Imaging of silkworm meiotic chromosome by atomic force microscopy

Although structural information of mitotic chromosomes has been accumulated, little information is available for meiotic chromosome structures. Here, we applied atomic force microscopy (AFM) to investigate the ultrastructures of the silkworm, Bombyx mori, meiotic pachytene chromosome in its native state with nanometer scale resolution. Two levels of DNA folding were observed on the meiotic chromosome surface, 50-70 nm granules, which were considered to be 30 nm chromatin fibers, and spherical protrusions of 400-600 nm, which were considered to be chromomeres and arranged on the surface of the chromosome parallel to the chromosome longitudinal axis. These observations suggested that AFM study is an excellent approach for obtaining information concerning the silkworm pachytene chromosome higher order structure.     

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.     

3-D morphological characterization of the liver parenchyma by atomic force microscopy and by scanning electron microscopy

A comparative study of atomic force microscopy (AFM) and scanning electron microscopy (SEM) imaging of the healthy human liver parenchyma was carried out to determine the similarities and the differences. In this study, we compared the fine hepatic structures as observed by SEM and AFM. Although AFM revealed such typical hepatic structures as bile canaliculi and hepatocytes, it also showed the location of the nucleus and chromatin granules in rough relief structure, which was not visible by SEM. By contrast, SEM visualized other structures, such as microvilli, the central vein, and collagenous fibers, none of which was visualized by AFM. For better orientation and confirmation of most of the structures imaged by SEM and AFM, Congo Red-stained specimens were also examined. Amyloid deposits in the Disse's spaces were shown especially clearly in these images. The differences between the SEM and AFM images reflected the characteristics of the detection systems and methods used for sample preparation. Our results reveal that more detailed information on hepatic morphology is obtained by exploiting the advantages of both SEM and AFM.     

Visualization of cytoskeletal elements by the atomic force microscope

We describe a novel application of atomic force microscopy (AFM) to directly visualize cytoskeletal fibers in human foreskin epithelial cells. The nonionic detergent Triton X-100 in a low concentration was used to remove the membrane, soluble proteins, and organelles from the cell. The remaining cytoskeleton can then be directly visualized in either liquid or air-dried ambient conditions. These two types of scanning provide complimentary information. Scanning in liquid visualizes the surface filaments of the cytoskeleton, whereas scanning in air shows both the surface filaments and the total "volume" of the cytoskeletal fibers. The smallest fibers observed were ca. 50 nm in diameter. The lateral resolution of this technique was ca.20 nm, which can be increased to a single nanometer level by choosing sharper AFM tips. Because the AFM is a true 3D technique, we are able to quantify the observed cytoskeleton by its density and volume. The types of fibers can be identified by their size, similar to electron microscopy.     

Silk fibers and silk‐producing organs of Harpactea rubicunda (C. L. Koch 1838) (Araneae,Dysderidae)

Scanning electron microscopy and atomic force microscopy were used to study the silk spinning apparatus and silks of Harpactea rubicunda spiders. Three types of silk secretions that are produced by three kinds of silk spinning glands (ampullate, piriform, and pseudaciniform) and released through three types of spigots, were confirmed for both adult and juvenile spiders. Silk secretions for the construction of spider webs for shelter or retreat are produced by the pseudaciniform silk glands. Silk secretions that are released from spigots in the course of web construction are not processed by the legs during the subsequent process of hardening. Pairs of nanofibril bundles seemed to be part of the basic microarchitecture of the web silk fibers as revealed by AFM. These fiber bundles frequently not only overlap one another, but occasionally also interweave. This structural variability may strengthen the spider web. High‐resolution AFM scans of individual nanofibrils show a distinctly segmented nanostructure. Each globular segment is ～30–40 nm long along the longitudinal axis of the fiber, and resembles a nanosegment of artificial fibroin described by Perez‐Rigueiro et al. (2007). Microsc. Res. Tech., 2013. © 2012 Wiley Periodicals, Inc.     

Nano-scale imaging of chromosomes and DNA by scanning near-field optical/atomic force microscopy

Nano-scale structures of the YOYO-1-stained barley chromosomes and lambda-phage DNA were investigated by scanning near-field optical/atomic force microscopy (SNOM/AFM). This technique enabled precise analysis of fluorescence structural images in relation to the morphology of the biomaterials. The results suggested that the fluorescence intensity does not always correspond to topographic height of the chromosomes, but roughly reflects the local amount and/or density of DNA. Various sizes of the bright fluorescence spots were clearly observed in fluorescence banding-treated chromosomes. Furthermore, fluorescence-stained lambda-phage DNA analysis by SNOM/AFM demonstrated the possibility of nanometer-scale imaging for a novel technique termed nano-fluorescence in situ hybridization (nano-FISH). Thus, SNOM/AFM is a powerful tool for analyzing the structure and the function of biomaterials with higher resolution than conventional optical microscopes.     

