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At least 1,423 records · Page 79Linked to original sources
PubMed2001

Quantification of red blood cells using atomic force microscopy.

For humans the sizes and shapes of their red blood cells are important indicators of well being. In this study, the feasibility of using the atomic force microscope (AFM) to provide the sizes and shapes of red blood cells has been investigated. An immobilisation procedure has been developed that enabled red blood cells to be reliably imaged by contact AFM in air. The shapes of the red blood cells were readily apparent in the AFM images. Various cell quantification parameters were investigated, including thickness, width, surface area and volume. Excellent correlation was found between the AFM-derived immobilised mean cell volume (IMCV) parameter and the mean cell volume (MCV) parameter used in current haematological practice. The correlation between MCV and IMCV values has validated the immobilisation procedure by demonstrating that the significant cell shrinkage that occurs during immobilisation and drying does not introduce quantification artifacts. Reliable IMCV values were obtained by quantifying 100 red blood cells and this typically required 3-5 AFM images of 100 microm x 100 microm area. This work has demonstrated that the AFM can provide in a single test the red blood cell size and shape data needed in the assessment of human health.

Anemia, Macrocytic↗
PubMed2006

Anionic polyelectrolyte adsorption on mica mediated by multivalent cations: a solution to DNA imaging by atomic force microscopy under high ionic strengths.

Adsorption of DNA molecules on mica, a highly negatively charged surface, mediated by divalent or trivalent cations is considered. By analyzing atomic force microscope (AFM) images of DNA molecules adsorbed on mica, phase diagrams of DNA molecules interacting with a mica surface are established in terms of concentrations of monovalent salt (NaCl) and divalent (MgCl2) or multivalent (spermidine, cobalt hexamine) salts. These diagrams show two transitions between nonadsorption and adsorption. The first one arises when the concentration of multivalent counterions is larger than a limit value, which is not sensitive to the monovalent salt concentration. The second transition is due to the binding competition between monovalent and multivalent counterions. In addition, we develop a model of polyelectrolyte adsorption on like-charged surfaces with multivalent counterions. This model shows that the correlations of the multivalent counterions at the interface between DNA and mica play a critical role. Furthermore, it appears that DNA adsorption takes place when the energy gain in counterion correlations overcomes an energy barrier. This barrier is induced by the entropy loss in confining DNA in a thin adsorbed layer, the entropy loss in the interpenetration of the clouds of mica and DNA counterions, and the electrostatic repulsion between DNA and mica. The analysis of the experimental results provides an estimation of this energy barrier. We then discuss some important issues, including DNA adsorption under physiological conditions.

Adsorption↗
PubMed1996

Chaperonins GroEL and GroES: views from atomic force microscopy.

The Escherichia coli chaperonins, GroEL and GroES, as well as their complexes in the presence of a nonhydrolyzable nucleotide AMP-PNP, have been imaged with the atomic force microscope (AFM). We demonstrate that both GroEL and GroES that have been adsorbed to a mica surface can be resolved directly by the AFM in aqueous solution at room temperature. However, with glutaraldehyde fixation of already adsorbed molecules, the resolution of both GroEL and GroES was further improved, as all seven subunits were well resolved without any image processing. We also found that chemical fixation was necessary for the contact mode AFM to image GroEL/ES complexes, and in the AFM images. GroEL with GroES bound can be clearly distinguished from those without. The GroEL/ES complex was about 5 nm higher than GroEL alone, indicating a 2 nm upward movement of the apical domains of GroEL. Using a slightly larger probe force, unfixed GroEL could be dissected: the upper heptamer was removed to expose the contact surface of the two heptamers. These results clearly demonstrate the usefulness of cross-linking agents for the determination of molecular structures with the AFM. They also pave the way for using the AFM to study the structural basis for the function of GroE system and other molecular chaperones.

Adenylyl Imidodiphosphate↗
PubMed1994

Imaging of the membrane surface of MDCK cells by atomic force microscopy.

The membrane surface of polarized renal epithelial cells (MDCK cells) grown as a monolayer was imaged with the atomic force microscope. The surface topography of dried cells determined by this approach was consistent with electron microscopy images previously reported. Fixed and living cells in aqueous medium gave more fuzzy images, likely because of the presence of the cell glycocalix. Treatment of living cells with neuraminidase, an enzyme that partly degrades the glycocalix, allowed sub-micrometer imaging. Protruding particles, 10 to 60 nm xy size, occupy most of the membrane surface. Protease treatment markedly reduced the size of these particles, indicating that they corresponded to proteins. Tip structure effects were probably involved in the exaggerated size of imaged membrane proteins. Although further improvements in the imaging conditions, including tip sharpness, are required, atomic force microscope already offers the unique possibility to image proteins at the membrane surface of living cells.

