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.     

Using the atomic force microscope to observe and study the ultrastructure of the living BIU-87 cells of the human bladder cancer

In this study, the ultrastructure of living BIU-87 cells of human bladder cancer was mapped using atomic force microscopy to reveal the dynamic change of single cancerous cell division. Simultaneously, the feasibility and functional reliability of the atomic force microscope (AFM) were established and a laboratory model using AFM to study living cancerous cells was created. In this experiment, BIU-87 cells of human bladder cancer were cultured by conventional methods and grown in gelatin-treated dishes. A thermostat was used for preserving the cell's living temperature. Scanning of these cells using AFM was carried out in physiologic condition. The AFM images of the ultrastructure of living BIU-87 cells as well as those of the cell's membrane and cytoskeleton were very clear. The dynamic phenomenon of single cell division was observed. It was concluded that the AFM was able to observe and depict the ultrastructure of living cells of human bladder cancer directly and in real time. This experimental model is expected to play an important role in elucidating the cancerous mechanism of bladder normal cells at the atomic or nanometer level.     

Papain‐gel degrades intact nonmineralized type I collagen fibrils

Papain‐gel has been utilized as a chemo‐mechanical material for caries removal due to its ability to preserve underlying sound dentin. However, little is known about the effect of the papain enzyme on intact type I collagen fibrils that compose the dentin matrix. Here we sought to define structural changes that occur in intact type I collagen fibrils after an enzymatic treatment with a papain‐gel. Intact and nonmineralized type I collagen fibrils from rat tail were obtained and treated with a papain‐gel (Papacarie) for 30 s, rinsed with water and imaged using an atomic force microscope (AFM). Additionally, polished healthy dentin specimens were also treated using the same protocol described above and had their elastic modulus (E) and hardness (H) measured by means of AFM‐based nanoindentation. AFM images showed that the papain‐gel induced partial degradation of the fibrils surface, yet no rupture of fibrils was noticed. The distinction between gap and overlap zones of fibrils vanished in most regions after treatment, and overlap zones appeared to be generally more affected. Mechanical data suggested a gradual decrease in E and H after treatments. A significant two‐fold drop from the values of normal dentin (E=20±1.9, H=0.8±0.08 GPa) was found after four applications (E=9.7±3.2, H=0.24±0.1 GPa) (P<0.001), which may be attributed to the degradation of proteoglycans of the matrix. In summary, this study provided novel evidence that intact nonmineralized type I collagen fibrils are partially degraded by a papain‐gel. SCANNING 31: 253–258, 2009. © 2010 Wiley Periodicals, Inc.     

Artesunate induced morphological and mechanical changes of Jurkat cell studied by AFM

In this study, we have used atomic force microscopy (AFM) to study the morphology and mechanical property changes of Jurkat cells exposed to different concentrations of Artesunate (ART) for 24 h at single cellular level. Cell viability and proliferation assays were performed by using the Cell Counting Kit‐8. The concentration of ART, which resulted in the inhibition rate >50% was selected. The AFM images revealed that the cell membrane changed and the ultrastructure also became complex. Mechanical properties of individual cell were tracked with AFM‐based force spectroscopy. The force curves revealed that when a cell was exposed to the ART, the mechanical properties changed obviously. Treated cells had a lower adhesion force of 416.8±37.9 pN, whereas control group had a higher adhesion force of 1064.2±97.0 pN. The Young's modulus decreased to nearly one‐third, from control group of 0.648±0.037 kPa to treated group of 0.254±0.035 kPa and the stiffness increased to nearly 1.5 times, from control group of 1.231±0.084 mN/m to treated group of 1.917±0.137 mN/m. These results suggest that ART can inhibit the proliferation of Jurkat and induce changes in the morphological structure and mechanical properties of Jurkat cells. The high resolution and high sensitivity of AFM can be used to detect morphological and mechanical properties of cells exposed to ART. The AFM may be developed to be a useful tool for detecting the cell death and evaluating the anti‐carcinogen efficacy against tumor cell. SCANNING 31: 83–89, 2009. © 2009 Wiley Periodicals, Inc.     

Third ventricular pineal gland grafts in the gerbil: an electron microscopical and immunohistochemical investigation.

