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PubMed2006

Nanostructural analysis of starch components by atomic force microscopy.

Morphological and structural features of starch from potato (Solanum tuberosa) and rice (Oryza sativa) have been examined using atomic force microscopy. Amylose from potato and rice was observed in aggregated structures, which are suggested to be a result of retrogradation during sample preparation. The degrees of polymerization of amylose from potato and rice starches were calculated from the mean contour lengths of the observed structures to be approximately 1440 and 1860, respectively. Potato amylopectin appeared as a highly branched and extended molecule. Our results show that atomic force microscopy provides a useful method for examining the fine structural features and estimating the dimensions of starch molecules.

Amylopectin↗
PubMed2005

Atomic force microscopy and laser confocal scanning microscopy analysis of callose fibers developed from protoplasts of embryogenic cells of a conifer.

Efficiency of novel fiber formation was much improved in protoplast culture of embryogenic cells (ECs) of a conifer, Larix leptolepis (Sieb. et Zucc.) Gord., by pre-culturing ECs in a medium containing a high concentration of glutamine (13.7 mM). The fibrillar substructures of large and elongated fibers of protoplasts isolated from Larix ECs were investigated by laser confocal scanning microscopy (LCSM) after Aniline Blue staining and atomic force microscopy (AFM) using a micromanipulator without any pre-treatment. Fibers were composed of bundles of fibrils and subfibrils, whose diameters were defined as 0.7 and 0.17 mum, respectively, by image analysis after LCSM and AFM. These fibers were proven to be composed of callose by using specific degrading enzymes for beta-1,4-glucan and beta-1,3-glucan.

Carbohydrate Conformation↗
PubMed1998

High resolution mapping DNAs by R-loop atomic force microscopy.

R-loops formed by short RNA transcripts have been imaged by atomic force microscopy (AFM) at a constant force in the height mode. The technique was applied to mapping the human endogenous retrovirus K10 family (HERV-K10) long terminal repeats (LTR) within individual plasmids and cosmids. RNA probes specific for the U3 (384 nt) and U5 (375 nt) LTR regions separated by a span of 200 bp were used for R-loop formation with LTRs located within plasmid (3.8 kb) or cosmid ( approximately 40 kb) DNAs. R-loops stabilized by glyoxal treatment and adsorbed onto the mica surface in the presence of magnesium ions looked like looped out segments of RNA:DNA hybrids. The total yield of R-loops was usually approximately 95%. The RNA:DNA hybrids were found to be 12-15% shorter than the corresponding DNA:DNA duplex. The two regions of the LTR could be easily discerned in the AFM images as clearly separated loops. R-loop positions determined on cosmids by AFM were accurate to approximately 0.5% of the cosmid length. This technique might be easily adapted for mapping various sequences such as gene exons or regulatory regions and for detecting insertions, deletions and rearrangements that cause human genetic diseases.

Cosmids↗
PubMed2004

Imaging of all dangling bonds and their potential on the Ge/Si105 surface by noncontact atomic force microscopy.

High-resolution noncontact atomic force microscope (AFM) images were successfully taken on the Ge105-(1 x 2) structure formed on the Si105 substrate and revealed all dangling bonds of the surface regardless of their electronic situation, surpassing scanning tunneling microscopy, whose images strongly deviated from the atomic structure by the electronic states involved. An atomically resolved electrostatic potential profile by a Kelvin-probe method with AFM shows potential variations among the dangling bond states, directly observing a charge transfer between them. These results clearly demonstrate that high-resolution noncontact AFM with a Kelvin-probe method is an ideal tool for analysis of atomic structures and electronic properties of surfaces.

Journal Article↗
PubMed2002

A novel sample holder allowing atomic force microscopy on transmission electron microscopy specimen grids: repetitive, direct correlation between AFM and TEM images.

A novel sample holder that allows atomic force microscopy (AFM) to be performed on transmission electron microscope (TEM) grids is described. Consequently, AFM and TEM images were repeatedly obtained on exactly the same sample area. For both techniques, a thin carbon film was used as the imaging substrate. Although these techniques have been previously used in conjunction, AFM and TEM images on exactly the same area have not been repeatedly obtained for any system. Correlation of AFM and TEM images is useful for work where the three-dimensional topographical information provided by the AFM could be used to better interpret the two-dimensional images provided by the TEM and vice versa. To demonstrate the applicability of such correlation, new results pertaining to a fibrillar collagen system are summarized.

Fibrillar Collagens↗
PubMedDate not supplied

Imaging the surface of Staphylococcus aureus by atomic force microscopy.