Atomic force microscopy study of chromosome surface structure changed by protein extraction

We applied atomic force microscopy (AFM) to investigate the surface structure of barley chromosome in combination with a chemical treatment method. As a result, we have obtained high-resolution topographic images of granular structures with a diameter of ca. 50 nm on the surface of critical-point dried metaphase chromosomes. Treatment with 2M NaCl significantly modified the chromosome surface structure: surface roughness was increased and chromosome thickness was decreased. The NaCl treatment extracted two major proteins with molecular weights of 4000 and 20,000 Da. These proteins might be belonging to non-histone protein families that do not contain any aromatic amino acid. The results demonstrate the advantage of the combined method of high-resolution AFM imaging and chemical treatments for understanding nano-scale surface structures of the chromosome.     

Human chromosome structure studied by scanning force microscopy after an enzymatic digestion of the covering cell material

In standard preparations, metaphase human chromosomes are covered by a cell material film composed mainly of proteins and RNA. This film (approximately 30 nm thickness) hides the chromosome structure to the tip of a scanning force microscope. In this work, a mild enzymatic treatment is applied to remove the cell material film. After treatment, the individual chromatin fibers at the surface were resolved. Furthermore, the chromosome shows a thickness modulation, in which thicker/thinner regions could be associated with G/R bands. Finally, the topography of the chromosomes in solution is presented. The chromosome volume swelled about five-fold and chromatin packaging in bands and coils was observed.     

Molecular dynamics of DNA and nucleosomes in solution studied by fast-scanning atomic force microscopy

Nucleosome is a fundamental structural unit of chromatin, and the exposure from or occlusion into chromatin of genomic DNA is closely related to the regulation of gene expression. In this study, we analyzed the molecular dynamics of poly-nucleosomal arrays in solution by fast-scanning atomic force microscopy (AFM) to obtain a visual glimpse of nucleosome dynamics on chromatin fiber at single molecule level. The influence of the high-speed scanning probe on nucleosome dynamics can be neglected since bending elastic energy of DNA molecule showed similar probability distributions at different scan rates. In the sequential images of poly-nucleosomal arrays, the sliding of the nucleosome core particle and the dissociation of histone particle were visualized. The sliding showed limited fluctuation within ∼50 nm along the DNA strand. The histone dissociation occurs by at least two distinct ways: a dissociation of histone octamer or sequential dissociations of tetramers. These observations help us to develop the molecular mechanisms of nucleosome dynamics and also demonstrate the ability of fast-scanning AFM for the analysis of dynamic protein–DNA interaction in sub-seconds time scale.     

Scanning force microscopy of chromatin fibers in air and in liquid

We have adapted specimen preparation techniques of conventional electron microscopy for visualizing chromatin structures in the scanning force microscope (SFM) in air and in liquid. The beaded substructure of the nucleoprotein filament was obtained after hypotonic lysis of chicken erythrocytes and air drying, whereas supranucleosomal structures were preserved after treatment of cell nuclei with detergent. In the latter case, the nucleosomes were still distinct but appeared more condensed. A modified droplet diffusion-spreading technique of chromatin from Namalwa cells (a human B-lymphoid line) yielded a uniform filamentous morphology and similar fiber appearance. A reversible swelling of spread chromatin was observed upon exposure of air-dried samples to solutions differing in salt concentrations.     

Dynamic force microscopy imaging of native membranes

We employed magnetic ACmode atomic force microscopy (MACmode AFM) as a novel dynamic force microscopy method to image surfaces of biological membranes in their native environments. The lateral resolution achieved under optimized imaging conditions was in the nanometer range, even when the sample was only weakly attached to the support. Purple membranes (PM) from Halobacterium salinarum were used as a test standard for topographical imaging. The hexagonal arrangement of the bacteriorhodopsin trimers on the cytoplasmic side of PM was resolved with 1.5nm lateral accuracy, a resolution similar to images obtained in contact and tapping-mode AFM. Human rhinovirus 2 (HRV2) particles were attached to mica surfaces via nonspecific interactions. The capsid structure and 2nm sized protein loops of HRV2 were routinely obtained without any displacement of the virus. Globular and filamentous structures on living and fixed endothelial cells were observed with a resolution of 5-20nm. These examples show that MACmode AFM is a favorable method in studying the topography of soft and weakly attached biological samples with high resolution under physiological conditions.     