The superficial pineal gland was grafted into the third ventricle of adult Mongolian gerbils. Donor pineal glands from both neonatal and 3-4 week old gerbils were able to survive for at least 6 months. The pinealocytes of the grafted superficial pineal glands maintained the morphology and the S-antigen immunoreactivity of the in situ pineal complex. Synaptic ribbons and spherules were present but rare. Unlike the in situ pinealocytes, glycogen accumulations were common in the graft pinealocytes. Site specific modulation of structure was indicated as the ventricular surface of the grafts became covered with cerebrospinal fluid (CSF)-contacting pinealocytes typical of those seen in the deep pineal. The CSF-contacting pinealocytes of the graft had numerous processes that extended along the ventricular surface of the graft. The blood vessels of the grafts had non-fenestrated endothelium and wide perivascular areas typical of those seen in the in situ pineal complex. Tyrosine hydroxylase-immunopositive nerve fibers were present in the grafted tissue indicating reinnervation of the graft. The source of the fibers was not determined. The nerve fibers were present both within the perivascular area and within the parenchyma where they were associated with pinealocytes. The results demonstrate that the cerebral ventricles are an ideal location for the survival of superficial pineal gland grafts. It is suggested that pineal grafts may be a means to further study pineal development and innervation.     

Light microscopy evaluation of polyurethane vascular grafts porosity by Sudan Black B staining

In small‐diameter vascular grafts, the porosity of the internal surface plays an important role because it affects initial thrombus deposition and therefore the graft’s patency. As well as many other studies reported in the literature we have carried out a study of the relationship between porosity and the manufacturing parameters of polyurethane (PU) grafts by standard scanning electron microscopy (SEM) analysis. However, SEM was not completely satisfactory for evaluating the ‘sponge‐like’ uptake of water by the graft due to the unavoidable water loss and metal coating during preparation. In fact this preparation produces artefacts of the three‐dimensional porous structure. To avoid this problem we investigated the possibility of observing the graft’s internal surface through a stereomicroscope after it had absorbed water. We looked into a simple staining procedure which preferentially colours the PU graft fibres with respect to the void areas. After testing different kinds of stains, we eventually found that Sudan Black B, which usually stains for all kinds of lipid, turned out to be an excellent stain for the water‐loaded PU grafts when diluted with ethanol. This staining procedure, coupled with a computerized image analysis system, allowed us to evaluate the degree of void and average void size of the graft internal surface and to correlate these data with graft density and manufacturing parameters.     

Investigations on the leaf surface ultrastructure in grapevine (Vitis vinifera L.) by scanning microscopy

Several Scanning microscopy techniques were used to investigate the leaf surface ultrastructure in the local “Razegui” grapevine cultivar (Vitis vinifera L.). Conventional scanning electron microscopy performed on glutaraldehyde‐fixed samples allowed observation of well‐preserved epidermal cells with an overlaying waxy layer. At a high magnification, the waxy layer exhibited crystalline projections in the form of horizontal and vertical platelets. Also, to avoid eventual ultrastructural alterations inherent in the use of solvents during sample preparation, fresh leaf blade samples were directly observed by environmental scanning electron microscopy. A classical image of convex living epidermal cells was observed. At 2400× magnification, epicuticular waxes exhibited a granular structure. However, high‐magnification images were not obtained with this device. The atomic force microscopy (AFM) performed on fresh leaf blade samples allowed observation of a textured surface and heterogeneous profiles attributed to epicuticular wax deposits. AFM topography images confirmed further, the presence of irregular crystalloid wax projections as multishaped platelets on the adaxial surface of grapevine leaf. SCANNING 31: 127–131, 2009. © 2009 Wiley Periodicals, Inc.     

Effect of twisting hamstring tendon grafts on strength and joint laxity through the range of knee movement

Twisting or braiding of hamstring tendon grafts for use in anterior cruciate ligament reconstructions has been advocated to increase their strength and stiffness under load. In this study, a two-dimensional model was used to determine the failure strength of twisted and parallel grafts and associated knee laxity under simulated physiological loading conditions. For validation, mechanical tests of tendon grafts were also simulated with these models. The simulated physiological loading of the graft models showed that knees with twisted grafts had greater laxity than knees with parallel grafts, although there was little difference in failure load between the two graft configurations. The tensile loading of the graft models showed little difference in failure load when the tendons were modelled using line segments. When the tendons were considered as three-dimensional helical elements, which more accurately describe the tendon structure, the failure load of the twisted graft decreased significantly. This research provided no evidence to support the belief that a twisted tendon graft is a superior graft configuration relative to a parallel tendon graft.     

