Removal of histone tails from nucleosome dissects the physical mechanisms of salt-induced aggregation,linker histone H1-induced compaction,and 30-nm fiber formation of the nucleosome array

In order to reveal the roles of histone tails in the formation of higher-order chromatin structures, we employed atomic force microscopy (AFM), and an in vitro reconstitution system to examine the properties of reconstituted chromatin composed of tail-less histones and a long DNA (106-kb plasmid) template. The tail-less nucleosomes did not aggregate at high salt concentrations or with an excess amount of core histones, in contrast with the behavior of nucleosomal arrays composed of nucleosomes containing normal, N-terminal tails. Analysis of our nucleosome distributions reveals that the attractive interaction between tail-less nucleosomes is weakened. Addition of linker histone H1 into the tail-less nucleosomal array failed to promote the formation of 30 nm chromatin fibers that are usually formed in the normal nucleosomal array. These results demonstrate that the attractive interaction between nucleosomes via histone tails plays a critical role in the formation of the uniform 30-nm chromatin fiber.     

Sharing of secondary electrons by in-lens and out-lens detector in low-voltage scanning electron microscope equipped with immersion lens

To understand secondary electron (SE) image formation with in-lens and out-lens detector in low-voltage scanning electron microscopy (LV-SEM), we have evaluated SE signals of an in-lens and an out-lens detector in LV-SEM. From the energy distribution spectra of SEs with various boosting voltages of the immersion lens system, we revealed that the electrostatic field of the immersion lens mainly collects electrons with energy lower than 40 eV, acting as a low-pass filter. This effect is also observed as a contrast change in LV-SEM images taken by in-lens and out-lens detectors.     

Atomic force microscopy: Unraveling the fundamental principles governing secretion and membrane fusion in cells

The story of cell secretion and membrane fusion is as old as life itself. Without these fundamental cellular processes known to occur in yeast to humans, life would cease to exist. In the last 15 years, primarily using the atomic force microscope, a detailed understanding of the molecular process and of the molecular machinery and mechanism of secretion and membrane fusion in cells has come to light. This has led to a paradigm shift in our understanding of the underlying mechanism of cell secretion. The journey leading to the discovery of a new cellular structure the ‘porosome’,—the universal secretory machinery in cells, and the contributions of the AFM in our understanding of the general molecular machinery and mechanism of cell secretion and membrane fusion, is briefly discussed in this article.     

General three-dimensional image simulation and surface reconstruction in scanning probe microscopy using a dexel representation

The ability to image complex general three-dimensional (3D) structures, including reentrant surfaces and undercut features using scanning probe microscopy, is becoming increasing important in many small length-scale applications. This paper presents a dexel data representation and its algorithm implementation for scanning probe microscope (SPM) image simulation (morphological dilation) and surface reconstruction (erosion) on such general 3D structures. Validation using simulations, some of which are modeled upon actual atomic force microscope data, demonstrates that the dexel representation can efficiently simulate SPM imaging and reconstruct the sample surface from measured images, including those with reentrant surfaces and undercut features.     

High-resolution three-dimensional imaging of the rich membrane structures of bone marrow-derived mast cells

Atomic force microscopy (AFM) enables high-resolution three-dimensional (3D) imaging of cultured bone marrow-derived mast cells. Cells were immobilized by a quick centrifugation and fixation to preserve their transient cellular morphologies followed by AFM characterization in buffer. This "fix-and-look" approach preserves the structural integrity of individual cells. Well-known membrane morphologies, such as ridges and microvilli, are visualized, consistent with prior electron microscopy observations. Additional information including the 3D measurements of these characteristic features are attained from AFM topographs. Filopodia and lamellopodia, associated with cell spreading, were captured and visualized in three dimensions. New morphologies are also revealed, such as high-density ridges and micro-craters. This investigation demonstrates that the "fix-and-look" approach followed by AFM imaging provides an effective means to characterize the membrane structure of hydrated cells with high resolution. The quantitative imaging and measurements pave the way for systematic correlation of membrane structural features with the biological status of individual cells.     