Direct imaging in a water layer of human chromosome fibres composed of nucleosomes and their higher-order structures by laser-plasma X-ray contact microscopy

X-ray contact microscopy with a 300-ps-duration laser-plasma X-ray source has been used to image hydrated human chromosomes. Clearly imaged are individual nucleosomes and their higher-order particles (superbeads), elementary chromatin fibrils c. 30 nm in diameter and their higher-order fibres of various sizes up to c. 120 nm in diameter. The results demonstrate that X-ray microscopy is now capable of opening a new path of investigation into the detailed structures of hydrated chromosome fibres in their natural state.     

The mechanism of cellulase action on cotton fibers: evidence from atomic force microscopy

Two cellulases from Trichoderma reesei--an exoglucanase, CBH I, and an endoglucanase, EG II--alone and in combination were incubated with cotton fibers. The effects of the cellulases on the surfaces of the cotton fibers were examined by atomic force microscopy. At high magnification, the physical effects on the fibers caused by the two types of enzymes were considerably different. Treatment with CBH I resulted in the appearance of distinct pathways or tracks along the length of the macrofibril. Treatment with EG II appeared to cause peeling and smoothing of the fiber surface. In combination, their effect was observed to be greatest when both enzymes were present simultaneously. When fibers smoothed by treatment with EG II were treated subsequently with CBH I, further evidence of path way formation caused by the action of CBH I along the fibers was observed. Incubation with a cellulase from Thermotoga maritima that lacks a cellulose binding domain had no effect on the surface of cotton fibers. These images provide the first physical evidence of differences in the effect of cellulase components action on the surface of cotton fibers and provide evidence for the movement or tracking of CBH I along the fibers. The first AFM image of CBH I molecules are presented.     

Atomic force microscopy imaging and 3-D reconstructions of serial thin sections of a single cell and its interior structures

The thin sectioning has been widely applied in electron microscopy (EM), and successfully used for an in situ observation of inner ultrastructure of cells. This powerful technique has recently been extended to the research field of atomic force microscopy (AFM). However, there have been no reports describing AFM imaging of serial thin sections and three-dimensional (3-D) reconstruction of cells and their inner structures. In the present study, we used AFM to scan serial thin sections approximately 60 nm thick of a mouse embryonic stem (ES) cell, and to observe the in situ inner ultrastructure including cell membrane, cytoplasm, mitochondria, nucleus membrane, and linear chromatin. The high-magnification AFM imaging of single mitochondria clearly demonstrated the outer membrane, inner boundary membrane and cristal membrane of mitochondria in the cellular compartment. Importantly, AFM imaging on six serial thin sections of a single mouse ES cell showed that mitochondria underwent sequential changes in the number, morphology and distribution. These nanoscale images allowed us to perform 3-D surface reconstruction of interested interior structures in cells. Based on the serial in situ images, 3-D models of morphological characteristics, numbers and distributions of interior structures of the single ES cells were validated and reconstructed. Our results suggest that the combined AFM and serial-thin-section technique is useful for the nanoscale imaging and 3-D reconstruction of single cells and their inner structures. This technique may facilitate studies of proliferating and differentiating stages of stem cells or somatic cells at a nanoscale.     

C-banding visualized by atomic force microscopy

C-banding is a method used for studying chromosome rearrangements near centromeres and for investigating polymorphisms. In human chromosomes, the C-bands are located at the centromere of all the chromosomes and the distal long arm of the Y chromosome. In this study, we aimed to detect the structural changes in chromosomes during the stages of C-banding by atomic force microscopy. We observed crater-like structures in the chromosomes after 2xSSC (saline sodium citrate) treatment and measured the relative difference between the heights of chromatid and centromere of the chromosomes. Results showed that the relative difference was 3 nm in chromosomes 1, 9, 16, and Y, whereas in the other chromosomes this value was 11.6 nm. After Giemsa staining, the relative difference increased by a factor of 16 in chromosomes 1, 9, 16, and Y. The other chromosomes showed no such increase, which is in accordance with our suggestion that nonhiston proteins associated with DNA in constitutive heterochromatin can make the constitutive heterochromatin resistant to C-banding.     