The surfaces of four strains of Staphylococcus aureus, which differed in their expression of capsular polysaccharides, were examined using atomic force microscopy. The images show that it is possible to get information about surface characteristics of S. aureus using atomic force microscopy (AFM) following simple preparation. Strains Smith Diffuse (serotype 2), Reynolds (serotype 5), Wood-46 (capsule negative) and JL243 (capsule negative) were grown on medium known to promote the expression of capsular polysaccharides. The bacteria were air-dried prior to being imaged using tapping-mode AFM. Differences in the appearance of the bacterial surfaces were evident between the strains. The two capsule-negative strains exhibited a smooth regular surface, as opposed to the mucoid appearance of the two strains having polysaccharide capsules. Moreover, comparison of images of the heavily encapsulated serotype 2 strain and the serotype 5 strain indicates that a type 2 capsule can be distinguished from a type 5 microcapsule.

Microscopy, Atomic Force↗
PubMedDate not supplied

Imaging stretched single DNA molecules by pulsed-force-mode atomic force microscopy.

The effect of a surface water layer on DNA strands deposited on a substrate was studied by atomic force microscopy (AFM). DNA molecules were deposited and stretched on chemically modified glass coverslips by a molecular combing method. Lambda bacteriophage DNA molecules were aligned on the organosilane-modified substrate surfaces by chemical and physical adsorption during the molecular combing. The combed DNA molecules were observed in humidity-controlled air and in aqueous solutions by pulsed-force-mode AFM (PFM-AFM). Chemical modification of cantilevers with an Au-coated tip by organothiol compounds was also applied to DNA observation. Mapping adhesive forces in aqueous media was useful to discriminate chemically the DNA strands from the substrate surface. The results suggest that PFM-AFM can be used widely to image the stretched DNA molecules on the silane-modified substrates.

Adsorption↗
PubMed2001

Stepwise unfolding of titin under force-clamp atomic force microscopy.

Here we demonstrate the implementation of a single-molecule force clamp adapted for use with an atomic force microscope. We show that under force-clamp conditions, an engineered titin protein elongates in steps because of the unfolding of its modules and that the waiting times to unfold are exponentially distributed. Force-clamp measurements directly measure the force dependence of the unfolding probability and readily captures the different mechanical stability of the I27 and I28 modules of human cardiac titin. Force-clamp spectroscopy promises to be a direct way to probe the mechanical stability of elastic proteins such as those found in muscle, the extracellular matrix, and cell adhesion.

Animals↗
PubMed2002

Surface charge of Plasmodium falciparum merozoites as revealed by atomic force microscopy with surface potential spectroscopy.

Electric charges on the surface of Plasmodium falciparum merozoites and erythrocytes were investigated by atomic force microscopy with surface potential spectroscopy. The apical end of merozoites was positively charged, while the entire erythrocyte surface was negatively charged. Transmission electron microscopy also demonstrated that negatively charged nanogold particles attached to the apical end of merozoites, and cationized ferritin particles attached to the entire surface of the erythrocyte. This indicates that the surface charge at the apical end of the merozoite may play an important role in invasion of the erythrocyte.

Animals↗
PubMed2003

Analysis of insect holocentric chromosomes by atomic force microscopy.

In order to go in depth into the analysis of holocentric chromosome structure, atomic force microscopy (AFM) was applied to metaphase plates of the aphid Megoura viciae. AFM showed that aphid chromatids adhere to one another without any prominent structure detectable between them and without any evidence of chromosomal constrictions. AFM thus provided new and reliable evidences at a nanomolecular level concerning the holocentric structure of aphid chromosomes, without any of the artefacts due to sample staining or coating that are usually associated with electron microscopy.

Animals↗
PubMed1997

Application of atomic force microscopy to visualization of DNA, chromatin, and chromosomes.

The scanning force microscope (SFM, also called the atomic force microscope, AFM) provides a new and powerful method for visualization and manipulation of biological samples. Its high precision and sensitivity allow the investigator to interrogate samples at very high spatial resolution and simultaneously accumulate a variety of data types, including topography, viscoelasticity, chemical properties, and local friction. We provide here a brief review of the literature describing the current state of the art in the application of SFM to the study of DNA, chromatin and chromosomes, and some examples from this laboratory. Suggestions for future directions of this technology are also presented.

Animals↗
PubMed1988

Atomic force microscopy of an organic monolayer.

Atomic force microscope images of polymerized monolayers of n-(2-aminoethyl)-10,12-tricosadiynamide revealed parallel rows of molecules with a side-by-side spacing of approximately equal to 0.5 nanometer. Forces used for imaging (10(-8) newton) had no observable effect on the polymer strands. These results demonstrate that atomic force microscope images can be obtained for an organic system.

