early 1990s (1-3). The methodologies have now reached a stage of relative maturity (4). The principal merit of the AFM is as a nonintrusive local probe of live cells and their dynamics in the biofluid environment. As well as offering high spatial resolution imaging in one or more operational modes, the AFM can deliver characterization of mechanical properties and local chemistry through operation in the force-vs-distance (F-d) mode (e.g., ref. 5). The lateral resolution delivered by the AFM will in most cases, and especially for soft materials, be inferior to that obtained by electron-optical techniques, but the z-resolution is routinely in the nanometer range with a depth of focus equal to the dynamic range of the z-stage travel. The instrument may be operated in one of several modes, of which the most common ones are as follows: the contact mode, using a soft lever in which contours of constant strength of interaction are traced out; the intermittent-contact mode, in which a relatively stiff lever is vibrated at a frequency near that of a free-running resonance and in which contours of constant decrement of the free-running amplitude or a constant phase shift are mapped; and the F-d mode, in which the local stiffness of interaction between tip and specimen is determined over a range of applied force (lever deflection and z-stage travel being the two measurable variables). [ABSTRACT FROM AUTHOR]

During the last few years, a series of new detection methods in the field of biosensors have been developed. Biosensors are analytical devices that combine a biologically sensitive element with a physical or chemical transducer to selectively and quantitatively detect the presence of specific compounds in a given external environment. [ABSTRACT FROM AUTHOR]

Images taken with the atomic force microscope (AFM) originate in physical interactions that are totally different from those used for image formation in conventional light and electron microscopy. One of the effects is that a new series of artifacts can appear in images that may not be readily recognized by users accustomed to conventional microscopy. Because we are addressing our-selves to novices in this field, we would like to give an idea of what can happen while taking images with the AFM, how one can recognize the source of the artifact, and then try to avoid it or minimize it. Essentially, one can identify the following sources of artifacts in AFM images: the tip, the scanner, vibrations, the feedback circuit, and image-processing software. [ABSTRACT FROM AUTHOR]

One can easily distinguish between two general modes of operation of the atomic force microscope (AFM) depending on absence or presence in the instrumentation of an additional device that forces the cantilever to oscillate in the proximity of its resonant frequency. The first case is usually called static mode, or DC mode, because it records the static deflection of the cantilever, whereas the second takes a variety of names (some patented) among which we may point out the resonant or AC mode. In this case, the feedback loop will try to keep at a set value not the deflection but the amplitude of the oscillation of the cantilever while scanning the surface. To do this, additional electronics are necessary in the detection circuit, such as a lock-in or a phase-locked loop amplifier, and also in the cantilever holder to induce the oscillatory excitation. [ABSTRACT FROM AUTHOR]

Microscopes have always been one of the essential instruments for research in the biomedical field. Radiation-based microscopes (such as the light microscope and the electron microscope) have become trustworthy companions in the laboratory and have contributed greatly to our scientific knowledge. However, although digital techniques in recent years have still enhanced their performance, the limits of their inherent capabilities have been progressively reached. [ABSTRACT FROM AUTHOR]