Effect of a physical phase plate on contrast transfer in an aberration-corrected transmission electron microscope

In this theoretical study we analyze contrast transfer of weak-phase objects in a transmission electron microscope, which is equipped with an aberration corrector (C(s)-corrector) in the imaging lens system and a physical phase plate in the back focal plane of the objective lens. For a phase shift of pi/2 between scattered and unscattered electrons induced by a physical phase plate, the sine-type phase contrast transfer function is converted into a cosine-type function. Optimal imaging conditions could theoretically be achieved if the phase shifts caused by the objective lens defocus and lens aberrations would be equal to zero. In reality this situation is difficult to realize because of residual aberrations and varying, non-zero local defocus values, which in general result from an uneven sample surface topography. We explore the conditions--i.e. range of C(s)-values and defocus--for most favourable contrast transfer as a function of the information limit, which is only limited by the effect of partial coherence of the electron wave in C(s)-corrected transmission electron microscopes. Under high-resolution operation conditions we find that a physical phase plate improves strongly low- and medium-resolution object contrast, while improving tolerance to defocus and C(s)-variations, compared to a microscope without a phase plate.     

Sensitivity of surface roughness parameters to changes in the density of scanning points in multi-scale AFM studies. Application to a biomaterial surface

In the field of biomaterials surfaces, the ability of the atomic force microscope (AFM) to access the surface structure at unprecedented spatial (vertical and lateral) resolution, is helping in a better understanding on how topography affects the overall interaction of biological cells with the material surface. Since cells in a wide range of sizes are in contact with the biomaterial surface, a quantification of the surface structure in such a wide range of dimensional scales is needed. With the advent of the AFM, this can be routinely done in the lab. In this work, we show that even when it is clear that such a scale-dependent study is needed, AFM maps of the biomaterial surface taken at different scanning lengths are not completely consistent when they are taken at the same scanning resolution, as it is usually done: AFM images of different scanning areas have different point-to-point physical distances. We show that this effect influences the quantification of the average (R(a)) and rms (R(q)) roughness parameters determined at different length scales. This is the first time this inconsistency is reported and should be taken into account when roughness is measured in this way. Since differences will be in general in the range of nanometres, this is especially interesting for those processes involving the interaction of the biomaterial surface with small biocolloids as bacteria, while this effect should not represent any problems for larger animal cells.     

Blind estimation of general tip shape in AFM imaging

The use of flared tip and bi-directional servo control in some recent atomic force microscopes (AFM) has made it possible for these advanced AFMs to image structures of general shapes with undercut surfaces. AFM images are distorted representations of sample surfaces due to the dilation produced by the finite size of the tip. It is necessary to obtain the tip shape in order to correct such tip distortion. This paper presents a noise-tolerant approach that can for the first time estimate a general 3-dimensional (3D) tip shape from its scanned image in such AFMs. It extends an existing blind tip estimation method. With the samples, images, and tips described by dexels, a representation that can describe general 3D shapes, the new approach can estimate general tip shapes, including reentrant features such as undercut lines.     

Surface morphology and interdiffusion of LiF in Alq3-based organic light-emitting devices

Highly efficient organic light-emitting devices (OLEDs) have been realized by insertion of a thin insulating lithium fluoride (LiF) layer between aluminum (Al) cathode and an electron transport layer, tris-(8-hydroxyquinoline) aluminum (Alq(3)). In this paper, we study the surface morphology of LiF on Alq(3) by synchrotron X-ray scattering and atomic force microscopy (AFM) as a function of thickness of LiF. We also study the interdiffusion of LiF into Al cathode as well as into Alq(3) layer as a function of temperature. Initially, LiF molecules are distributed randomly as clusters on the Alq(3) layer and then gradually form a layer as increasing LiF thickness. The interdiffusion of LiF into Al occurs more actively than into Alq(3) in annealing process. LiF on Alq(3) induces the ordering of Al to (111) direction strongly with increasing LiF thickness.     