Amides↗
PubMed2006

Profiles of a high-aspect-ratio grating determined by spectroscopic scatterometry and atomic-force microscopy.

The new and fast scatterometry method called optical diffraction microscopy is compared with atomic-force microscopy by use of cross-section scanning-electron microscope images as references. The sample is a high-aspect-ratio grating with a period of approximately 1000 nm. To allow the atomic-force microscope to track all parts of the grating profile, the grating is investigated at different tilt angles. The measured quantities of the profile include sidewall angle gamma (approximately 90 degrees), groove height h (approximately 2000 nm), and degree of filling f (approximately 40%). The two methods, which respond to quite different material properties, give consistent results within standard uncertainties of u(gamma)</=0.8 degrees , u(h)</=15 nm, and u(f)</=1%.

Journal Article↗
PubMed2004

Imaging of human metaphase chromosomes by atomic force microscopy in liquid.

Human metaphase chromosomes were observed using an intermittent contact mode of atomic force microscopy (AFM) in a phosphate-buffered saline solution to clarify their conformation close to that in the physiological state. In the AFM images in liquid, symmetric alternating ridges and grooves were evident on their surface of the paired sister chromatids. The number of the ridges and grooves were rather specific to the type of the chromosome. The structural changes of chromosomes caused by trypsin treatment were also directly observable using AFM in liquid. These results suggest that the intermittent contact mode AFM is useful not only for analyzing the structure of chromosomes in a liquid condition but also for studying the effect of chemical treatments on chromosomes in relation to their structural changes.

Chromosomes, Human↗
PubMedDate not supplied

Use of atomic force microscopy to reveal sperm ultrastructure in HIV-patients on highly active antiretroviral therapy.

Atomic force microscopy (AFM) has been employed to examine morphological and topographical changes caused by human immunodeficiency virus (HIV) and the effects of highly active antiretroviral therapy (HAART) on spermatozoon of HIV infected patients. This powerful technique has allowed us to visualize morphological alterations present in the spermatozoa of patients either with or without treatment. In addition to this, even the minute details, such as viral particles, located on the membrane of the spermatozoa, and the merging of such particles on the surface of the spermatozoa were detected with precision. The most important aspect is that AFM, unlike electron microscopy, permits to image virions in their nearly natural environment. Excess of damage of spermatozoon is due to the chemicals involved in HAART rather than the damage made by virus.

Antiretroviral Therapy, Highly Active↗
PubMed1999

DNA bending by photolyase in specific and non-specific complexes studied by atomic force microscopy.

Specific and non-specific complexes of DNA and photolyase are visualised by atomic force microscopy. As a substrate for photolyase a 1150 bp DNA restriction fragment was UV-irradiated to produce damaged sites at random positions. Comparison with a 735 bp undamaged DNA fragment made it possible to separate populations of specific and non-specific photolyase complexes on the 1150 bp fragment, relieving the need for highly defined substrates. Thus it was possible to compare DNA bending for specific and non-specific interactions. Non-specific complexes show no significant bending but increased rigidity compared to naked DNA, whereas specific complexes show DNA bending of on average 36 degrees and higher flexibility. A model obtained by docking shows that photolyase can accommodate a 36 degrees bent DNA in the vicinity of the active site.

DNA↗
PubMed2004

Atomic force microscopy of cell growth and division in Staphylococcus aureus.

The growth and division of Staphylococcus aureus was monitored by atomic force microscopy (AFM) and thin-section transmission electron microscopy (TEM). A good correlation of the structural events of division was found using the two microscopies, and AFM was able to provide new additional information. AFM was performed under water, ensuring that all structures were in the hydrated condition. Sequential images on the same structure revealed progressive changes to surfaces, suggesting the cells were growing while images were being taken. Using AFM small depressions were seen around the septal annulus at the onset of division that could be attributed to so-called murosomes (Giesbrecht et al., Arch. Microbiol. 141:315-324, 1985). The new cell wall formed from the cross wall (i.e., completed septum) after cell separation and possessed concentric surface rings and a central depression; these structures could be correlated to a midline of reactive material in the developing septum that was seen by TEM. The older wall, that which was not derived from a newly formed cross wall, was partitioned into two different surface zones, smooth and gel-like zones, with different adhesive properties that could be attributed to cell wall turnover. The new and old wall topographies are equated to possible peptidoglycan arrangements, but no conclusion can be made regarding the planar or scaffolding models.

Cell Division↗