AFM and STM study of beta-amyloid aggregation on graphite

Atomic force microscopy (AFM) and scanning tunneling microscopy (STM) have been employed in situ and ex situ to directly study the aggregation of beta-amyloid(1-42) (Abeta42) peptide on hydrophobic graphite.From in situ AFM images, Abeta42 peptides were seen to aggregate into the sheets that preferred to three orientations with characteristic 3-fold symmetry (Proc. Natl. Acad. Sci. USA 96 (1999) 3688). The sheets were formed by parallel narrow lines with a height of 0.8-1.0nm and a width of 12-14nm. The narrow lines looked like beaded chains and have a right-handed axial periodicity.The high-resolution ex situ AFM and STM images showed that some fibrils of beta-amyloid had a characteristic domain texture, indicating they were formed through the association of protofibrils and monomers. The fibril containing lateral associated filaments that exhibited right-handed twist was clearly observed in the STM image.These results provide important clues to study the detailed structure of beta-amyloid aggregates and the mechanism of the Abeta fibrils formation on hydrophobic surface.     

Investigation of nanostructural changes following acute injury using atomic force microscopy in rabbit vocal folds

There continues to be a paucity of data regarding the nanostructural changes of vocal fold (VF) collagen after injury. The aim of this study is to investigate the nanostructural and morphological changes in the rabbit VF lamina propria following acute injury using atomic force microscopy (AFM). Unilateral VF injury was performed on 9 New Zealand breeder rabbits. Sacrifice and laryngeal harvest were performed at three time points: 1 day, 3 days, and 7 days after injury. Histology and immunohistochemistry data were collected to confirm extracellular matrix (ECM) changes in rabbit VF. The progressive changes in thickness and D‐spacing of VF collagen fibrils were investigated over a 7‐day postinjury period using AFM. At post‐injury day 1, a fibrin clot and inflammatory cell infiltration were observed at the injured VF. The inflammatory score at postinjury day 1 was highest in injured VF tissue, with a significant decrease at postinjury day 7. The immunoreactivity of inflammatory proteins (COX‐2, TNF‐α) was observed in VF up to day 7 after injury. AFM investigation showed clustered and disorganized collagen fibrils at the nanoscale resolution at post‐injury day 7. Collagen fibrils in injured VF at postinjury day 7 were significantly thicker than control and postinjury days 1 and 3 (P < 0.001). D‐spacing of collagen at postinjury day 7 was not studied due to loss of distinct edges resulting from immature collagen deposition. AFM investigation of VF could add valuable information to understanding micromechanical changes in VF scar tissue. Microsc. Res. Tech. 78:569–576, 2015. © 2015 Wiley Periodicals, Inc.     

The structure of proteoglycan aggregate determined by atomic force microscopy

Proteoglycan aggregate is the major extracellular matrix component in cartilage, comprising about 18% of the dry weight of hyaline cartilage. The proteoglycan aggregate is the major substance in cartilage which resists compression in the joint. The purpose of this study was to utilize the newly developed imaging technique, Atomic force Microscopy (AFM), to visualize the ultrastructure of proteoglycan aggregates. The proteoglycan aggregate molecules were imaged in air using the tapping mode of the AFM. The images illustrated the ultrastructure of the aggregates, especially the individual proteoglycan and the core hyaluronic acid. In addition to the length and width of each molecule, the height of the proteoglycan aggregates and the individual proteoglycans could be directly measured. The images of the ultrastructures of proteoglycan aggregates visualized from the AFM are comparable with those using conventional electron microscopy approaches. Nevertheless, the sample preparation for AFM imaging does not involve fixation, staining, coating, and other routine procedures required for traditional electron microscopy imaging. Thus, this technique could be a simple alternative approach for future analysis of proteoglycan aggregate and its assembly.     