Animals↗
PubMed2005

Quantitative atomic force microscopy image analysis of unusual filaments formed by the Acanthamoeba castellanii myosin II rod domain.

We describe a quantitative analysis of Acanthamoeba castellanii myosin II rod domain images collected from atomic force microscope experiments. These images reveal that the rod domain forms a novel filament structure, most likely requiring unusual head-to-tail interactions. Similar filaments are seen also in negatively stained electron microscopy images. Truncated myosins from Acanthamoeba and other model organisms have been visualized before, revealing laterally associated bipolar minifilaments. In contrast, the filament structures that we observe are dominated by axial rather than lateral polymerization. The unusually small features in this structure (1-5 nm) required the development of quantitative and statistical techniques for filament image analysis. These techniques enhance the extraction of features that hitherto have been difficult to ascertain from more qualitative imaging approaches. The heights of the filaments are observed to have a bimodal distribution consistent with the diameters of a single rod domain and a pair of close-packed rod domains. Further quantitative analysis indicates that in-plane association is limited to at most a pair of rod domains. Taken together, this implies that the filaments contain no more than four rod domains laterally associated with one another, somewhat less than that seen in bipolar minifilaments. Analysis of images of the filaments decorated with an anti-FLAG antibody reveals head-to-tail association with mean distances between the antibodies of 75 +/- 15 nm. We consider a set of molecular models to help interpret possible structures of the filaments.

Acanthamoeba castellanii↗
PubMed2001

Ultrastructure and assembly of segmental long spacing collagen studied by atomic force microscopy.

The in vitro formation of segmental long spacing (SLS) collagen as induced by the addition of ATP to acidified Type I collagen solutions has been examined with the atomic force microscope (AFM). AFM images obtained suggest that the assembly proceeds in a stepwise manner, through an intermediate stage of oligomers, which then associate laterally to form the so-called "SLS crystallites". Attempts to induce SLS formation by the addition of other polyanionic species to monomeric collagen solutions met with mixed success; ATP-gamma-S and GTP produced SLS crystallites, whereas inorganic phosphate and other polyanionic dyes did not. This indicates that the formation of SLS cannot simply be attributed to the negation of positive charges believed to be located on the end of the collagen monomer, but rather it is a complex function of the structure and charge of both the collagen monomer and polyanion.

Adenosine Triphosphate↗
PubMed2001

A study of fibrous long spacing collagen ultrastructure and assembly by atomic force microscopy.

Fibrous long spacing collagen (FLS) fibrils are collagen fibrils that display a banding with periodicity greater than the 67nm periodicity of native collagen. FLS fibrils can be formed in vitro by addition of alpha(1)-acid glycoprotein to an acidified solution of monomeric collagen, followed by dialysis of the resulting mixture. We have investigated the ultrastructure of FLS fibrils formed in vitro using the atomic force microscope (AFM). The majority of the fibrils imaged showed typical diameters of approximately 150nm and had a distinct banding pattern with a approximately 250nm periodicity. However, we have also observed an additional type of FLS fibril, which is characterized by a secondary banding pattern surrounding the primary bands. These results are compared with those obtained in past investigations of FLS ultrastructure carried out using the transmission electron microscope (TEM). The importance of the fibril's surface topography in TEM staining patterns is discussed. Images of FLS fibrils in various stages of assembly have also been collected, and the implications of these images in determining the mechanism of assembly and the formation of the characteristic banding pattern of the fibrils is discussed.

Animals↗
PubMed1994

Atomic force microscopy and scanning tunnelling microscopy: refining techniques for studying biomolecules.

The scanning tunnelling microscope and the atomic force microscope offer the prospect of real-time, nanometre-scale imaging of biomolecules and biosurfaces under physiological environments. Much effort is therefore being made to establish these techniques as routine biophysical tools. The considerable recent progress that has been made in biotechnological applications is reviewed, highlighting specific examples of the applications of this new and exciting method of analysis.

Biological Products↗
PubMedDate not supplied

Ultrastructural pathology of varicocele spermatozoa by using atomic force microscopy (AFM).