Effects of substrates on biofilm formation observed by atomic force microscopy

Formation of biofilm is known to be strongly dependent on substrates including topography, materials, and chemical treatment. In this study, a variety of substrates are tested for understanding biofilm formation. Sheets of aluminum, steel, rubber, and polypropylene have been used to examine their effects on formation of Pseudomonas aeruginosa biofilm. In particular, the morphological variation, transition, and adhesiveness of biofilm were investigated through local measurement by atomic force microscopy (AFM). Mechanism of removing biofilm from adhering to substrate is also analyzed, thus the understanding of the mechanism can be potentially useful to prevent the biofilm formation. The results reveal that formation of biofilm can remain on rough surface regardless of substrates in hot water, which may easily induce extra-polymeric substances detachment from bacterial surface. By probing using AFM, local force–distance characterization of extra-cellular materials extracted from the bacteria can exhibit the progress of the biofilm formation and functional complexities.     

Unfolding study of native bacteriorhodopsin under acidic condition

In this study, we measured the structural properties of “blue membrane”, which is specific formation of a purple membrane (PM) upon acid titration or removal of cations, by the force curve measurement mode of an atomic force microscopy. The PM fragments were immobilized on a glass substrate and force curve measurements were carried out on the fragments under neutral (pH 7.2) and acidic (pH 2.4) condition. The results revealed that peak positions of the unfolding spectra obtained under acidic condition were shifted to the shorter extension region than those at neutral pH and that the relative position of only the first peak was changed by about 5 nm. These results suggest the possibility that the specific secondary structure is formed at the site from its C-terminus to helices F in acidified PM.     

Atomic force microscopy study of the antibacterial effects of chitosans on Escherichia coli and Staphylococcus aureus

Chitosan has been reported to be a non-toxic, biodegradable antibacterial agent. The aim of this work was to elucidate the relationship between the molecular weight of chitosan and its antimicrobial activity upon two model microorganisms, one Gram-positive (Staphylococcus aureus) and one Gram-negative (Escherichia coli). Atomic force microscopy (AFM) imaging was used to obtain high-resolution images of the effect of chitosans on the bacterial morphology. The AFM measurements were correlated with viable cell numbers, which show that the two species reacted differently to the high- and low-molecular-weight chitosan derivatives. The images obtained revealed not only the antibacterial effects, but also the response strategies used by the bacteria; cell wall collapse and morphological changes reflected cell death, whereas clustering of bacteria appeared to be associated with cell survival. In addition, nanoindentation experiments with the AFM revealed mechanical changes in the bacterial cell wall induced by the treatment. The nanoindentation results suggested that despite little modification observed in the Gram-positive bacteria in morphological studies, cell wall damage had indeed occurred, since cell wall stiffness was reduced after chitooligosaccharide treatment.     

Bloch wave-based calculation of imaging properties of high-resolution scanning confocal electron microscopy

An efficient, Bloch wave-based method is presented for simulation of high-resolution scanning confocal electron microscopy (SCEM) images. The latter are predicted to have coherent nature, i.e. to exhibit atomic contrast reversals depending on the lens defocus settings and sample thickness. The optimal defocus settings are suggested and the 3D imaging capabilities of SCEM are analyzed in detail. In particular, by monitoring average image intensity as a function of the probe focus depth, it should be possible to accurately measure the depth of a heavy-atom layer embedded in a light-element matrix.     