Real-time enzymatic degradation of human dentin collagen fibrils exposed to exogenous collagenase: an AFM study in situ

Objective: This study was carried out to observe the enzymatic degradation of human dentin collagen fibrils exposed to exogenous collagenase in situ using atomic force microscopy, to understand the characteristics of the enzymatic degradation of collagen fibrils on dentin specimens. Methods: Polished dentin specimens from caries‐free third molars were etched with citric acid, and then treated with an aqueous solution of 6.5% NaOCl for 120 s. The specimen was then put into a fluid cell and treated with a mixed solution of collagenase I (MMP‐1) and collagenase II (MMP‐8) for 9 h. AFM with contact mode was performed in situ to monitor the enzymatic degradation process of the dentin collagen fibrils. The distinctly topographic changes of the dentin surface were recorded continuously during different stages of the enzymatic degradation process. Results: The mixed solution of exogenous collagenase I and collagenase II could degrade dentin organic matrix (mainly collagen) efficiently, and the structures of dentin substrate were clearly exposed. Conclusion: It is possible to carry out real‐time observations on the enzymatic biodegradation process of human dentin collagen fibrils on dentin specimens with atomic force microscopy in situ. By this means, the fine structures of the etched dentin substrate were clearly revealed, possibly contributing to the related study of human dentin in vitro.     

Ultrastructure of Trypanosoma cruzi revisited by atomic force microscopy

Most advances in atomic force microscopy (AFM) have been accomplished in recent years. Previous attempts to use AFM to analyze the organization of pathogenic protozoa did not significantly contribute with new structural information. In this work, we introduce a new perspective to the study of the ultrastructure of the epimastigote form of Trypanosoma cruzi by AFM. Images were compared with those obtained using field emission scanning electron microscopy of critical point dried cells and transmission electron microscopy of negative stained detergent-extracted and air-dried cells. AFM images of epimastigote forms showed a flagellum furrow separating the axoneme from the paraflagellar rod (PFR) present from the emergence of the flagellar pocket to the tip of the flagellum. At high magnification, a row of periodically organized structures, which probably correspond to the link between the axoneme, the PFR and the flagellar membrane were seen along the furrow. In the origin of the flagellum, two basal bodies were identified. Beyond the basal bodies, small periodically arranged protrusions, positioned at 400 nm from the flagellar basis were seen. This structure was formed by nine substructures distributed around the flagellar circumference and may correspond to the flagellar necklace. Altogether, our results demonstrate the importance of the application of AFM in the structural characterization of the surface components and cytoskeleton on protozoan parasites.     

生物大分子超微结构研究中的原子力显微术

原子力显微镜作为第三代显微探测工具,具有原子级的空间分辨率,其样品制备方法简单易行,可在离体的近生理条件下直接观测生物样品及其动态变化过程,能够对样品进行力学操纵,在观察生物大分子的结构和生物力学特性上具有显著的优势。本文尝试从蛋白质、核酸、多糖的超微结构和力学特性的研究角度入手,期望向读者展现出原子力显微镜在大分子生物学研究中的应用前景。     

Reconstruction of the anterior cruciate ligament.

Ligaments are strong collagenous structures that act as constraints on joint motion, thus confining the articular surfaces to more or less the same paths. In so doing they prevent arbitrary apposition of these surfaces from occurring and resulting in abnormal stresses which may damage the joint surfaces. Ligaments rupture due to excessive loads, particularly those resulting from trauma occurring during sporting events or motor vehicle accidents. Knee and ankle joints have the highest frequency of ligamentous injuries. This paper is a brief review of the current approaches to the reconstruction of the knee ligaments with specific reference to the anterior cruciate ligament (ACL) being the most frequently reconstructed. This is not only because it is frequently injured but also because of the debilitating consequences of such an injury. Approaches ranging from the conservative to those that advocate the use of frank prosthetic replacement have been adopted by surgeons at both ends of the spectrum. Following a discussion of the rationale for reconstruction of the ACL, the mechanical and biological considerations of the reconstructive procedure are discussed. The different methods of ACL reconstruction are reviewed. These include: (a) primary repair, (b) reconstruction with different tissues, including autogenous allografts and xenografts, (c) reconstruction employing different synthetic devices. A brief discussion of the procedures used for reconstruction with different types of tissue and of the surviving examples of the synthetic devices will follow.     

Imaging and studying human topoisomerase I on mica surfaces in air and in liquid by atomic force microscopy

In this study, the topography of human topoisomerase I (TOPO I) on mica surfaces in air and in liquid has been studied by atomic force microscopy (AFM). The average height of TOPO I on mica surface in air measured by AFM was 2.59±0.32 nm. After adsorption of the 0.3 U/µl TOPO I on mica surfaces for 2 h, and then imaged in liquid by AFM, well‐separated single TOPO I was observed. The average height of TOPO I on mica surfaces in liquid measured by AFM was 2.93±0.42 nm. After adsorption of the 4 U/µl TOPO I on mica surfaces for 1.5 h, TOPO I monolayer can be formed. The produced TOPO I monolayer on mica was flat and exhibited good stability. SCANNING 31: 160–166, 2009. © 2009 Wiley Periodicals, Inc.     