Ultrastructural investigation of pathological spermatozoa of patients having varicocele (grades I and III) in its natural environment (without fixation, dehydration, embedding, sectioning, etc.) was carried out by using Atomic Force Microscope (AFM) in tapping mode. This is a unique technique to examine the topographical structure with precision and to obtain 3-D images. It revealed structural alterations caused by the presence of varicocele. Not only neckpieces and flagella were defective, but also the head; particularly, dimensions and heights are altered.

Case-Control Studies↗
PubMed1997

Ku proteins join DNA fragments as shown by atomic force microscopy.

The binding of the Ku protein to DNA was investigated using the atomic force microscope. Ku was found to bind predominantly to the ends of double-stranded DNA. Experiments with plasmid DNA revealed that Ku does not bind to circular plasmids but does bind to plasmids that have been linearized by treatment with ionizing radiation. The binding of Ku to poly(dG-dC) x poly(dG-dC) polynucleotides and to a 400-bp DNA EcoRI fragment resulted in a shift in the fragment size distribution to include longer fragments, with internally binding Ku. Furthermore, we observed images consistent with fragments joined together by Ku, showing an interaction with two ends of DNA. These observations suggest that Ku may play a role in physically orienting DNA for ligation by binding the ends of adjacent DNA molecules.

Antigens, Nuclear↗
PubMed2003

Seeing the atomic orbital: first-principles study of the effect of tip termination on atomic force microscopy.

We perform extensive first-principles calculations to simulate the topographical atomic-force-microscope image of an adatom on the Si(111)-(7 x 7) surface, demonstrating the feasibility of imaging not only the atoms but also the atomic orbitals. Our comparative study of tip terminations shows that two subatomic features can appear for a single adatom when it is imaged by a Si(001)-type tip having two dangling bonds on its apex, while only one feature would appear if it were imaged by a Si(111)-type tip having one dangling bond on the apex. The key condition for seeing the atomic orbitals is to bring the tip so close to the surface that the angular-dependent force dominates the tip-surface interaction.

Journal Article↗
PubMed2002

Lipid membrane phase behaviour elucidated in real time by controlled environment atomic force microscopy.

Lipids are integral components of all biological membranes. Understanding the physical and chemical properties of these lipids is critical to our understanding of membrane functions. We developed a new atomic force microscope (AFM) approach to visualize in real time the temperature-induced lipid phase transition and domain separation processes in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) membranes and estimate the thermodynamics of the phase transition process. The gel and liquid crystalline phases of DMPC coexisted over a broad temperature range (approximately 10 degrees C). Equal partitioning into two phases occurred at a transition temperature (Tm) of 28.5 degrees C. We developed a mathematical model to analyse AFM-derived DMPC membrane height changes as multi-peak Gaussian distributions. This approach allowed us to estimate the DMPC domain size, N, as 18-75 molecules per leaflet corresponding to a -4.2 nm diameter circular nanodomain. Lipid nanodomains may organize into microdomains or rafts which, in concert with proteins and other lipid components, play an important dynamic role in many biomedically important processes.

Dimyristoylphosphatidylcholine↗
PubMed2006

Measurement of the mechanical properties of isolated tectorial membrane using atomic force microscopy.

The tectorial membrane (TM) is an extracellular matrix situated over the sensory cells of the cochlea. Its strategic location, together with the results of recent TM-specific mutation studies, suggests that it has an important role in the mechanism by which the cochlea transduces mechanical energy into neural excitation. A detailed characterization of TM mechanical properties is fundamental to understanding its role in cochlear mechanics. In this work, the mechanical properties of the TM are characterized in the radial and longitudinal directions using nano- and microindentation experiments conducted by using atomic force spectroscopy. We find that the stiffness in the main body region and in the spiral limbus attachment zone does not change significantly along the length of the cochlea. The main body of the TM is the softest region, whereas the spiral limbus attachment zone is stiffer, with the two areas having averaged Young's modulus values of 37 +/- 3 and 135 +/- 14 kPa, respectively. By contrast, we find that the stiffness of the TM in the region above the outer hair cells (OHCs) increases by one order of magnitude in the longitudinal direction, from 24 +/- 4 kPa in the apical region to 210 +/- 15 kPa at the basilar end of the TM. Scanning electron microscopy analysis shows differences in the collagen fiber arrangements in the OHC zone of the TM that correspond to the observed variations in mechanical properties. The longitudinal increase in TM stiffness is similar to that found for the OHC stereocilia, which supports the existence of mechanical coupling between these two structures.

Animals↗
PubMed1996

The chromatin structure of well-spread demembranated human sperm nuclei revealed by atomic force microscopy.