Near-field scanning optical microscopy using polymethylmethacrylate optical fiber probes

We report the first use of polymethylmethacrylate (PMMA) optical fiber-made probes for scanning near-field optical microscopy (SNOM). The sharp tips were prepared by chemical etching of the fibers in ethyl acetate, and the probes were prepared by proper gluing of sharpened fibers onto the tuning fork in the conditions of the double resonance (working frequency of a tuning fork coincides with the resonance frequency of dithering of the free-standing part of the fiber) reported earlier for the case of glass fibers. Quality factors of the probes in the range 2000–6000 were obtained, which enables the realization of an excellent topographical resolution including state-of-art imaging of single DNA molecules. Near-field optical performance of the microscope is illustrated by the Photon Scanning Tunneling Microscope images of fluorescent beads with a diameter of 100 nm. The preparation of these plastic fiber probes proved to be easy, needs no hazardous material and/or procedures, and typical lifetime of a probe essentially exceeds that characteristic for the glass fiber probe.     

Numerical chromosomal abnormalities detected by atomic force microscopy.

The numerical abnormalities of human metaphase chromosomes, fixed according to standard procedures for optical microscopy but not treated for banding, were detected by atomic force microscopy (AFM). High-resolution AFM imaging of chromosomes in trisomy 13, 21, and Klinefelter syndrome can be compared directly with the traditional optical image. The unbanded metaphase chromosomes, including the extra ones in trisomic patients showed a structural pattern very similar to G-banding. Comparison of AFM images with light microscopic data allows the identification of specific chromosomes, and images of chromosomes showing numerical and structural abnormalities can then be analysed.     

Investigation of human hair fibers using lateral force microscopy

Atomic force microscopy (AFM) and lateral force microscopy (LFM) were used to investigate the morphologic and surface changes associated with various surface modifications to human hair. These included extraction with a series of solvents, bleaching, and treatment with a cationic copolymer. The study assessed the ability of these techniques to distinguish the changes in surface properties, including morphology and friction coefficient, as manifested in changes brought about by the indicated surface modifications. While topographic morphology can easily be investigated with contact AFM. LFM offers an additional tool for probing the surface distribution of oils and waxes. The removal of surface lipids from the fiber surface was accomplished using soxhlet extraction with t-butanol and n-hexane, while the free internal lipids (within the fiber structure) were removed by extraction with a mixture of chloroform and methanol (70:30, v/v). In addition, the surface of hair was modified with the cationic polymer, co(vinyl pyrrolidone-methacrylamidopropyl trimethylammonium chloride [PVP/MAPTAC]), and its distribution on the surface was monitored. Ambient AFM and LFM studies of surface modified and native fibers clearly indicate that when investigated as a function of tip loading force, the different modifications result in changes of the friction coefficient, which increase in this order: native, bleached, solvent extracted, and polymer-treated hair. Friction images show surface variations that are interpreted as areas of varying lipid film coverage. In addition, topographic images of the fibers show the presence of small pores, which become increasingly prevalent upon solvent extraction.     

The effect of colcemid and aluminum on the ultrastructure of human metaphase chromosomes

Recently, we have reported new basic information on the ultrastructure of human metaphase chromosomes using both scanning and transmission electron microscopy. This includes the observation of a bipartite chromatid structure (BCS) for some metaphase chromatids, a "zipper-like" configuration (ZC) between chromatids that likely resulted from chromatin coiling, and a "brush-like" border (BB) that was observed primarily on chromosomes that were not exposed to colcemid. Now we have examined the effects of colcemid and several metals on the occurrence of the BCS, the ZC, and the BB. Although we do not as yet know the function of the zipper-like and bipartite chromatid configurations, we have found that colcemid clearly caused a significant increase in the occurrence of chromosomes with a BCS or ZC. We also have confirmed our original observation of increased occurrence of the BB on chromosomes not exposed to colcemid and finally, have shown that aluminum and other metals had some effect on the frequencies of the BCS, the ZC, and the BB with and without exposure to colcemid.     

Visualization of microfibrillar elements in cross‐section of polyacrylonitrile fiber along the fiber spinning line

The microfibrils served as the structural elements in polyacrylonitrile (PAN) fiber, which played an important role in the quality of the PAN precursor fibers. Their morphologies were examined by the scanning electron microscopy (SEM), atomic force microscopy (AFM) and high‐resolution transmission electron microscope (HRTEM). The microfibrils existed in all of PAN fibers and arranged evenly in the cross‐sections. Furthermore, the pores existed between the microfibrils. The unoriented microfibrillar network was already formed in nascent fiber during coagulated process. Although the microfibrillar network was elongated and the microfibrils oriented along the fiber longitudinal direction during the spinning process, the interconnected microfibrillar network was still existed in the fiber transverse section. Furthermore, the transverse connection of the microfibrils was reinforced and the small microfibrils were tended to aggregate into the large fibrils. For mechanical performance of PAN fibers, their tensile strength increased to 708 MPa and the elongation at break decreased to 15.5%. PAN fibers exhibited ductile rupture during the mechanical test and the microfibrils served as reinforcing elements.