Metal vs bioabsorbable interference screws: initial fixation

Interference screws are used for graft fixation in anterior cruciate ligament reconstruction. There is current debate as to whether metal or bioabsorbable varieties provide the optimal results. This paper provides a critique of the single load-to-failure uniaxial tensile test data available in the literature, which provide the initial postoperative characteristics. Both metal and bioabsorbable screws were found to give similar levels of pull-out force and stiffness when used on similar types of graft material. Screws with bone-patellar tendon-bone grafts provided the greatest values, exceeding the forces transmitted through the native anterior cruciate ligament during normal daily activities. Hamstring grafts provided lower values, only being comparable with the forces of daily living. Consequently, this raises the question as to whether the rehabilitation regime of the hamstring patient must not be aggressive in the early postoperative stages when using either metal or bioabsorbable screws. This would ensure that the graft will not fail in the initial postoperative period before biological fixation occurs, which will then boost the strength to an acceptable level.     

Cemented cup stability during lever-out testing after acetabular bone impaction grafting with bone graft substitutes mixes containing morselized cancellous bone and tricalcium phosphate--hydroxyapatite granules

Bone defects after failed total hip arthroplasty can be reconstructed with impacted morselized bone grafts and a cemented cup. In the near future the amount of bone grafts available for surgical purposes will be insufficient. Ceramic calcium phosphates [tricalcium phosphate (TCP) and hydroxyapatite (HA)] have been widely considered as potential bone graft substitutes or bone graft extenders. In the past, mechanical experiments have been performed to determine implant stability of bone grafts and ceramic TCP-HA granules mixes under a compressive load. However, in-vivo migration studies suggest that shear loading may be equally important. This in-vitro study investigated the initial stability of cups reconstructed with various mixes of bone grafts and ceramic TCP-HA granules in a lever-out situation, where shearing is the predominant loading mode. It was found that the cups reconstructed with mixes of bone graft and TCP-HA granules exhibited greater mechanical stability than the cups reconstructed with bone grafts only. It is concluded that from a mechanical standpoint, when considering shear force resistance, 50-50 per cent volume mix and 25-75 per cent volume mix of morselized cancellous bone graft and TCP-HA granules both provide adequate initial cup stability and can be used for acetabular reconstructions with the bone impaction grafting technique.     

Assessment of pressure drop flowrate relationship of axillobifemoral bypass grafts.

An in vitro study was conducted to measure the pressure distributions in knitted polyester axillobifemoral bypass grafts under steady and pulsatile flow of a Newtonian fluid. The relationship between pressure drop and flowrate was determined across the outflow branches of two types of axillobifemoral grafts. Pressure losses were greater across the 90 degrees side branch of the T-type graft than in the Y-type graft at all Reynolds numbers studied. Mean pulsatile pressure drops in the T graft were greater than those measured in steady flow at the same Reynolds number. Estimates of the likely in vivo pressure drops are made under resting and exercise flow conditions. It is suggested that the Y graft is less likely to have a significant effect on blood supply to the lower limbs, even at high blood flowrates.     

Atomic force microscopy of freeze-fracture replicas of rat atrial tissue

Atomic force microscopy (AFM) has provided three-dimensional (3-D) surface images of many biological specimens at molecular resolution. In the absence of spectroscopic capability for AFM, it is often difficult to distinguish individual components if the specimen contains a population of mixed structures such as in a cellular membrane. In an effort to understand the AFM images better, a correlative study between AFM and the well-established technique of transmission electron microscopy (TEM) was performed. Freeze-fractured replicas of adult rat atrial tissue were examined by both TEM and AFM. The same replicas were analysed and the same details were identified, which allowed a critical comparison of surface topography by both techniques. AFM images of large-scale subcellular structures (nuclei, mitochondria, granules) correlated well with TEM images. AFM images of smaller features and surface textures appeared somewhat different from the TEM images. This presumably reflects the difference in the surface sensitivity of AFM versus TEM, as well as the nature of images in AFM (3-D surface contour) and TEM (2-D projection). AFM images also provided new information about the replica itself. Unlike TEM, it was possible to examine both sides of the replica with AFM; the resolution on one side was significantly greater compared with the other side. It was also possible to obtain quantitative height information which is not readily available with TEM.