Explanation and correction of false step heights in amplitude modulation atomic force microscopy measurements on alkane films

We use a prototypical alkane film (n-C(32)H(66) or C32) adsorbed on a SiO(2) surface to compare step heights measured by amplitude modulation atomic force microscopy (AM-AFM) with those measured in the contact mode. The C32 film exhibits layers in which the molecules are oriented with their long axis parallel to the SiO(2) surface followed by partial layers of perpendicular molecules. We show that step heights measured in the AM and contact modes agree in all cases except where the step is between a surface formed by a layer of parallel molecules and one of perpendicular molecules. In this case, the AM mode gives a false step height that is as much as 20% lower than that measured in the contact mode and inferred from synchrotron X-ray specular reflectivity measurements. We propose that the weaker van der Waals forces between the AFM tip and a perpendicular layer compared to a parallel layer causes this discrepancy. We show how to correct the false step height by using the approximately linear relationship observed between phase angle (cantilever oscillation relative to the drive signal) and cantilever height measured in an approach curve.     

A method to correlate optical properties and structures of metallic nanoparticles

The optical response of individual nanoparticles is strongly influenced by their structures. In this report, we present a quick and simple pattern-matching based approach in which optical images of nanoparticles from localized surface plasmon resonance and single-molecule surface-enhanced Raman spectroscopy were used in conjunction with transmission electron microscopy for correlation of optical responses and the nanostructures of exactly the same nanoparticles or clusters of nanoparticles.     

Direct electron imaging in electron microscopy with monolithic active pixel sensors

A new imaging device for dynamic electron microscopy is in great demand. The detector should provide the experimenter with images having sufficient spatial resolution at high speed. Immunity to radiation damage, accumulated during exposures, is critical. Photographic film, a traditional medium, is not adequate for studies that require large volumes of data or rapid recording and charge coupled device (CCD) cameras have limited resolution, due to phosphor screen coupling. CCD chips are not suitable for direct recording due to their extreme sensitivity to radiation damage. This paper discusses characterization of monolithic active pixel sensors (MAPS) in a scanning electron microscope (SEM) as well as in a transmission electron microscope (TEM). The tested devices were two versions of the MIMOSA V (MV) chip. This 1M pixel device features pixel size of 17 x 17 microm(2) and was designed in a 0.6 microm CMOS process. The active layer for detection is a thin (less than 20 microm) epitaxial layer, limiting the broadening of the electron beam. The first version of the detector was a standard imager with electronics, passivation and interconnection layers on top of the active region; the second one was bottom-thinned, reaching the epitaxial layer from the bottom. The electron energies used range from a few keV to 30 keV for SEM and from 40 to 400 keV for TEM. Deterioration of the image resolution due to backscattering was quantified for different energies and both detector versions.     

Study of the antibacterial effects of chitosans on Bacillus cereus (and its spores) by atomic force microscopy imaging and nanoindentation

Bacillus cereus is a Gram-positive, spore-forming bacterium that is widely distributed in nature. Its intrinsic thermal resistance coupled with the extraordinary resistance against common food preservation techniques makes it one of the most frequent food-poisoning microorganisms causing both intoxications and infections. In order to control B. cereus growth/sporulation, and hence minimize the aforementioned hazards, several antimicrobial compounds have been tested. The aim of this work was to assess by atomic force microscopy (AFM) the relationship between the molecular weight (MW) of chitosan and its antimicrobial activity upon both vegetative and resistance forms of B. cereus. The use of AFM imaging studies helped us to understand how chitosans with different MW act differently upon B. cereus. Higher MW chitosans (628 and 100 kDa) surrounded both forms of B. cereus cells by forming a polymer layer—which eventually led to the death of the vegetative form by preventing the uptake of nutrients yet did not affect the spores since these can survive for extended periods without nutrients. Chitooligosaccharides (COS) (<3 kDa), on the other hand, provoked more visible damages in the B. cereus vegetative form—most probably due to the penetration of the cells by the COS. The use of COS by itself on B. cereus spores was not enough for the destruction of a large number of cells, but it may well weaken the spore structure and its ability to contaminate, by inducing exosporium loss.     