The fundamental structure formed when genomic DNA is packaged by protamine in the human sperm nucleus still remains essentially unresolved. It is known that the binding of protamine, a small arginine-rich protein, to DNA generates a large dense, hydrophobic complex making the sperm chromatin structure difficult to study microscopically. To visualize the internal nuclear structures, isolated human sperm nuclei were swollen extensively in saline buffer using only a reducing agent. The nuclei were swollen during deposition onto coverglass and then imaged in the atomic force microscope (AFM). The two main results obtained from imaging individual well-spread nuclei indicate that native human sperm chromatin is: (1) particulate, consisting primarily of large nodular structures averaging 98 nm in diameter, and (2) also composed of smaller, nucleosome-like particles observed to form linear chains near the nuclear periphery. These two types of chromatin particles imaged by AFM are remarkably similar to other AFM measurements made on native and reconstituted sperm and somatic chromatin.

Cell Nucleus↗
PubMed2001

Single-molecule imaging by atomic force microscopy of the native chaperonin complex of the thermophilic archaeon Sulfolobus solfataricus.

The chaperonin of the extremely thermophilic archaeon Sulfolobus solfataricus has been imaged for the first time under native conditions using the atomic force microscope. This technique allows to visualize the structure of biomolecules in solution under physiological conditions providing a nanometer resolution topographic image of the sample. Single molecule studies can reveal fine structural details, providing a powerful insight into the active conformation of a macromolecule, and also allowing to detect different conformational states corresponding to functional changes.

Archaeal Proteins↗
PubMed2000

Energy landscape of streptavidin-biotin complexes measured by atomic force microscopy.

The dissociation of ligand and receptor involves multiple transitions between intermediate states formed during the unbinding process. In this paper, we explored the energy landscape of the streptavidin-biotin interaction by using the atomic force microscope (AFM) to measure the unbinding dynamics of individual ligand-receptor complexes. The rupture force of the streptavidin-biotin bond increased more than 2-fold over a range of loading rates between 100 and 5000 pN/s. Moreover, the force measurements showed two regimes of loading in the streptavidin-biotin force spectrum, revealing the presence of two activation barriers in the unbinding process. Parallel experiments carried out with a streptavidin mutant (W120F) were used to investigate the molecular determinants of the activation barriers. From these experiments, we attributed the outer activation barrier in the energy landscape to the molecular interaction of the '3-4' loop of streptavidin that closes behind biotin.

Biotin↗
PubMed2003

Raman-atomic force microscopy of the ommatidial surfaces of Dipteran compound eyes.

The ommatidial lens surfaces of the compound eyes in several species of files (Insecta: Diptera) and a related order (Mecoptera) were analyzed using a recently developed Raman-atomic force microscope. We demonstrate in this work that the atomic force microscope (AFM) is a potentially useful instrument for gathering phylogenetic data and that the newly developed Raman-AFM may extend this application by revealing nanometer-scale surface chemistry. This is the first demonstration of apertureless near-field Raman spectroscopy on an intact biological surface. For Chrysopilus testaceipes Bigot (Rhagionidae), this reveals unique cerebral cortex-like surface ridges with periodic variation in height and surface chemistry. Most other Brachyceran flies, and the "Nematoceran" Sylvicola fenestralis (Scopoli) (Anisopodidae), displayed the same morphology, while other taxa displayed various other characteristics, such as a nodule-like (Tipula (Triplicitipula) sp. (Tipulidae)) or coalescing nodule-like (Tabanus punctifer Osten Sacken (Tabanidae)) morphology, a smooth morphology with distinct pits and grooves (Dilophus orbatus (Say) (Bibionidae)), or an entirely smooth surface (Bittacus chlorostigma MacLachlan (Mecoptera: Bittacidae)). The variation in submicrometer structure and surface chemistry provides a new information source of potential phylogenetic importance, suggesting the Raman-atomic force microscope could provide a new tool useful to systematic and evolutionary inquiry.

Animals↗
PubMed1988

Atomic force microscopy of purple membranes.

The surface structure of purple membranes was imaged using an atomic force probe mounted in a scanning tunnelling microscope. One of the two different membrane surfaces showed protruding, disc-shaped features forming an hexagonal lattice with about 6 nm centre to centre spacing. These are identified as the cytoplasmic surfaces of trimers of bacteriorhodopsin molecules and are correlated with the structural information on bacteriorhodopsin obtained from numerous earlier electron microscope and diffraction studies.

Bacteriorhodopsins↗