High-resolution wave field reconstruction using a through-focus series of images taken under limited electron dose

The three-dimensional Fourier filtering method and Schiske's Wiener filtering method are compared with the aim of high-resolution wave field reconstruction of an unstained deoxyribonucleic acid (DNA) molecular fiber using a through-focus series of images taken under a limited electron dose. There were some definite differences between the two reconstructed images, although the two kinds of processing are essentially equivalent except for the dimension and the filter used for processing. Through theoretical analyses together with computer simulations, the differences were proved to be primarily due to specimen drift during the experiment. Although the observed structure of the DNA molecular fiber was heavily damaged by electron beam irradiation, reconstructed images by the three-dimensional Fourier filtering method provided higher resolution information on the molecular structure even when relatively large specimen drift was included in the through-focus series. In contrast, in Schiske's Wiener filtering method, the detailed information of the structure was lost because of the drift, although the reconstructed image showed a higher signal-to-noise ratio. The three dimensional Fourier filtering method seems to be more applicable for observing radiation-sensitive materials under an extremely low electron dose, because specimen drift cannot be completely avoided.     

Quantitative EFTEM mapping of near physiological calcium concentrations in biological specimens

Although electron energy-loss spectroscopy (EELS) in the scanning transmission electron microscope (STEM) provides high sensitivity for measuring the important element, calcium, in biological specimens, the technique has been difficult to apply routinely, because of long acquisition times required. Here we describe a refinement of the complementary analytical technique of energy-filtered transmission electron microscopy (EFTEM), which enables rapid imaging of large cellular regions and measurement of calcium concentrations approaching physiological levels. Extraction of precise quantitative information is possible by averaging large numbers of pixels that are contained in organelles of interest. We employ a modified two-window approach in which the behavior of the background signal in the EELS spectrum can be modeled as a function of specimen thickness t expressed in terms of the inelastic mean free path λ. By acquiring pairs of images, one above and one below the Ca L_2,3 edge, together with zero-loss and unfiltered images, which are used to determine a relative thickness (t/λ) map, it is possible to correct the Ca L_2,3 signal for plural scattering. We have evaluated the detection limits of this technique by considering several sources of systematic errors and applied this method to determine mitochondrial total calcium concentrations in freeze-dried cryosections of rapidly frozen stimulated neurons. By analyzing 0.1 μm² areas of specimen regions that do not contain calcium, it was found that the standard deviation in the measurement of Ca concentrations was about 20 mmol/kg dry weight, corresponding to a Ca:C atomic fraction of approximately 2×10⁻⁴. Calcium concentrations in peripheral mitochondria of recently depolarized, and therefore stimulated and Ca loaded, frog sympathetic neurons were in reasonable agreement with previous data.     

Imaging of intracellular spherical lamellar structures and tissue gross morphology by a focused ion beam/scanning electron microscope (FIB/SEM)

We report the use of a focused ion beam/scanning electron microscope (FIB/SEM) for simultaneous investigation of digestive gland epithelium gross morphology and ultrastructure of multilamellar intracellular structures. Digestive glands of a terrestrial isopod (Porcellio scaber, Isopoda, Crustacea) were examined by FIB/SEM and by transmission electron microscopy (TEM). The results obtained by FIB/SEM and by TEM are comparable and complementary. The FIB/SEM shows the same ultrastructural complexity of multilamellar intracellular structures as indicated by TEM. The term lamellar bodies was used for the multillamellar structures in the digestive glands of P. scaber due to their structural similarity to the lamellar bodies found in vertebrate lungs. Lamellar bodies in digestive glands of different animals vary in their abundance, and number as well as the thickness of concentric lamellae per lamellar body. FIB/SEM revealed a connection between digestive gland gross morphological features and the structure of lamellar bodies. Serial slicing and imaging of cells enables easy identification of the contact between a lamellar body and a lipid droplet. There are frequent reports of multilamellar intracellular structures in different vertebrate as well as invertebrate cells, but laminated cellular structures are still poorly known. The FIB/SEM can significantly contribute to the structural knowledge and is always recommended when a link between gross morphology and ultrastructure is investigated, especially when cells or cellular inclusions have a dynamic nature due to normal, stressed or pathological conditions.